Toxicon 93 (2015) 144e154

Contents lists available at ScienceDirect

Toxicon
journal homepage: www.elsevier.com/locate/toxicon

First crotoxin-like phospholipase A2 complex from a New World nonrattlesnake species: Nigroviriditoxin, from the arboreal Neotropical
snake Bothriechis nigroviridis

n Ferna

ndez a, Libia Sanz b, Davinia Pla b,
Bruno Lomonte a, *, Diana Mora-Obando a, Julia
a
b

Mara Gutie

rrez , Juan J. Calvete
Jose
a
b

Instituto Clodomiro Picado, Facultad de Microbiologa, Universidad de Costa Rica, San Jos

e 11501, Costa Rica
Instituto de Biomedicina de Valencia, CSIC, Jaume Roig 11, 46010 Valencia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 19 September 2014
Received in revised form
21 November 2014
Accepted 27 November 2014
Available online 28 November 2014

. A previous
Bothriechis nigroviridis is an arboreal Neotropical pitviper found in Costa Rica and Panama
proteomic proling of its venom revealed the presence of proteins with homology to the A and B subunits of crotoxin/Mojave toxin, a heterodimeric phospholipase A2 (PLA2) complex only described in
rattlesnake venoms (genera Crotalus and Sistrurus). The native crotoxin-like heterodimer, named
nigroviriditoxin, and its A and B subunits were isolated in the present work, and the complete amino acid
sequence of the B subunit was determined. The puried A and B components were demonstrated to form
a complex when reconstituted under native conditions. Nigroviriditoxin presents features similar to
crotoxin, albeit displaying lower toxicity: the A component decreases the PLA2 activity of the B
component, and increases its lethal potency in mice. Also in similarity to crotoxin B, nigroviriditoxin B
induces myonecrosis. Its 122 amino acid sequence presents 81% identity with crotoxin B. Accordingly,
nigroviriditoxin B was cross-recognized by equine antibodies from a Crotalus durissus terricus antivenom. Phylogenetic analysis shows that the novel PLA2 from B. nigroviridis venom is basal to the branch
including all the homologous PLA2 enzymes described in rattlesnakes, and more distant from PLA2s from
Bothriechis species. Nigroviriditoxin is the rst heterodimeric PLA2 complex found in a non-rattlesnake,
Neotropical viperid venom, which displays structural, functional, and immunochemical similarities to
crotoxin. The present ndings are compatible with the existence of the particular structural trait of
crotoxin-like molecules in New World pitvipers before the split of the Meso-South American and the
Nearctic clades.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Snake venom
Viperidae
Phospholipase A2
Crotoxin
Bothriechis nigroviridis

1. Introduction
The black-speckled palm snake, Bothriechis nigroviridis, is an
arboreal Neotropical pit viper that inhabits subtropical rainforests
and temperate forests at medium to high elevations

n (highlands of Mon(1150e3000 m) on the Cordillera de Tilara
teverde) and Cordillera Volc

anica Central in Costa Rica, southeastward through the Cordillera de Talamanca to the Chiriqu province

(Savage, 2002; Campbell and Lamar, 2004; Solo

rzano,
in Panama
2004). Its Latin name originates from its distinctive speckled color pattern combining black (nigro) and emerald green (viridis)

* Corresponding author.
E-mail address: bruno.lomonte@ucr.ac.cr (B. Lomonte).
http://dx.doi.org/10.1016/j.toxicon.2014.11.235
0041-0101/ 2014 Elsevier Ltd. All rights reserved.

spots. Adults have been described to prey on small rodents, lizards,

rzano, 2004). Owing to its
frogs, and occasionally small birds (Solo
connement to undisturbed habitats with little human contact,
envenomings by this pitviper species appear to be extremely uncommon, as no documented cases could be found in a literature
search.
In a previous study, the venom of B. nigroviridis was analyzed by

ndez et al.,
a combined proteomic and toxicological approach (Ferna
2010), revealing remarkable differences with the venoms of three
other Bothriechis species found in Costa Rica: Bothriechis lateralis,
Bothriechis schlegelii (Lomonte et al., 2008) and Bothriechis supraciliaris (Lomonte et al., 2012). Surprisingly, B. nigroviridis venom is

ndez et al., 2010), a widespread
devoid of metalloproteinases (Ferna
and often predominant protein family in viperid venoms (Calvete
et al., 2007; Lomonte et al., 2014). In agreement with its lack of

B. Lomonte et al. / Toxicon 93 (2015) 144e154

145

metalloproteinases, B. nigroviridis venom does not induce hemorrhage in the mouse skin assay (Fern

andez et al., 2010). Another
striking feature revealed by the proteomic study of this venom was
the identication of a major phospholipase A2 (PLA2; EC 3.1.1.4)
component which showed signicant amino acid sequence ho
ndez et al., 2010). In
mology to crotoxin B from rattlesnakes (Ferna
addition, this venom presented a protein with homology with the
acidic subunit of crotoxin, i.e. crotoxin A (Fern

andez et al., 2010).
Crotoxin is a well characterized heterodimeric PLA2 complex (Aird
et al., 1986; Faure et al., 1994; Marchi-Salvador et al., 2008; Sampaio
et al., 2010) thus far only found in the venom of rattlesnakes
(genera Crotalus and Sistrurus). The unusual presence of crotoxinlike components outside rattlesnake venoms prompted us to
isolate the native crotoxin-like complex, and characterize its
functional properties, the amino acid sequence of the PLA2
component, and its phylogenetic relationships to homologous
proteins found in other crotalid snake venoms.

2.4. SDS-PAGE and nESI-mass spectrometry

2. Materials and methods

2.5. Complex formation between nigroviriditoxin A and B subunits

2.1. Venom

The A and B subunits of nigroviriditoxin were incubated

together or alone for 15 min at room temperature, and then subjected to agarose gel electrophoresis under native conditions. The
agarose was dissolved at 1% (w/v) in 0.116 M Tris, 0.3 M glycine, pH
8.6 buffer. Electrophoresis was performed at 2 mA/cm for 90 min,
and proteins were visualized by Coomassie R-250 staining.

Venom was collected and pooled from three adult specimens of

B. nigroviridis kept at the serpentarium of Instituto Clodomiro Picado, University of Costa Rica. After centrifugation to remove
insoluble matter, the venom was lyophilized and stored at
20  C
until use.

2.2. Isolation and characterization of native nigroviriditoxin

heterodimer
To demonstrate the existence of the nigroviriditoxin AB heterodimer, 50 mg of whole venom in 100 mL of 0.1 M ammonium acetate, pH 6.9, were fractionated using an ETTAN-LC chromatograph
and a Bio-Sil SEC 250 (300  7.8 mm) size-exclusion chromatographic column (Bio-Rad) equilibrated in the same buffer. The
eluate was monitored at 215 nm and the fractions, collected
manually, were analyzed by LC-MS using a nano-Acquity UltraPerformance LC (UPLC) using a BEH130 C18 (100 mm  100 mm,
1.7 mm particle size) column in-line with a Waters SYNAPT G2 High
Denition Mass Spectrometry System. The ow rate was set to
0.6 ml/min and the column was developed with a linear gradient of
0.1% formic acid in water (solution A) and 0.1% formic acid in
acetonitrile (solution B), isocratically 1% B for 1 min, followed by
1e12% B for 1 min, 12e40% B for 15 min, and 40e85% B for 2 min.

2.3. Isolation of nigroviriditoxin A and B subunits

Venom aliquots of 2.0e2.5 mg were dissolved in 220 mL of solution A (0.1% triuoroacetic acid in water) and centrifuged at
3000  g for 5 min. The clear supernatant was fractionated by
reverse-phase HPLC, using an Agilent Model 1200 chromatograph.
Two-hundred microliters of venom were injected to a C18 column
(Teknochroma; 250  4.6 mm; 5 mm particle size) equilibrated with
solution A, and elution was then carried out at a ow rate of 1 mL/
min by applying the following gradient toward solution B (0.1% TFA
in acetonitrile): 0% B for 5 min, 0e15% B over 10 min, 15e45% B over
60 min, 45e70% B over 10 min, and 70% B over 9 min. The eluant
was monitored at 215 nm, and the fractions of interest were
collected manually, dried by vacuum centrifugation at 45  C, and
stored at
20  C.

Peaks corresponding to the A and B subunits of nigroviriditoxin

obtained from RP-HPLC were redissolved in water, and their protein
concentration was estimated by measuring the absorbance at
280 nm in a NanoDrop 2000c instrument (Thermo Scientic).
Samples were then analyzed by SDS-PAGE using pre-cast gradient
gels (4e20%; Bio-Rad) under reducing or non-reducing conditions.
Proteins were visualized by Coomassie blue R-250 staining, and
recorded using a ChemiDoc gel imager with ImageLab software
(Bio-Rad). Molecular mass markers were run in parallel. The
isotope-averaged molecular mass of puried A and B subunits,
respectively, was determined by nESI-MS on a QTrap-3200 mass
spectrometer (Applied Biosystems). The proteins were diluted in
50% acetonitrile containing 0.1% formic acid, and directly infused
into a nanospray source for ionization at 1200 V. Analysis was
performed in positive enhanced multicharge mode in the range
600e1700 m/z, aided by the Analyst v.1.5 software (ABSciex).

2.6. Amino acid sequencing of nigroviriditoxin B

The N-terminal amino acid sequence of nigroviriditoxin B was
obtained by automated direct Edman degradation in a Procise instrument (Applied Biosystems) following manufacturer's instructions (Fern

andez et al., 2010). The protein sequence was
completed by tandem mass spectrometry of peptides obtained after the digestion of the DTT-reduced and iodoacetamide-alkylated
protein with trypsin or chymotrypsin. Peptides were diluted 1:2
with a saturated a-cyanohydroxycinnamic acid solution in 50%
acetonitrile and 0.1% triuoroacetic acid, spotted onto an Opti-TOF384 plate, dried, and analyzed by MALDI-TOF-TOF on a Model
4800-Plus Proteomics Analyzer (Applied Biosystems). Spectra were
acquired in positive reector mode at 2 kV in the 875e4000 m/z
range using a laser intensity of 3000 and 1500 shots (Lomonte et al.,
2012). CalMix standards (ABSciex) spotted on the same plate were
used for external calibration. Few peptides were analyzed by nESIMS/MS on a QTrap2000 mass spectrometer (Applied Biosystems)
by direct infusion into a nanospray source. Selected doubly- or
triply-charged ions from spectra obtained in enhanced resolution
mode (250 amu/s) were subjected to fragmentation using the
enhanced product ion option with Q0 trapping. Settings were: Q1,
unit resolution; collision energy, 25e40 eV; linear ion trap Q3 ll
time, 250 ms; and Q3 scan rate, 1000 amu/s (Calvete et al., 2007).
All fragmentation spectra obtained by MALDI- or ESI-mass spectrometry were interpreted manually to derive de novo amino acid
sequences.
2.7. Phospholipase A2 activity
The PLA2 activity of nigroviriditoxin B was determined on the
monodisperse synthetic substrate 4-nitro-3-octanoyl-benzoic acid
(NOBA) (Holzer and Mackessy, 1996). Various amounts of the toxin,
dissolved in 25 mL of 10 mM Tris, 10 mM CaCl2, 0.1 M NaCl, pH 8.0
buffer, were added to 200 mL of this buffer in triplicate wells of a
microplate. After mixing, 25 mL of NOBA (1 mg/mL in acetonitrile)
were added, to achieve a nal substrate concentration of 0.32 mM.

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B. Lomonte et al. / Toxicon 93 (2015) 144e154

The mixtures were incubated for 60 min at 37  C, and absorbances

at 405 nm were recorded. PLA2 activity was expressed as the nal
change in absorbance  1000. For comparison, puried B subunit of
crotoxin from Crotalus durissus terricus (kindly provided by Prof.
^nia, Brazil) was
Andreimar Soares, Universidade Federal de Rondo
included in the assay. In another experiment, the effect of the A
component of nigroviriditoxin on the PLA2 activity of the B
component was tested by mixing both at 1.5:1 or 3:1 M ratios (A:B)
and then comparing enzyme activity to that of component B alone,
as described above.
2.8. Myotoxic activity
The ability of nigroviriditoxin B to induce skeletal muscle necrosis was evaluated in groups of ve CD-1 mice of 18e20 g of body
weight, following protocols approved by the Institutional Committee for the Use and Care of Animals (CICUA) at University of
Costa Rica. Twenty mg of the toxin were injected intramuscularly in
the gastrocnemius, in a volume of 100 mL of PBS (0.12 M NaCl,
0.04 M sodium phosphate buffer, pH 7.2). A control group received
an identical injection of PBS alone. After 3 h, a blood sample was
obtained from the tail of each animal into a heparinized capillary
tube, and after centrifugation, 4 mL of plasma were used to determine the creatine kinase (CK) activity using a UV-kinetic assay (CKNac, Biocon Diagnostik). CK activity was expressed in units/L. For
comparison, 20 mg of crotoxin B was injected into another group of
mice and the plasma CK activity was assessed as described.
2.9. Lethal activity of nigroviriditoxin B and potentiation by
nigroviriditoxin A
Variable doses of nigroviriditoxin B (10e80 mg) were injected
intravenously (i.v.) in the tail vein in groups of four mice of 16e18 g
of body weight, in 100 mL of PBS. Deaths were recorded after 48 h
and the median lethal dose (LD50 S.E.) was calculated by probits
(Finney, 1971) using the BioStat v.2009 software (AnalystSoft). A
similar assay was performed for the isolated subunit A component,
using doses up to 70 mg. For evaluation of synergy, the natural
proportion of the A and B components of nigroviriditoxin in the
venom was estimated by integrating their corresponding chromatographic peak areas in the RP-HPLC signal at 215 nm, roughly
corresponding to peptide bond abundance. The molar proportion of
A:B components was estimated to be approximately 1:2. On this
basis, the lethality of mixtures of both components was tested in
mice at 1:2 M ratio, and compared to the results obtained when
each component was assayed independently.
2.10. Immunochemical cross-recognition of nigroviriditoxin B by
antivenom against C. d. terricus
Puried nigroviriditoxin A subunit, B subunit, or crotoxin B,
respectively, were adsorbed onto wells of an ELISA plate (Nunc
Maxisorp) by incubating 0.2 mg/well of the proteins in 100 mL of
0.1 M Tris, 0.15 M NaCl, pH 9.0 buffer overnight at 4  C. After
washing ve times with PBS, free sites were blocked with PBS
containing 1% bovine serum albumin (BSA) for 1 h. Then, serial
dilutions of equine antivenom against C. d. terricus venom (Instituto Butantan, batch 0309-38), or normal equine serum as a
negative control, were added and incubated 1 h. Plates were then
washed with 0.05 M Tris, 0.15 M NaCl, 20 mM ZnCl2, 1 mM MgCl2
(pH 7.4), and bound antibodies were detected by incubation with
an anti-horse total IgG-alkaline phosphatase conjugate (1:3000;
Sigma) for 1 h, followed by washing and color development with pnitrophenylphosphate (1 mg/mL) in diethanolamine buffer (pH

9.8). Absorbances were recorded on a microplate reader (Labsystems) at 405 nm. Assays were performed in triplicate wells.
2.11. Phylogenetic analysis of nigroviriditoxin B
Proteins showing amino acid sequence identity values of at least
75% and a score of at least 191 in comparison to nigroviriditoxin B
were retrieved after a BLAST search (http://blast.ncbi.nlm.gov).
These proteins, together with three available sequences corresponding to Asp49-PLA2s characterized from the genus Bothriechis
(Q6EER4, C0HJC1, A8E2V4), were aligned with the program MUSCLE (Edgar, 2004) using the MEGA6 software (Tamura et al., 2013).
The evolutionary history was inferred with MEGA6, by using the
maximum likelihood method based on the JTT matrix-based model
(Jones et al., 1992). Three sequences corresponding to PLA2s from
elapid snake species (P81167 from Micrurus nigrocinctus, P00605
from Naja nigricollis and P00614 from Oxyuranus scutellatus scutellatus) were used as an outgroup in this analysis, which involved a
total of 22 amino acid sequences.
2.12. Homology modeling of nigroviriditoxin B
The Swiss-Model automated homology modeler (http://
swissmodel.expasy.org/; Biasini et al., 2014; Arnold et al., 2006)
was used to predict the three-dimensional structure of nigroviriditoxin B. The modeler selected crotoxin B (PDB access code 3R0L;
Faure et al., 2011) as the best template, with a resolution of 1.35
and 80.5% sequence identity with the target. Swiss-PDB viewer
v.4.1 was used to superimpose the obtained model to the template,
and to calculate r.m.s.d values for main chain a-carbons and backbones. Electrostatic surface potential of the proteins were represented with the DS ViewerPro v.6.0 software (Accelrys).
3. Results
Fractionation of B. nigroviridis venom by RP-HPLC on C18 is
shown in Fig. 1. The peaks eluting at ~42 and ~51 min had been
previously identied to have homology to the acidic (A) and basic
(B) subunits of crotoxin, respectively, in a proteomic study on this

ndez et al., 2010). Therefore, we explored if these two
venom (Ferna
proteins could form a crotoxin-like complex, hereby named
nigroviriditoxin. By SDS-PAGE, the PLA2 B subunit migrated as a
band of ~15 kDa under reducing conditions, whereas the A-chain
migrated as a main band of ~9 kDa (Fig. 2). Under non-reducing
conditions, the A chain was observed at ~15 kDa, suggesting a
possible dimerization or an anomalous migration. Similarly, the B
chain appeared to aggregate, as evidenced by a continuous smear in
the 15e25 kDa range (Fig. 2), commonly observed under non-

Fig. 1. Isolation of the A and B subunits of nigroviriditoxin from the venom of

Bothriechis nigroviridis. Crude venom (~2 mg) was fractionated by RP-HPLC on a C18
column (4.6  250 mm) and monitored at 215 nm. Protein elution was performed with
an acetonitrile gradient (dashed line) as described in Materials and methods. The
peaks corresponding to A (acidic chain) and B (PLA2) subunits are labeled.

B. Lomonte et al. / Toxicon 93 (2015) 144e154

Fig. 2. Electrophoretic mobility of the A and B subunits of nigroviriditoxin by SDSPAGE (4e20% gradient gel). Samples were analyzed after reducing (r) or nonreducing (nr) conditions) and visualized by Coomassie blue R-250 staining. Values
for the molecular mass markers (m) are shown to the right, in kDa. (For interpretation
of the references to color in this gure legend, the reader is referred to the web version
of this article.)

reducing conditions in group II PLA2s of viperid snake venoms

~ ez et al., 2004),
(Soares et al., 2000; Angulo et al., 2000; Nn

147

including crotoxin (Faure et al., 1994). By ESI-MS analysis, the A

subunit presented an isotope-averaged molecular mass of 9607 Da
(1), whereas the B subunit showed a main molecular mass of
14,083 Da, and an additional mass of 14,113 Da (2) (Fig. 3). In the
latter case, the observed mass heterogeneity likely correspond to
the presence of PLA2 isoforms, a frequent nding in snake venoms
(Doley et al., 2010) and in crotoxin preparations, due to the complex
multigene nature of this protein family (Faure et al., 1994; Faure and
Bon, 1988).
We explored the capability of the two proteins to associate into
a crotoxin-like complex using two different approaches. On the one
hand, B. nigroviridis venom was fractionated by size-exclusion
chromatography and the composition of the resulting fractions
was analyzed by mass spectrometry. A major peak eluted at 10.1 mL
(Fig. 4A), corresponding to the position expected for a 22.9 kDa
standard protein (Fig. 4B), and contained components of 9605.6 Da
(A1), 9421.5 Da (A2), and 14,081.3 Da (B) (Fig. 4C, upper panel).
Reverse-phase chromatography prior to MS analysis separated the
14 kDa (Fig. 4C, middle panel) from the 9 kDa (Fig. 4C, lower panel)
proteins. As a whole, these data clearly indicate that the crotoxinlike proteins of B. nigroviridis associate into heterodimers, hereby
named nigroviriditoxin A1B and A2B.
The capability of the RP-HPLC-dissociated and isolated A and B
components of nigroviriditoxin to reconstitute the heterodimer(s)
was also analyzed by agarose electrophoresis under native conditions. The free A component of nigroviriditoxin migrated rapidly
towards the anode, indicating its acidic character, whereas the B

Fig. 3. Molecular mass of the A and B subunits of nigroviriditoxin as determined by nano-electrospray ionization mass spectrometry (ESI-MS). The multiply-charged ion series for
the A (A) and B (C) subunits are deconvoluted in panels (B) and (D), respectively.

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B. Lomonte et al. / Toxicon 93 (2015) 144e154

Fig. 4. Panel A, fractionation of B. nigroviridis venom (green trace) by size-exclusion chromatography. The column was calibrated with a mixture of dextran blue (2000 kDa), bovine
serum albumin (66.4 kDa), equine cytochrome c (12.3 kDa), and vitamin B12 (1.35 kDa), and used for estimating the apparent molecular mass of nigroviriditoxin (peak labeled with
an asterisk) as 22.9 kDa (panel B). The heterodimeric association of nigroviriditoxin A and B subunits was demonstrated by mass spectrometry (panel C). The major peak eluting at
10.1 mL contained components of 9605.6 Da (A1), 9421.5 Da (A2) and 14,081.3 Da (B) (upper panel). Reverse-phase chromatographic separation prior to MS analysis separated the
14 kDa B-subunit (middle panel) from the 9 kDa A1 and A2 subunits (lower panel). (For interpretation of the references to color in this gure legend, the reader is referred to the
web version of this article.)

B. Lomonte et al. / Toxicon 93 (2015) 144e154

Fig. 5. Complex formation between the A and B subunits of nigroviriditoxin evaluated

by native agarose gel electrophoresis at pH 8.6. Lane 1: A subunit; lane 2: B subunit;
lane 3: mixture of A and B subunits, at 1.5:1 (A:B) molar ratio; lane 4: crotoxin B; lane
5: mixture of nigroviriditoxin A subunit and crotoxin B, at 1.5:1 (A:B) molar ratio.
Anode () and cathode (
) positions are indicated. Proteins were loaded at 6 mg/lane
and visualized by Coomassie R-250 staining after electrophoresis.

component migrated toward the cathode, indicating its basic pI

(Fig. 5). When these two components were mixed, a new band
appeared at half-way the migration distance between both free
components (Fig. 5), clearly indicating the formation of an A B
complex. Accordingly, an evident reduction in the intensity of the A
subunit was observed, and no free B subunit was detected. Further
evidence for the similarity of the A component of nigroviriditoxin
to crotoxin A was obtained in this native gel assay, by observing that
a mixture of the former with crotoxin B also results in the formation
of a complex, concomitant with the disappearance of free crotoxin
B (Fig. 5).
Due to the limited availability of B. nigroviridis venom, which
yielded only small amounts of isolated nigroviriditoxin A (Fig. 1),
and considering its expected structural complexity (assuming
similarity to crotoxin A, formed by three covalently-linked chains
generated by proteolytic processing at the same polypeptide bonds

ndez et al., 2010), the determination of the amino
as crotoxin; Ferna
acid sequence of this component could not be attempted. On the
other hand, the complete sequence of nigroviriditoxin B was
determined by the combination of N-terminal Edman degradation
and tandem MS of proteolytic peptides (Fig. 6). Only the C-terminal

149

cysteine residue of this sequence could not be directly observed in

the tryptic peptide ion of m/z 946.13 due to the preceding lysine,
but was inferred on the basis of its absolute conservation in group II
PLA2s from snake venoms (Arni and Ward, 1996), and the isotopeaveraged molecular mass determined for the native protein. The
sequence of nigroviriditoxin B consists of 122 amino acid residues,
with all canonical cysteine positions of group II PLA2s being
conserved. This sequence will appear in UniProt (http://www.
uniprot.org/) under the accession number C0HJL8. The theoretical
molecular mass predicted by the sequence is 14,112 Da, which
presents a difference of ~29e30 Da with the most abundant form
(14,083 Da) or coincides, within instrumental error, with the second form observed (14,113 Da) by ESI-MS analysis (Fig. 3). According to its sequence, the theoretically expected pI of this protein
would be 8.5, in agreement with its observed cathodic migration in
the native agarose gel system (Fig. 5).
Nigroviriditoxin B presents sequence homology to crotoxin B
isoforms described in the venom of C. d. terricus (Fig. 7), with
identity values ranging from 77 to 80%. The highest identity (81%)
was observed in comparison to a PLA2 from Sistrurus catenatus
tergeminus (Q6EER2; Chen et al., 2004). By multiple alignment of
nigroviriditoxin B with PLA2s having 75% identity, and with three
Asp49-PLA2s described in the genus Bothriechis, a phylogenetic tree
was inferred (Fig. 8). Elapid PLA2s served as an outgroup, as they
belong to the group I classication of this enzyme superfamily.
Noteworthy, nigroviriditoxin B appears basal to the group containing all the different crotoxin B variants described in rattlesnakes, i.e. Crotalus and Sistrurus species, and does not cluster
together with other PLA2s characterized from venoms of species of
the genus Bothriechis (Fig. 8). The branch of nigroviriditoxin B stems
from acidic PLA2s of Old-world crotalid species (Gloydius halys,
Ovophis monticola) and three acidic PLA2s isolated from B. lateralis
and B. schlegelii (Fig. 8).
The homology of nigroviriditoxin B to the various crotoxin B
isoforms found in the venoms of rattlesnakes is in agreement with
its functional characteristics. As shown in Fig. 9A, isolated nigroviriditoxin B displays functional PLA2 activity, only slightly lower in
comparison to crotoxin B. Also in similarity with the latter, nigroviriditoxin B induced myonecrosis when injected intramuscularly
in mice, although this effect was markedly lower in comparison to
crotoxin B (Fig. 9B). Regarding lethal activity, nigroviriditoxin B
alone showed an LD50 of 50 (44.9e55.6) mg/mouse (i.e., 2.9 mg/g
body weight) when injected intravenously. Mice showed signs of
respiratory paralysis before death. When nigroviriditoxin A was
added to the B component, at 1:2 (A:B) molar ratio, the potency of

Fig. 6. Amino acid sequence of nigroviriditoxin B. Peptides generated by the digestion of the protein with trypsin (T) or chymotrypsin (C) were de novo sequenced by mass
spectrometry. The rst 60 amino acid residues were determined by N-terminal Edman degradation.

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B. Lomonte et al. / Toxicon 93 (2015) 144e154

Fig. 7. Multiple alignment of the amino acid sequence of nigroviriditoxin B with crotalid venom phospholipases A2 selected on the basis of sequence identity values of at least 75%
and a BLAST score of at least 191. Three available sequences of phospholipases A2 from Bothriechis venoms were additionally included. Sequences were aligned using MUSCLE (Edgar,
2004) with the MEGA6 software (Tamura et al., 2013), as described in Materials and methods. Protein access codes correspond to the UniProtKB database at the ExPASy Proteomics
Server. Identical amino acid positions are shaded in gray, with cysteine residues in boldface. Isoelectric points (pI) were calculated with the compute pI/MW tool (http://web.expasy.
org/compute_pi/), and the basic or acidic proteins are represented in blue or red colors, respectively. (For interpretation of the references to color in this gure legend, the reader is
referred to the web version of this article.)

this effect increased by 60%, to an LD50 of 31 (27.5e33.9) mg/mouse

(2.2 mg/g body weight). Higher amounts of A subunit could not be
tested due to limitations in venom availability. The isolated A
subunit was not lethal per se, and did not cause any evident alterations, when injected at doses up to 70 mg/mouse (4.1 mg/g body
weight).
The addition of the acidic A component to nigroviriditoxin B
caused a signicant reduction of its PLA2 activity in vitro, nearly by
half (Fig. 10). On the other hand, in ELISA experiments nigroviriditoxin B was clearly immunorecognized by an equine antivenom
raised against the venom of C. d. terricus (Fig. 11A), with a signal
nearly as intense as that generated by puried crotoxin B (Fig. 11C).
Recognition of nigroviriditoxin A by this antivenom was negligible
(Fig. 11B), although in this case the unavailability of crotoxin A did
not allow to control for the level of antibodies against the homologous antigen in the antivenom.
The three-dimensional structure of nigroviriditoxin B was
modeled using the crystal structure of crotoxin B as a template. The
obtained model appears virtually superimposable to the latter
structure, with r.m.s.d. values for a-carbon and backbone atoms of
0.06 and 0.08 , respectively (Fig. 12). Both proteins differ at 24
out of their 122 amino acid residues, and the corresponding side

chains of these are represented in Fig. 12B. Out of the 22 amino acid
residues of crotoxin B proposed to interact with crotoxin A in the
crystallized complex (Faure et al., 2011), nigroviriditoxin B conserves 18. The only four amino acid residues that change from
crotoxin B to nigroviriditoxin B in this particular set include His1/
Asn, Lys14/Arg, Trp61/Ser, and Pro111/Leu. The high structural similarity between nigroviriditoxin B and crotoxin B is also reected by
their conserved surface charge distributions, compared in Fig. 12C
and D).
4. Discussion
A previous proteomic study on the venom of B. nigroviridis

ndez et al., 2010).
revealed several unusual characteristics (Ferna
Among them, the unexpected occurrence of a predominant PLA2
with similarity to crotoxin B, together with a protein resembling
the acidic A subunit of crotoxin, prompted us to investigate
whether this venom would contain a crotoxin-like complex, to date
only found in the venoms of rattlesnakes (Crotalus and Sistrurus)
among New World pitvipers. Crotoxin, rst isolated from the South
American rattlesnake C. d. terricus, is the major toxic component in
the venom of several rattlesnake species. It is a heterodimeric

B. Lomonte et al. / Toxicon 93 (2015) 144e154

151

Fig. 8. Inferred phylogenetic relationships of nigroviriditoxin B. The amino acid sequences aligned in Fig. 7, together with three sequences corresponding to phospholipases A2 from
elapids used as an outgroup (P81167 from Micrurus nigrocinctus, P00605 from Naja nigricollis and P00614 from Oxyuranus s. scutellatus), were used to construct a tree using the
maximum likelihood method based on the JTT matrix-based model, implemented in MEGA6. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Codes for basic and acidic proteins are represented in blue and red, respectively. (For interpretation of the references to color in this gure legend, the reader is
referred to the web version of this article.)

complex composed of a basic PLA2 subunit (crotoxin B) and an

acidic, non-enzymatic subunit (crotoxin A, crotapotin), held
together non-covalently (Sampaio et al., 2010). A similar complex
found in various North American rattlesnakes such as Crotalus
scutulatus scutulatus has been named Mojave toxin (Hendon, 1975).
Although PLA2s with homology to crotoxin B have been found in
non-rattlesnake venoms (for example, agkistrodotoxin; Chen et al.,
2004), none have been shown to form a complex with a subunit Alike component from the same venom. However, reconstitution
experiments of agkistrodotoxin from Agkistrodon blomhof brevicaudus with crotoxin A from C. d. terricus, have shown that this
potential exists (Choumet et al., 1993). Thus, the term crotoxinlike has been used until now to indicate amino acid sequence
similarity between a given PLA2 and crotoxin B (Chen et al., 2004),
but not similarity regarding the formation of its characteristic A B
heterodimeric complex.
This study conrms that B. nigroviridis venom contains a heterodimeric PLA2 built by A and B components with homology to
crotoxin A and B subunits. In addition, our work demonstrates the
ability of the isolated A and B subunits to reconstitute the complex
under native conditions. The ability of the nigroviriditoxin A
component to form an inter-species complex with crotoxin B as
well, strongly suggests a high degree of conservation of the structural determinants needed for subunit interaction in these two
related heterodimeric toxins.
The low yield of subunit A, and its likely multichain structure,
precluded attempts to determine its amino acid sequence. On the
other hand, the complete sequence of nigroviriditoxin B was
determined. Identity values from 75 to 81% were observed by its
comparison with basic PLA2s from Crotalus and Sistrurus venoms,
although at least two acidic PLA2s from the Asian genera Ovophis
and Gloydius showed sequence identities of 75%. Interestingly,
three available Asp49-PLA2 sequences from other Bothriechis

venoms showed lower identity (52e68%) with nigroviriditoxin B,

than PLA2s from rattlesnakes. The phylogenetic tree inferred by
these sequence comparisons positioned nigroviriditoxin B basal to
the branch that includes all members of the group of PLA2s from
rattlesnakes. Consistent with hypotheses on the invasion of viperid
ancestors from the Old World into North America (Van Devender
and Conant, 1990; Douglas et al., 2006; Castoe and Parkinson,
2006; Castoe et al., 2009), the two PLA2s from Asian genera are
basal to nigroviriditoxin B in the tree, which is in concordance with
the proposal of Old World genera (Protobothrops, Ovophis, Trimeresurus, and Gloydius) as potential sister taxa to the New World clade
(Malhotra and Thorpe, 2004; Wster et al., 2008). The closer
phylogenetic relationship of nigroviriditoxin B to PLA2s from rattlesnakes, and its separation from other PLA2s found in Bothriechis
species to date, suggest the divergence of these enzymes before the
diversication of this genus across Middle America, estimated to
have occurred 12e16 Mya (Castoe et al., 2009). Our ndings are
compatible with the existence of the particular structural trait of
crotoxin-like molecules in New World pitvipers, prior to the split of
the Meso-South American and the Neartic clades (Castoe and
Parkinson, 2006).
Evident structural similarities between nigroviriditoxin B and
crotoxin B are additionally supported at the functional level.
Nigroviriditoxin B displays PLA2 activity in vitro, only slightly lower
than that of crotoxin B. Although weaker than crotoxin B, nigroviriditoxin B is also myotoxic in vivo. Furthermore, as observed for
crotoxin (Faure et al., 1993), the acidic A component of nigroviriditoxin modulates both the enzymatic and toxic activities of the
basic PLA2 subunit, albeit the effect of complex formation on these
two activities appears to be less pronounced than in the case of
crotoxin. The lethal potency of nigroviriditoxin B by the i.v. route is
weaker than that of crotoxin B, whose i.v. LD50 has been reported at
0.7 mg/g body weight (Faure et al., 1993). Altogether, these results

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B. Lomonte et al. / Toxicon 93 (2015) 144e154

Fig. 11. Immunochemical cross-recognition of nigroviriditoxin B by an equine antivenom to Crotalus d. terricus, evaluated by ELISA. Nigroviriditoxin B (A), nigroviriditoxin A (B), or crotoxin B (C) were adsorbed onto solid-phase and the binding of
antibodies from antivenom (C) or normal horse serum (B) was detected as described
in Materials and methods. Each point represents mean SD of triplicate wells.
Fig. 9. (A) Phospholipase A2 activity of nigroviriditoxin B (B), compared to crotoxin B
from Crotalus durissus terricus (C), on the synthetic substrate 4-nitro-3-octanoylbenzoic acid. Each point represents mean SD of triplicates. The differences between
both enzymes are statistically signicant (p < 0.05; Student's t-test) at 2.5, 5, and 10 mg.
(B) Myotoxic activity of nigroviriditoxin B (Ngvtx B) and crotoxin B (Ctx B) after
intramuscular injection of 20 mg in mice. Plasma creatine kinase activity was determined 3 h after injection. Phosphate-buffered saline (PBS) was injected to a control
group of mice. Each bar represents mean SD of ve mice. Differences between all
groups are statistically signicant (p < 0.05; ANOVA, followed by TukeyeKramer test).

indicate that nigroviriditoxin B displays a similar prole of activities

as crotoxin B, although with a general lower potency. Finally,
nigroviriditoxin B was strongly recognized by equine antibodies

Fig. 10. Modulation of the phospholipase A2 activity of nigroviriditoxin B by nigroviriditoxin A. Enzyme activity of the B subunit alone, or in the presence of A subunit at
the indicated molar ratios, was assayed on 4-nitro-3-octanoyl-benzoic acid. Each bar
represents mean SD of triplicate assays. The difference in enzymatic activity of
mixtures at both ratios, compared to nigroviriditoxin B alone is statistically signicant
(p < 0.05; ANOVA, followed by TukeyeKramer test).

from an antivenom raised against the venom of C. d. terricus,

demonstrating its antigenic similarity with crotoxin B, in agreement with their high sequence identity.
From a structural point of view, it is noteworthy that the amino
acid sequence differences between nigroviriditoxin B and crotoxin
B, consisting of 24 out of 122 positions, do not predict any major
deviations in the modeled three-dimensional structure of the
former. In the crystal structure of crotoxin analyzed by Faure et al.
(2011), 22 amino acid residues of the B subunit were found to be
engaged in its interaction with the A subunit. Of these, 18 are
conserved in nigroviriditoxin B. Given that nigroviriditoxin A was
shown to form a complex with crotoxin B, the high conservation of
amino acid residues between the latter and nigroviriditoxin B
would also predict a high conservation of key residues in the A
subunits from both snake species. Among the four amino acid
residues relevant for heterodimerization that differ between crotoxin B and nigroviriditoxin B, the Trp61/Ser substitution (Trp70/Ser
in the numbering system used by Faure et al. (2011)) deserves
special attention. Trp30 and Trp61 are considered to be critical to the
stability and toxicity of the crotoxin complex, by establishing
intermolecular contacts with the b-chain of crotoxin A (Faure et al.,
2011). Trp30 is conserved in nigroviriditoxin B, but Trp61 is replaced
by Ser, and this could inuence either its binding afnity to
nigroviriditoxin A, its toxicity, or both. Interestingly, inspection of
the multiple alignment of PLA2s homologous to nigroviriditoxin B
(Fig. 4), shows that Trp61 (Trp70) is exclusively present in the
enzymes from rattlesnake species, whereas nigroviriditoxin B and
the two PLA2s from Old World species (positioned basal to the
rattlesnake group in the phylogenetic tree; Fig. 5), present Ser at
this position. Considering that nigroviriditoxin B showed weaker
toxicity than crotoxin B, it is therefore tempting to speculate that
the mutation leading to the emergence of Trp61 (Trp70) instead of

B. Lomonte et al. / Toxicon 93 (2015) 144e154

153

Fig. 12. (A) Modeled three-dimensional structure of nigroviriditoxin B (red-orange), superposed to the crystal structure of crotoxin B (green; PDB access code 3R0L; Faure et al.,
2011). (B) The superposed structures are shown with the side chains of the 24 amino acid residues that differ, represented as sticks with their parent colors. These differences
are highlighted with the same colors in the alignment shown. The twenty-two amino acid residues proposed to participate in the interaction of crotoxin B with its A-subunit (Faure
et al., 2011) are pointed out in the alignment by triangles. Positions indicated by black triangles are conserved in nigroviriditoxin B, whereas pink triangles indicate four amino acid
positions that differ. Electrostatic surface potentials of the nigroviriditoxin B model (C) and crotoxin B (D) are represented with blue (positive) and red (negative) colors. Molecular
representations were prepared with DS ViewerPro (Accelrys) software. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of
this article.)

Ser in a common ancestor to Crotalus and Sistrurus rattlesnakes

might be related to the enhancement of toxic potency.
Although toxic PLA2s having homology with crotoxin B chain
have been found in a variety of viperid snake venoms (Chen et al.,
2004), the simultaneous presence of A and B subunits of crotoxin or
Mojave toxin has been described so far only in rattlesnakes, i.e.
genera Crotalus and Sistrurus, among New World pitvipers. Our
description of a crotoxin-like heterodimer in the venom of
B. nigroviridis represents, to the best of our knowledge, the rst
nding of such a complex in a non-rattlesnake New World pitviper
venom. This raises a number of intriguing issues regarding the
molecular evolution of this neurotoxic trait. As evidenced by its
lethal potency, the toxicity of nigroviriditoxin is considerably lower
than that of crotoxin and Mojave toxin, suggesting that it may
represent a less differentiated, and hence less toxic stage in the
molecular evolution towards neurotoxicity, although the structural
paradigm of this heterodimeric design is clearly already present in
B. nigroviridis venom. These ndings suggest that the emergence of
this particular structural trait, involving a non-covalent A B
complex between an acidic, non-toxic chaperone component and a
toxic, basic PLA2, occurred earlier than previously thought in the
evolution of pit viper venoms.

Conict of interests statement

The authors declare that there are no conicts of interest related
to this study.
Acknowledgments
We thank the International Centre for Genetic Engineering and
Biotechnology (ICGEB, Italy; CRP/COS13-01), Vicerrectora de

n, Universidad de Costa Rica (UCR; 741-B4-100 and
Investigacio
741-B3-760), CYTED (BioTox, P211RT0412), and Ministerio de

n (currently, Ministerio de Economa y ComCiencia e Innovacio
petitividad, Madrid; BFU2010-17373) for their nancial support, as
well as Dr Mahmood Sasa for fruitful discussions on this study, and
Dr Andreimar Soares for providing crotoxin B. The valuable

n and Aaro

n Go

mez at
contribution of Jazmn Arias, Danilo Chaco
the serpentarium of Instituto Clodomiro Picado is also gratefully
acknowledged.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.toxicon.2014.11.235.

154

B. Lomonte et al. / Toxicon 93 (2015) 144e154

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