toxins

Article
Varying Intensities of Introgression Obscure Incipient
Venom-Associated Speciation in the Timber Rattlesnake
(Crotalus horridus)
Mark J. Margres 1, * , Kenneth P. Wray 2 , Dragana Sanader 2 , Preston J. McDonald 1 , Lauren M. Trumbull 1 ,
Austin H. Patton 3 and Darin R. Rokyta 2

1 Department of Integrative Biology, University of South Florida, Tampa, FL 33620, USA;

[email protected] (P.J.M.); [email protected] (L.M.T.)
2 Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA;
[email protected] (K.P.W.); [email protected] (D.S.); [email protected] (D.R.R.)
3 Department of Integrative Biology, University of California, Berkeley, CA 94720, USA;
[email protected]
* Correspondence: [email protected]

Abstract: Ecologically divergent selection can lead to the evolution of reproductive isolation through
the process of ecological speciation, but the balance of responsible evolutionary forces is often ob-
scured by an inadequate assessment of demographic history and the genetics of traits under selection.
Snake venoms have emerged as a system for studying the genetic basis of adaptation because of
their genetic tractability and contributions to fitness, and speciation in venomous snakes can be
associated with ecological diversification such as dietary shifts and corresponding venom changes.
Here, we explored the neurotoxic (type A)–hemotoxic (type B) venom dichotomy and the potential for





 ecological speciation among Timber Rattlesnake (Crotalus horridus) populations. Previous work iden-
tified the genetic basis of this phenotypic difference, enabling us to characterize the roles geography,
Citation: Margres, M.J.; Wray, K.P.;
history, ecology, selection, and chance play in determining when and why new species emerge or are
Sanader, D.; McDonald, P.J.;
Trumbull, L.M.; Patton, A.H.;
absorbed. We identified significant genetic, proteomic, morphological, and ecological/environmental
Rokyta, D.R. Varying Intensities of differences at smaller spatial scales, suggestive of incipient ecological speciation between type A and
Introgression Obscure Incipient type B C. horridus. Range-wide analyses, however, rejected the reciprocal monophyly of venom type,
Venom-Associated Speciation in the indicative of varying intensities of introgression and a lack of reproductive isolation across the range.
Timber Rattlesnake (Crotalus horridus). Given that we have now established the phenotypic distributions and ecological niche models of
Toxins 2021, 13, 782. https://doi.org/ type A and B populations, genome-wide data are needed and capable of determining whether type A
10.3390/toxins13110782 and type B C. horridus represent distinct, reproductively isolated lineages due to incipient ecological
speciation or differentiated populations within a single species.
Received: 23 September 2021
Accepted: 25 October 2021
Keywords: adaptation; venom; genome; ecological speciation
Published: 5 November 2021

Key Contribution: Genetic, proteomic, morphological, and environmental data show that incipient
Publisher’s Note: MDPI stays neutral
ecological speciation and putative reproductive isolation among Timber Rattlesnakes (Crotalus
with regard to jurisdictional claims in
horridus) with different venom phenotypes vary substantially across the range.
published maps and institutional affil-
iations.

1. Introduction

Copyright: © 2021 by the authors.

Natural selection can promote local adaptation and determine the geographical distri-
Licensee MDPI, Basel, Switzerland.
butions of phenotypes within species [1,2]. Ecological speciation is the process by which
This article is an open access article
populations become reproductively isolated due to divergent, ecologically based natural
distributed under the terms and selection [3], and these divergent selective pressures can be due to differences in the abiotic
conditions of the Creative Commons environment [4], assemblages of interacting species [5], or other ecological factors. For
Attribution (CC BY) license (https:// example, a reduced immigrant or hybrid fitness mediated through ecological factors could
creativecommons.org/licenses/by/ reduce gene flow between populations [6,7], and reproductive isolation would evolve as a
4.0/). result. Evidence in support of ecological speciation is widespread from diverse taxa [3,8,9],

Toxins 2021, 13, 782. https://doi.org/10.3390/toxins13110782 https://www.mdpi.com/journal/toxins

Toxins 2021, 13, 782 2 of 17

but within species, divergence can occur without leading to speciation [10]. Selection
can also reduce the extent of reproductive isolation by favoring introgression of mutually
beneficial alleles across population and species boundaries (i.e., increased hybrid fitness
via heterozygote advantage) [11,12]. Geography, history, ecology, genetics, selection, and
chance determine, through unknown relative contributions, whether new species emerge
or are absorbed, but the balance of evolutionary forces responsible is generally obscured
by the inadequate assessment of demographic history and the genetics of traits under
selection [13].
Snake venoms have emerged as a system for studying adaptive diversification because
of their genetic tractability [14,15], contributions to fitness [16–18], and high evolutionary
rates [19–21]. Speciation in venomous snakes is often associated with ecological diversifica-
tion such as dietary shifts and corresponding changes in venom expression [22,23], and
gene expression changes have been implicated in ecological speciation in other systems [24].
Rattlesnake venoms are broadly classified into two categories [25]: neurotoxic, which we
refer to as type A, and hemorrhagic, which we refer to as type B [26,27]. This dichotomy
reflects an inverse relationship between toxicity and tissue-damaging effects [28]. Type A
venoms have high toxicity resulting from the presence of a set of homologous potent presy-
naptic heterodimeric phospholipase A2 (PLA2 ) neurotoxins (e.g., crotoxin, Mojave toxin,
and canebrake toxin) and low levels of tissue-damaging snake-venom metalloproteinases
(SVMPs). Type B venoms completely lack heterodimeric PLA2 s and express high levels
of SVMPs. Dowell et al. [29,30] showed that different sets of PLA2 genes, rather than the
differential regulation of shared loci, explained much of the differentiation between A and
B venoms (but not all; see [15]).
Most of the >30 rattlesnake species have type B venoms, but type A venoms are
known from at least ten species [25]. Nine of these ten species, however, are polymor-
phic for both venom types. The consistency of the A–B venom dichotomy suggests that
intermediate venoms are disfavored by selection (although introgression, albeit rare, has
been documented in some cases [31–33]), providing a mechanism for intrinsic or extrinsic
postzygotic reproductive isolation. Although the selective pressures favoring one venom
type over the other are unknown, venoms are costly to produce [34]; selection often favors
reduced venom expenditures [18,35], suggesting that type A venoms might be generally
advantageous due to their heightened toxicity. The predigestive benefits of type B venoms
and SVMPs, however, may allow for the consumption of larger prey or digestion at lower
temperatures [25,28], potentially expanding the range, altitude, and diet of type B species
and populations. The distributions of A-B venoms in some species have been shown
to be correlated with the annual environmental temperature [33], suggesting abiotic as
well as biotic (e.g., prey) sources of selection may determine phenotype distributions in
this system.
The Timber Rattlesnake (Crotalus horridus) occurs in the eastern half of North Amer-
ica from southern Ontario, southward to northern Florida, and westward to Texas and
Minnesota [36], exhibiting both A and B venoms [26,27,30,37]. We previously examined
venom-gland transcriptomic and venom proteomic divergence between one type A C. hor-
ridus from Florida (Osceola National Forest, herein referred to as ONF population) and
one type B C. horridus from Georgia (Jones Center at Ichuaway, herein referred to as JC
population) [37]. We found that complete changes in venom composition, rather than
point mutations affecting venom gene coding sequences, explained much of the differences
between venom types, consistent with the work of Dowell et al. [30]. Dowell et al. [30] used
BAC cloning to show that type A C. horridus possessed six PLA2 venom genes (including
the acidic and basic subunits of canebrake toxin, the defining neurotoxin of type A venoms)
whereas type B individuals possessed only one functional PLA2 venom gene, highlighting
the role gene loss can play in venom evolution. For SVMPs, however, type B C. horridus
possessed 13 SVMP paralogs; type A C. horridus possessed nearly all of the same loci,
indicating that, in this region, the differential gene regulation accounts for the divergent
phenotypes, consistent with other type A species [15].
Toxins 2021, 13, 782 3 of 17

Given that the genetic bases of both venom phenotypes have been characterized in
C. horridus [30,37], we can now begin to determine the roles geography, history, ecology, se-
lection, and chance play in determining when and why new species emerge or are absorbed.
Here, we aimed to (1) establish the phenotypic distributions of A and B venoms, (2) deter-
mine whether morphological characters were also associated with feeding such as head
shape and fang length [33,38,39] vary by venom type, (3) identify the environmental differ-
ences between regions harboring different phenotypes, and (4) determine to what extent
the venom dichotomy impacts gene flow. To accomplish these goals, we first sampled indi-
viduals from adjacent populations in northern Florida (ONF; type A transcriptome locality)
and southern Georgia (JC; type B transcriptome locality) [37] and used proteomic, genetic,
and morphological data to extensively characterize microgeographic A–B divergence (i.e.,
populations were ∼225 km apart). We then sampled C. horridus across their entire range
to determine the spatial assortment of these phenotypes, explore abiotic characters that
may explain these distributions, and test for reciprocal monophyly by phenotype. Overall,
we used a combination of data to determine whether type A and type B C. horridus may
represent distinct, reproductively isolated lineages due to incipient ecological speciation or
differentiated populations within a single species. We recognize that mitochondrial-based
inferences about species divergence and boundaries can be problematic (see [40]) and
consider this study a first-step in exploring the evolutionary relationships between type A
and type B C. horridus.

2. Materials and Methods

2.1. Sampling
We collected and/or were provided venom and blood samples from 22 C. horridus
across Florida (n = 7), Georgia (n = 7), Texas (n = 2), Oklahoma, (n = 1), Alabama
(n = 1), and Ohio (n = 4). Snout–vent length (SVL) and total length (TL) were recorded
for each individual prior to release. Additional tissue was obtained from 236 preserved
specimens that were collected by the authors and/or donated to us for the study (see
Acknowledgments section). Samples collected by the authors were conducted under per-
mits LSSC-13-00004, LSSC-09-0399, and LSSC-20-00037 from the Florida Fish and Wildlife
Conservation Commission (FWC). All procedures were approved by the Florida State
University Institutional Animal Care and Use Committee (IACUC) under protocols #0924
and #1333 and the University of South Florida IACUC under protocol #IS00008815.

2.2. Reversed-Phase High-Performance Liquid Chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed
on a Beckman System Gold HPLC (Beckman Coulter, Fullerton, CA, USA) as previously
described [41,42] for the 22 C. horridus venom samples described above. In total, 50 µg
of venom protein was injected onto a Jupiter C18 column (250 × 4.6 mm; Phenomenex,
Torrence, CA, USA) using a solvent system of 0.1% trifluoroacetic acid (TFA) in water
(solvent A) and 0.075% TFA in acetonitrile (solvent B). Following five minutes at 5% B, a
1% per minute linear gradient of A and B was run to 25% B; this was followed by a 0.25%
per minute gradient from 25% to 65% B at a flow rate of 1 mL per minute. Column effluent
was monitored at 220 and 280 nm.

2.3. Canebrake Toxin PCR Assay

Genomic DNA was extracted from whole blood samples drawn from the caudal
vein or muscle/liver/scale tissue from 258 specimens using the Omega bio-tek E.Z.N.A
Tissue DNA Kit according to the manufacturer’s protocol; scale extraction procedures were
modified (e.g., increased tissue amounts and addition of 1 M diothiothreitol) as previously
described [43]. All extractions were visualized on a 2% agarose gel and run for 30–60 min
at 110 V to ensure the presence of high-quality DNA. The acidic and basic subunits of
canebrake toxin were amplified from template DNA in 25–50 µL PCR reactions using
the acidic subunit-sense (50 GGT ATT TCG TAC TAC AGC TCT TAC GGA 30 ), acidic
Toxins 2021, 13, 782 4 of 17

subunit-antisense (50 TGA TTC CCC CTG GCA ATT 30 ), basic subunit-sense (50 AAC GCT
ATT CCC TTC TAT GCC TTT TAC 30 ), and basic subunit-antisense (50 CCT GTC GCA
CTC ACA AAT CTG TTC C 30 ) primers, respectively, under the following thermal cycling
protocol: 95 ◦ C for five minutes, 35 cycles of 95 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for
five minutes, followed by 72 ◦ C for ten minutes [37,44]. Evidence for amplification was
visualized by means of a 0.7% agarose gel and ethidium bromide imaged on a Bio Rad Gel
Doc using Quantity One Version 4.1.1 or SYBR Safe DNA gel stain and photographed on
Dual LED Blue/White Light Transilluminator.
Because these subunits have been shown to be present in type A individuals but absent
in type B individuals [30], we were able to phenotype individuals from only genetic samples.
If an assay for an individual amplified both subunits, that individual was considered type A.
If an assay for an individual did not lead to amplification of either subunit, that individual
was considered type B. No individuals amplified only a single subunit which, based on the
genomic location of these genes [29,30], was expected. Because all DNA extractions were
visualized on a 2% agarose gel to ensure the presence of high-quality DNA and reduce
our false-negative rate, we were confident in all type B classifications; in some instances,
amplification of cytochrome b (see below) was used to confirm the presence of amplifiable
DNA for type B individuals. We do note, however, that this assay could not distinguish
between type A and type A+B hybrid venoms, which we discuss below.

2.4. Morphological Analysis

We collected data for 5 morphological characters from 33 preserved C. horridus spec-
imens (Table S1) as previously described [38]: (1) SVL, (distance from tip of snout to
posterior edge of cloaca), (2) head length (HL, distance from tip of snout to articular-
quadrate joint), (3) head width (HW, distance across widest point of head behind the eyes),
(4) interfang distance (IF, distance between fangs at maxillae), and (5) fang length (FL,
distance from anterior end of maxilla to the tip of fang as folded against roof of mouth).
Snout–vent length was measured to the nearest 0.5 cm using a tailor’s tape, and HL, HW, IF,
and FL were each measured to the nearest 0.01 mm using 150 mm digital calipers. Venom
type was ascribed based on origin from one of the two focal populations (i.e., JC or ONF).
To remove the effect of size prior to statistical analysis, we subtracted each of the
four natural-log-transformed head and fang measurements above (2–4) from the natural-
log-transformed SVL [45]. Note that for these transformed values, larger transformed
values represent smaller raw values and vice versa. We performed Kruskal–Wallis tests
on the transformed data for each variable to determine whether type A and type B indi-
viduals significantly differed for any character. We then performed a linear discriminant
function analysis using the lda function and leave-one-out crossvalidation from the MASS
package [46] in R to assess group membership placement probabilities based solely on
morphology across venom type. All statistical analyses were conducted using R v. 3.6.1 [47].

2.5. Niche Modeling

To determine if differences in venom type distributions were correlated with differ-
ences in abiotic environmental variables, we followed the approach of Strickland et al. [33]
and constructed environmental niche models for each venom type independently using
MaxEnt 3.4.1 [48] as implemented in dismo 1.3–3 [49]. We included locality information for
86 type A and 172 type B C. horridus and 19 bioclimatic variables as well as elevation at
30 arc second resolution [50]. All predictor variable rasters were cropped to the extent of
the presence records and masked with the IUCN species range [51]. To address collinearity,
we performed a principal component analysis and removed principal components with
eigenvalues <1 [52]. The two highest loading predictors for each of the remaining principal
components were included in the model; these variables comprised Minimum Temperature
of Coldest Month, Maximum Temperature of Warmest Month, Precipitation of Warmest
Quarter, Mean Temperature of Wettest Quarter, Mean Diurnal Range, Temperature Sea-
sonality, Precipitation of Driest Month, Precipitation of Wettest Quarter, and Precipitation
Toxins 2021, 13, 782 5 of 17

of Coldest Quarter. Models were evaluated by AUC with k = 4 (i.e., 25% of the data)
crossvalidation. To reduce the effects of sampling bias on the model, presence records
occurring in the same grid cell were removed. After this reduction, the Type A model used
51 training and 18 testing records, and the Type B model used 85 training and 29 testing
records. Variable contribution was assessed by permutation and jackknife.
To test niche equivalency across venom types, we used the niche identity test [53]
implemented in ENMTools 1.0.5 [54] to determine whether the distribution of type A and
type B individuals was identical in geographic and environmental space. Here, the test
computed three measures of similarity between our two environmental niche models,
Schoener’s D, Warren’s I, and Spearman’s rank correlation, and compared these measures
across both geographic and environmental space. For each statistic, null distributions
were generated using 99 permutation replicates where the occurrence points of type A
and type B individuals were randomly reassigned to one of the two models. The test
statistic calculated using the original, unpermuted data was then compared with the null
distribution to test for significance using a one-tailed test.

2.6. mtDNA Sequencing and Phylogenetic Reconstruction

DNA was extracted and quality checked as described above. For 56 C. horridus, a
1079 bp fragment of cytochrome b was amplified in 25 µL PCR runs using the H16064 and
L14910 primers and thermal cycling protocol previously described [55]. PCR products
were purified using the QIagen QIAquick PCR Purification Kit. Sequencing was performed
on the Applied Biosystems 3730 Genetic Analyzer.
Sequences were aligned and editing using Geneious Prime and adjusted manually.
We then constructed two phylogenies using the same approach outlined below: (1) a
ONF (n = 17) and JC (n = 9) phylogeny to match our initial venom and morphological
comparisons, and (2) a range-wide phylogeny (n = 56). For each, we fit 88 candidate
models of nucleotide substitution using jModelTest 2.1 [56] using the CIPRES portal [57];
the best fit model was determined according to the Bayesian Information Criterion. Using
BEAST2 [58], we inferred time-calibrated phylogenies. We used a birth–death tree prior as
this prior leads to greater accuracy in the estimation of node times when datasets contain
a mixture of intra- and inter-specific sampling [59]. For our clock model, we used an
uncorrelated lognormal, relaxed molecular clock [60], and estimated, rather than specified,
the rate. We specified two fossil calibrations [61] as previously described [38]. Here, we set
the age as a mean of 0, SD of 1, and offset these prior distributions using the early boundary
of the time period in which the fossils were confirmed. We constrained the outgroup,
C. adamanteus (sequences from [38]), to be monophyletic, and specified their earliest time
to the Most Recent Common Ancestor (MRCA) as 0.781 Ma, using a fossil sample from
the Early Pleistocene (Irvingtonian I NALMA: [62]). We constrained the earliest time to
MRCA for C. horridus to 0.126 Ma based on a fossil sample of this species from the Middle
Pleistocene (Irvingtonian II NALMA; [62]). We conducted three independent runs for a
length of 50 million generations each, sampling every 5000 generations following an initial
burn-in of 10,000 generations. We visually assessed convergence and stationarity for all
parameters in Tracer [63]. The three independent runs were subsequently combined, resam-
pling from each every 50,000 generations for a final posterior distribution of 3000 samples.
All effective sample sizes for these combined runs exceeded 300. We summarized the
posterior distribution of trees using TreeAnnotator to obtain a maximum clade consensus
tree using median clade heights for node heights. Trees were visualized in FigTree; clades
with posterior probabilities >0.70 were annotated.

3. Results and Discussion

3.1. Fixed Venom Differences, Deep Mitochondrial Genetic Divergence, and Distinct Morphologies
over Small Spatial Scales
To characterize A–B divergence at the microgeographic scale, we used proteomic,
genetic, and morphological data to compare the ONF and JC populations, which were only
Toxins 2021, 13, 782 6 of 17

∼225 km apart. We used RP-HPLC to show that each population was monomorphic for
their respective venom phenotype (Figure 1A), with all individuals from JC possessing type
B venoms, and all individuals from ONF possessing type A venoms. These fixed differences
in venom phenotype were consistent with previous results showing that the two canebrake
toxin subunits were present in the genomes of animals from ONF but not in the genomes
of animals from JC [37]. Overall, the fixed venom phenotypes between JC and ONF
populations suggested little-to-no gene flow, reinforcing the hypothesis that intermediate
venoms may be disfavored by selection in this system (but see [31–33]) and, therefore, may
provide a mechanism for intrinsic or extrinsic postzygotic reproductive isolation.

A Type A Type B
B KW0654-FL-A
KW0874-FL-A
0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8 0.0 0.4 0.8

FL (KW1089) GA (KW1298) KW0917-FL-A

KW1089-FL-A
KW0526-FL-A
0.99 KW0923-FL-A
FL (KW1209) GA (KW1328) KW0871-FL-A
Absorbance (220 nm) relative to maximum

KW1211-FL-A
KW1242-FL-A
0.99 KW0854-FL-A
FL (KW1210) GA (KW1329)
0.734 Mya: KW1452-FL-A

[0.0798 - 1.72] KW0892-FL-A

KW1241-FL-A
KW1210-FL-A
FL (KW1211) GA (KW1330)
KW1299-FL-A
KW1209-FL-A
1.0 KW1451-FL-A

FL (KW1241) GA (KW1331) 1.23 Mya: KW1329-GA-B

[0.249 - 2.37] KW1404-GA-B
KW1331-GA-B
KW1298-GA-B

FL (KW1242) GA (KW1404) KW1406-GA-B

KW1330-GA-B
KW1369-GA-B
0.649 Mya: KW1328-GA-B
FL (KW1299) GA (KW1406) [0.0245 - 1.63] KW1334-GA-B
1.0 Cadam-KW0529

1.087 Mya: [0.806 - 1.75] Cadam-KW1144

Canebrake toxin SVMPs Canebrake toxin SVMPs

20 40 60 80 100 120 20 40 60 80 100 120 1.5 1.25 1 0.75 0.5 0.25 0 Mya
Time (minutes)

Figure 1. The type B Jones Center (JC) population showed significant venom expression and genetic
divergence from the type A Osceola National Forest (ONF) population, suggesting incipient venom-
associated speciation. (A) Reversed-phase high-performance liquid chromatography (RP-HPLC)
profiles for seven adult individuals from each population are shown. The highlighted region from
∼100–120 min includes the snake venom metalloproteinases (SVMPs), and the two phospholipase
A2 subunits of canebrake toxin were found prior to 60 min [37]. All individuals from JC were
monomorphic for type B venom, and all individuals from ONF were monomorphic for type A
venom. (B) A time-calibrated BEAST analysis of cytochrome b sequences from both populations
showed reciprocal monophyly and deep divergence between the populations. Median divergence
time estimates (with 95% credible intervals) and clade support values (posterior probabilities) are
shown for nodes where the posterior probability >0.70. Crotalus adamanteus (Cadam) was used as the
outgroup. Type A individuals are represented by red dots, and type B individuals are represented by
blue dots.

To determine whether A–B venom differences reflected population structure based on

neutral markers at this scale, we constructed a time-calibrated Bayesian phylogeny based
on cytochrome b sequence for 17 ONF (type A) and 9 JC (type B) individuals (Figure 1B).
The venom phenotypes were reciprocally monophyletic with a median divergence time
estimate between type A and type B individuals of ∼1.23 Ma (95% CI = 0.249–2.37 Ma).
Type A ONF individuals comprised a strongly supported monophyletic clade (posterior
probability = 0.99) distinct from that of the type B individuals (for which relationships
were less well supported). Based on mtDNA, these populations were genetically distinct
and putatively reproductively isolated.
To determine whether venom expression differences were integrated with morpho-
logical characters also associated with feeding, we measured HL, HW, IF, FL and SVL for
33 preserved C. horridus (18 JC and 15 ONF individuals) and performed Kruskal–Wallis
rank sum tests on each morphological character by venom type as previously described [38].
Type A individuals had significantly narrower heads (p = 0.023) and significantly shorter
fangs (p < 0.001) than type B individuals (Figure 2A). Although type A snakes also had
Toxins 2021, 13, 782 7 of 17

shorter heads, the difference was not significant (p = 0.096); we identified no differences
in IF (p = 0.745). A linear discriminant function analysis based on these characters alone
accurately placed 80.0% of type A snakes and 94.4% of type B snakes (Figure 2B), indicating
a strong association between head morphology and venom type, consistent with previous
results in rattlesnakes [38]. Coefficients of linear discriminants indicated that fang length
(−12.859) was the most important predictor when discriminating among groups (IF = 8.059,
HW = −4.217, HL = 6.278). Although venom plays a primary role in prey incapacitation
and predation in rattlesnakes, morphological characters such as gape and fang length
may also determine dietary breadth and feeding ecology [33,38,39]. Margres et al. [38]
previously showed that fang length positively covaried with myotoxin (i.e., crotamine)
expression in Crotalus adamanteus and posited that the increased depth of injection for
myotoxin-rich venoms may increase predation success. Here, the significant reductions
in head size and fang length were inversely correlated with overall toxicity, suggesting
that the (1) depth of injection may not matter for type A venoms (or at least may be less
important relative to type B venoms) and (2) narrower heads may be a consequence of prey
differences and/or a reduction in the volume of venom needed given the increased toxicity
of type A venoms. Interestingly, similar work in type A and type B Mojave Rattlesnakes
(C. scutulatus) did not find significant differences in HW, HL, IF, or FL between type A
and type B individuals, although trends did exist for type A individuals to have longer
fangs and potentially wider heads [33], opposite of what was found here. The discordance
between A–B C. horridus and C. scutulatus individuals suggests that these morphological
differences may be prey specific rather than venom-type specific.

A * B Type A (FL)
0.8
4.5

Placement probability = 0.80

0.4
4.0
ln(SVL)-ln(X)

0.0

* 3 2 1 0 1 2
3.5

Type B (GA)
0.8

Placement probability = 0.94

3.0

0.4
0.0

A B A B A B A B
Interfang Head Head Fang
distance width length length 3 2 1 0 1 2

Figure 2. The type B Jones Center (JC) and the type A Osceola National Forest (ONF) populations
differed significantly in head morphology. We measured interfang distance, head width, head length,
and fang length and adjusted the values on the basis of snout-to-vent length. (A) We found significant
differences in head width and fang length. The type A population had shorter fangs and narrower
heads. Note that due to our transformation of the data, larger values on the y-axis represent smaller
raw values. (B) A linear discriminant function analysis clearly distinguished the two populations
on the basis of head morphology. Representative head and fang morphology for each venom type
are shown. * p < 0.05.

Overall, we identified fixed venom differences, a deep phylogenetic split dating

back >1 Ma (although we recognize that mitochondrial gene trees can be misleading
when delimiting species (see [40]) and distinct head morphologies across a rather small
geographic area. Previous work by ourselves [37] and others [30] showed that many of the
major venom differences between these populations, such as the lack of heterodimeric PLA2
neurotoxins in type B venoms, reflected an absence in the genome of the corresponding
genes rather than the differential regulation of shared genes. Together, these results suggest
that type A ONF and type B JC C. horridus may represent independently evolving lineages,
perhaps due to incipient ecological speciation. Alternatively, these differences could reflect
incipient allopatric speciation as these populations occur on either side of the Suwannee
River, a known biogeographic barrier for continental and peninsular Florida organisms [64],
Toxins 2021, 13, 782 8 of 17

including rattlesnakes [13,38]. The Suwannee Straits ran through the Okefenokee Trough
from the Gulf of Mexico to the Atlantic Ocean, repeatedly separating peninsular Florida
from the continent [65]. Although the Straits likely closed ∼4 Ma, some evidence suggests
that the Straits may have opened briefly as recently as ∼1.75 Ma [38,64]. With our current
dataset, we could not distinguish between incipient allopatric and ecological speciation at
this scale. Crotalus horridus, however, has a very large geographic range, and ecological
speciation comes with explicit predictions. Isolation by environment (IBE) should be much
greater than isolation by distance (IBD) if ecological differences are leading to reproductive
isolation [66]. To determine whether phenotypes were spatially distinct or overlapping, we
next sampled more broadly across the range to identify the distributions of each phenotype.

3.2. Type A Venom Dominates along the Southern Periphery of the Range
We assayed 258 individuals across the range of C. horridus and detected 86 type A
individuals and 172 type B individuals (Figure 3). Type A venoms were largely present
in the southern periphery of the range, and type B venoms dominated the northern and
interior parts of the range. Type A venoms may be common along the western range
edge as well, but sampling in this region was too sparse to make a determination. Both
phenotypes co-occurred in the coastal plain regions of South Carolina, Georgia (see inset in
Figure 3), and Orleans Parish, Louisiana, although sampling in the latter was sparse (three
type Bs and one type A). We note that our assay could not distinguish between type A
and type A+B “hybrid” venoms as both venom types would possess the canebrake toxin
subunits. Therefore, some of the individuals classified as type A in Figure 3 could represent
A+B venoms, and one type A individual included here from Jekyll Island, Georgia was a
C. horridus × C. adamanteus hybrid [67].

Type A venom (n=86)

Type B venom (n=172)

JC population
ONF population

Figure 3. Type A venoms were found largely along the southern periphery of the range. We
used a PCR assay to detect the presence of the two subunits of canebrake toxin to establish the
presence of type A venom. The grey shading indicates the historical range of C. horridus. Our two
focal populations (JC and ONF) are indicated. Some points were jittered in an attempt to show all
individuals from a single locality. The inset map in the bottom right corner shows a clear contact zone
between type A and type B individuals in the coastal plain region of South Carolina and Georgia.
Toxins 2021, 13, 782 9 of 17

Venom-based analyses showed that both type A and type B phenotypes were similar
across the range (e.g., Florida and Texas type A venoms were very similar; Figures 1 and 4),
although there appeared to be more variation within type B venoms based on our limited
venom-based sampling (Figure 4). Given (1) the much larger geographic distribution of type
B venoms and, therefore, greater variation in abiotic and biotic selective pressures and (2)
that type B venoms are much more complex than type A venoms (Figures 1 and 4; [15,23])
and have many more axes on which they can vary, this result was expected.
Absorbance (220 nm) relative to maximum
0.8

TX (KW1238) Type A OH (KW1936) Type B

0.4
0.0

Type A Type B
0.8

TX (KW1253) OH (KW1937)
0.4
0.0
0.8

OK (KW1067) Type B OH (KW1938) Type B

0.4
0.0
0.8

AL (KW1412) Type B OH (KW1939) Type B

0.4
0.0

Canebrake toxin SVMPs Canebrake toxin SVMPs

20 40 60 80 100 120 20 40 60 80 100 120

Time (minutes)
Figure 4. Representative reversed-phase high-performance liquid chromatography (RP-HPLC)
profiles of venoms from additional populations showed that the A/B dichotomy was consistent
across the range. Venoms either had peaks after 100 min and were therefore type B or peaks before
60 min that corresponded to the two canebrake toxin subunits.

Overall, venom types were spatially sorted across the range, suggesting an ecological
divergence between type A and type B individuals. To test whether differences in venom-
type distributions were correlated with differences in abiotic environmental variables, we
next constructed environmental niche models for each venom type independently.

3.3. Type A and B Individuals Occupy Significantly Different Niches

The distributions of type A and type B individuals were significantly different in
both geographic (DGeo = 0.29, IGeo = 0.58, Spearman’s rank correlation = −0.03; p < 0.01
for all tests) and environmental (DEnv = 0.19, IEnv = 0.44, Spearman’s rank correlation
= 0.33; p < 0.01 for all tests) space (Figure 5). For the type A model (AUC: training 0.948,
test 0.960; Figure 5A), the mean temperature of the wettest quarter (72.2%) and minimum
temperature of the coldest month (14.2%) were the most informative environmental vari-
ables. Permutation importance (mean temperature of the wettest quarter perm = 26.1%;
minimum temperature of the coldest month perm = 60.8%) and jackknife testing confirmed
these results, with mean temperature of the wettest quarter being the most informative
variable during jackknife testing. For the type B model (AUC: training 0.826, test 0.810;
Figure 5B), precipitation of warmest quarter (47.4%) and temperature seasonality (24.1%)
were the most informative environmental variables. Results were again confirmed by
permutation importance (precipitation of warmest quarter perm = 37.3%; temperature
seasonality perm = 26.4%) and jackknife testing, with precipitation of warmest quarter being
the most informative variable during jackknife testing.
Toxins 2021, 13, 782 10 of 17

A B

Figure 5. Environmental niche models for Type A and Type B individuals revealed significant
differences in geographic and environmental distributions. Habitat suitability predictions produced
by MaxEnt environmental niche models using 69 Type A (A) and 114 Type B (B) presences, indicated
by points, are shown. The heatmap corresponds to habitat suitability scores and can be interpreted
as a percentage.

Temperature appeared to be a key factor in determining the distributions of type A

and B venoms in C. horridus. Type A habitat suitability was positively correlated with
mean temperature of the wettest quarter and minimum temperature of the coldest month,
the two most informative variables in the model, whereas these variables had relatively
low contributions in the type B model (<10%) and were negatively associated with type B
habitat suitability. Previous work showed that the distributions of A–B venoms in Mojave
Rattlesnakes (Crotalus scutulatus) was correlated with minimum temperature of the coldest
quarter and mean temperature of wettest quarter [33], consistent with our results here.
Given that type A venoms within polymorphic species appear to occur in warmer parts of
the range, perhaps the beneficial predigestive benefits of type B venoms and SVMPs are no
longer required for effective predation/digestion along the southern periphery [25,28]. In
this case, the more toxic type A venoms may be advantageous in the absence of digestive
constraints, but when these constraints do apply, type B venoms may be favored, thus
limiting the northward expansion of the type A phenotype.
Type A and B venoms, however, could also be locally adapted for different prey
species or genetically distinct populations of the same species, and the relationship with
temperature may simply correlate with local prey community composition. For example,
previous work in Pacific Rattlesnakes (C. oreganus) found that biotic information about local
prey species explained more variation in venom expression than abiotic environmental
data [68]. These hypotheses, however, are not mutually exclusive; here, type B venoms may
be adapted for a certain species of prey and pro-digestive effects whereas type A venoms
may be adapted for a different species of prey and also be free of digestive constraint.
Although diet information for most pit vipers is sparse, we examined 22 published articles,
books, and conference proceedings [69–90] containing dietary information for C. horridus
across their range to compare prey items in regions where C. horridus was monomorphic for
type B to regions where C. horridus was monomorphic for type A (regions where C. horridus
was polymorphic in venom type were excluded). We found 130 type B records but only
10 type A records (Table S2). Mammals (∼76% of type B records, 60% of type A records)
and birds (∼17% of type B records, 30% of type A records) comprised the majority of the
diet for each venom type. Although the small sample size in type A prevented statistical
comparisons, the largest presence/absence difference was found in rodents belonging to
the family Cricetidae. Cricetid rodents (e.g., Peromyscus, Microtus, and Sigmodon) were
∼33% of all prey items recorded for type B animals and were notably absent from the
type A records. Type B venoms, therefore, may be locally adapted for Cricetid rodents,
and perhaps type A venoms are locally adapted to a different prey species and/or abiotic
conditions. Toxicity and digestive assays quantifying these differences in natural prey
Toxins 2021, 13, 782 11 of 17

items are needed to test these hypotheses; although interesting and ultimately necessary to
characterize the source of ecological divergence (if present), such work was beyond the
scope of the present study.
The identified significant differences in niche space, accompanied with differences in
multiple, putatively adaptive phenotypes in venom and head morphology, again suggested
incipient ecological speciation. Assessing the degree of reproductive isolation is required
when investigating putative species boundaries in wide-ranging taxa, especially along
contact zones [40,91]. Therefore, we next sequenced cytochrome b across the range, with a
focus on the contact zone in the southeastern coastal plain, to determine whether venom
types retained reciprocal monophyly (as found in the JC and ONF populations) or if
reproductive isolation between venom types broke down at larger geographic scales.

3.4. A Lack of Reciprocal Monophyly Suggests Gene Flow between Venom Types across the Range
To determine whether the A–B reciprocal monophyly detected for the ONF and JC
populations (Figure 1B) was found range wide, we sequenced cytochrome b for 30 type A
and 26 type B individuals across the range and constructed a Bayesian phylogeny (Figure 6).
Although we identified two well-supported monophyletic clades of type A animals (one
including individuals from Florida, Georgia, and South Carolina; one including all three
Texas type As), neither venom type was monophyletic, and no well-supported mono-
phyletic clades of only type B animals were identified (Figure 6). This pattern suggested
that venom types were not reproductively isolated and is consistent with introgression
occurring primarily from type A populations into type B populations. However, more
extensive sampling of nuclear genetic data is necessary to test this hypothesis.
Given the lack of reciprocal monophyly based on venom type, we next looked at the
spatial distribution of the population structure identified in the phylogeny (Figure 6). We
identified six clades with varying levels of support: (1) a well-supported type A-only clade
containing all 17 ONF individuals, as well as one individual from Georgia and one from
South Carolina (pink clade in Figure 6), (2) a well-supported western clade of mostly type
B animals (seven type Bs and two type As) along the Mississippi drainage (cyan clade
in Figure 6), (3) a well-supported type A-only clade containing three Texas individuals
(black clade in Figure 6), (4) a less supported Georgia–South Carolina clade containing
all JC individuals and four other individuals (11 type Bs and 2 type As; orange clade in
Figure 6), (5) a well-supported South Carolina clade (three type Bs and four type As; gray
clade in Figure 6), and (6) a less supported type B only clade in the southeast (yellow
clade in Figure 6). Surprisingly, the type A-only pink clade that contained all 17 ONF
individuals and 1 individual each from Georgia and South Carolina was more closely
related to the western lineages (cyan Mississippi Drainage and black Texas lineages in
Figure 6) than the other three lineages identified in the southeast (yellow, orange, and gray
lineages in Figure 6); this relationship, however, was not strongly supported (posterior
probability = 0.74). While this could indicate that the type A phenotype arose in the west
and has since spread east, denser sampling across the genome and the range is needed
to determine the degree of reproductive isolation between venom types/populations
as well as the origin(s) of the type A phenotype in C. horridus. Overall, these results
revealed substantial geographic population structure within C. horridus that was only
weakly associated with venom type.
Toxins 2021, 13, 782 12 of 17

Type A venom (n = 30)

Type B venom (n = 26)

CH239
CH241

MM0020

CH393 CH447
CH245
CH414
CH407 CH274A
KW0216
CH364
CH271A
CH227 CH389 CH268
CH224 CH388
CH406
KW1067 KW1290

KW1295
CH233
CH296 KW1412
KW1253 CH298 CH265
KW1238
CH297 KW0526
MM0020
KW0654
KW1284 KW0854
KW0871 CH393 CH447
KW0874 CH414
KW1298 KW0892 CH407 CH274A
KW1328 KW0917
KW0923 KW0216
KW1329
KW1330 KW1089
KW1209 CH364
KW1331
KW1210 CH271A
KW1334 CH389 CH268
KW1369 KW1211
KW1241 CH388
KW1404 CH406
KW1406 KW1242
KW1299
KW1451
KW0892-FL-A
KW1452
KW1451-FL-A
CH265-GA-A
KW1210-FL-A
KW0854-FL-A
KW1209-FL-A
KW1241-FL-A
KW1299-FL-A
CH447-SC-A
KW1452-FL-A
0.99 KW0874-FL-A
KW0917-FL-A
KW1242-FL-A
KW0654-FL-A
KW0923-FL-A
0.99 KW1211-FL-A
KW1089-FL-A
KW0526-FL-A
KW0871-FL-A
CH241-IL-B
CH296-LA-A
CH239-IL-B
CH245-IL-B
0.74 KW1290-AR-B
0.97 CH227-AR-B
0.646 Mya: CH298-MS-B
[0.0834-1.98] CH297-LA-A
KW1067-OK-B
0.99 KW1238-TX-A
KW1253-TX-A
KW1284-TX-A
KW1330-GA-B
KW1406-GA-B
KW1334-GA-B
KW1369-GA-B
KW1329-GA-B
1.0 KW1404-GA-B
KW1298-GA-B
1.16 Mya: KW1331-GA-B
[0.269 - 3.26] KW1328-GA-B
CH271A-SC-A
0.99 CH388-GA-B
CH406-GA-B
CH389-GA-A
0.80 CH268-SC-A
KW0216-SC-A
CH393-SC-B
CH274A-SC-B
0.99 MM0020-SC-A
CH414-SC-B
0.646 Mya: CH407-SC-A
[0.0984-1.97] CH224-NC-B
CH233-GA-B
CH364-GA-B
KW1295-AL-B
KW1412-AL-B
1.0 Cadam-KW0529
Cadam-KW1144
1.083 Mya: [0.811 - 1.75]
4 1 0 Mya

Figure 6. Venom types were not reciprocally monophyletic, suggesting gene flow between venom
types in some geographic regions. We sequenced cytochrome b from 30 type A and 26 type B
individuals and constructed a time-calibrated BEAST phylogeny. We identified two well-supported
monophyletic clades of only type A animals, but we found no well-supported pure type B clades.
The dot to the right of each tip corresponds to venom type (red type A, blue type B), and branch
color corresponds to clade (distribution colored on map above). Top map inset shows clade color;
bottom inset shows venom type. Median divergence time estimates (with 95% credible intervals) are
shown at key nodes. Posterior probabilities >0.70 are shown. Crotalus adamanteus (Cadam) was used
as the outgroup.
Toxins 2021, 13, 782 13 of 17

How putatively distinct lineages interact along contact zones and potential species
boundaries is essential for determining whether such lineages are evolving independently
as distinct species or collectively as geographically differentiated populations [40]. Based
on a mitochondrial marker, type A and type B C. horridus were not independently evolving
lineages and appeared to readily interbreed in coastal Georgia and South Carolina (Figure 6)
despite isolation elsewhere (Figure 1). These equivocal results support varying intensities
of introgression between type A and type B individuals across the range, and this variance
may be the result of variance in ecologically divergent selective pressures. Here, ecologically
divergent selection would be strong between the ONF and JC populations, thus producing
substantial genetic divergence and putative reproductive isolation, whereas selection would
be weaker in the coastal plain of South Carolina and Georgia (i.e., orange and gray clades
in Figure 6) where the evidence of introgression was strong. Although the ecologically
divergent selection pressures may act on the venom phenotype as predicted in this study,
we could not rule out the possibility of other loci in linkage disequilibrium with the PLA2
venom genes being the actual targets of selection, perhaps for a trait not related to predation
and feeding ecology. Additionally, neutral processes due to historical biogeographic events
(e.g., the formation of the Suwannee Straits as discussed above [65]) could also produce
similar patterns. Given that mitochondrial gene trees are often misleading when making
inferences about species boundaries [40], nuclear genomic and ecological data across the
range with targeted, dense sampling along contact zones is required to better understand
the population structure and adequately assess reproductive isolation. Nevertheless, our
current mitochondrial data do not support type A and type B C. horridus as distinct lineages
range-wide.

3.5. Conclusions
We used a combination of genetic, proteomic, morphological, and environmental
data to test whether type A and type B C. horridus may represent distinct, reproductively
isolated lineages due to incipient ecological speciation or differentiated populations within
a single species. Although we found significant genetic, proteomic, morphological, and
environmental differences between type A and type B C. horridus at microgeographic
scales (i.e., between ONF and JC populations), the lack of reciprocal monophyly among
venom types range-wide suggested varying intensities of gene flow and an overall lack
of reproductive isolation across the range. Speciation is a continuous process [92], and
speciation with gene flow may be common [93], but resolving species relationships among
lineages with complex, reticulate evolutionary histories remains challenging [91]. To
address this issue, Marshall et al. [40] recently proposed the use of genome-wide nuclear
data and intensive sampling along contact zones to determine whether the lineages of
interest are evolving mostly independently (i.e., reproductively isolated with narrow zones
of F1 hybrids) or as geographically differentiated populations (i.e., intergradation across
wider zones with F2+ hybrids). Such an approach here with increased sampling in the
coastal plain regions of South Carolina and Georgia as well as along the Gulf Coast is
necessary to disentangle the complex evolutionary relationships between type A and type
B C. horridus.

Supplementary Materials: The following is available online at https://www.mdpi.com/article/10

.3390/toxins13110782/s1, Table S1: morphology data; Table S2: diet data. The data used to construct
the niche models are available on request from the corresponding author. The data are not publicly
available to protect specific locality information for a species that is protected and imperiled across
much of its range.
Author Contributions: M.J.M., D.R.R. and K.P.W. conceived of and designed the study. D.S., P.J.M.,
L.M.T., K.P.W. and M.J.M. generated the data. M.J.M., K.P.W., P.J.M., A.H.P. and D.R.R. analyzed the
data. All authors wrote the manuscript. All authors have read and agreed to the published version of
the manuscript.
Toxins 2021, 13, 782 14 of 17

Funding: This work was funded by the National Science Foundation (DEB 1145987 to DRR) and the
University of South Florida (to MJM).
Institutional Review Board Statement: This study was conducted under permits LSSC-13-00004,
LSSC-09-0399, and LSSC-20-00037 from the Florida Fish and Wildlife Conservation Commission
(FWC). All procedures were approved by the Florida State University Institutional Animal Care
and Use Committee (IACUC) under protocols #0924 and #1333 and the University of South Florida
IACUC under protocol #IS00008815.
Informed Consent Statement: Not applicable.
Data Availability Statement: All cytochrome b sequences used in our phylogenetic analyses were
deposited on NCBI under accessions MZ394520–MZ394575.
Acknowledgments: We thank Jennifer and Daniel Abney, Richard Bartlett, Joseph Colbert and the
Jekyll Island Authority, Brian Crother, Matthew Holding, Mark S. and Robin Margres, and Lora
Smith and Jennifer Howze at the Joseph W. Jones Ecological Research Center at Ichuaway for help in
acquiring venom and tissue samples. We thank Paul Moler for donating hundreds of tissue samples
to the project as well as Max Nickerson and the Florida Museum of Natural History for access to
preserved specimens for morphological analysis. We thank Rhett M. Rautsaw and Erich P. Hofmann
for assistance in collating diet information.
Conflicts of Interest: The authors declare no conflict of interest.

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