Molecular Phylogenetics and Evolution xxx (xxxx) 107313

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

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Genome-wide data reveal extensive gene flow during the diversification of
the western rattlesnakes (Viperidae: Crotalinae: Crotalus)
Edward A. Myers ⁎

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Department of Herpetology, American Museum of Natural History, New York, NY, USA
Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC, USA

ARTICLE INFO ABSTRACT

Keywords: Hybridization and introgression are important, but often overlooked processes when inferring phylogenies.
Reticulation When these processes are not accounted for and a strictly diverging phylogenetic model is applied to groups with
Hybridization

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a history of hybridization, phylogenetic inference and parameter estimation can be inaccurate. Recent develop-
Introgression
ments in phylogenetic network approaches coupled with the increasing availability of genomic data allow infer-
Crotalus
ences of reticulate evolutionary histories across the tree of life. The western rattlesnake species group (C. viridis
Multispecies network coalescent
species complex, C. mitchellii species complex, C. scutulutas, and C. tigris) is an iconic snake lineage that is wide-
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spread across western North America. This group is composed of several species complexes with unclear species
limits, likely the result of ongoing gene flow among nascent lineages. Here I generate reduced representation ge-
nomic data and test for a history of reticulation within this group. I demonstrate that all species have undergone
hybridization with at least one other lineage, suggesting introgression is widespread in this group. Topologies
differ between phylogenies estimated under the multispecies coalescent and multispecies network coalescent
methods, indicating that gene flow has obscured phylogenetic relationships within this group. These past intro-
gression events are predominantly restricted to species that co-occur geographically. However, within species
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that have a history of introgression, this signature is detected regardless of specimen sampling across geography.
Overall, my results suggest the accumulation of reproductive isolating barriers occurs slowly in rattlesnakes
which likely leads to the difficulty in delimiting species, furthermore, the results of this study have implications
for trait evolution in this group.
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1. Introduction Introgression has been documented in a number of taxa, yet we have

Contrary to the long-held believe that reproductive isolation is re-
quired to form new species (Mayr, 1963), evolutionary biologists are in-
creasingly finding that gene flow between lineages occurs during speci-
ation (Kautt et al., 2020; Mallet et al., 2016; Nosil, 2008; Roux et al.,
2016). These findings imply there is a period of time during which re-
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a limited understanding of its prevalence during the process of species
diversification (Pulido-Santacruz et al., 2020). However, methods to
test for divergence with gene flow have recently been developed. In-
complete lineage sorting (ILS) and gene flow both result in shared ge-
netic variation among species. Multispecies coalescent approaches as-
sume that incongruence among gene-trees is the result of incomplete

productive barriers are incomplete, allowing for hybridization and in-
trogression between diverging lineages (Singhal and Moritz, 2013).
During this protracted period of time, the intensity of gene flow may de-
crease as divergence time increases between a pair of species (Pulido-
Santacruz et al., 2020). Moreover, the timescale at which reproductive
barriers evolve between taxa with stable species boundaries may be >
10 mya (Barth et al., 2020). An extended period in which reproductive
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lineage sorting alone (Edwards, 2009). These models may be inappro-
priate for reconstructing the evolutionary history of a group of hy-
bridizing species where both ILS and gene flow must be considered
(Ferreira et al., 2020; Malinsky et al., 2018). Recently developed multi-
species network coalescent (MSNC) approaches allow for the recon-
struction of phylogenies that are not strictly diverging and incorporate
reticulation (Hejase et al., 2018; Solís-Lemus et al., 2017; Wen et al.,

barriers are incomplete may allow for even non-sister species to hy- 2018; Zhang et al., 2018). These methods are computationally intensive
bridize (Jónsson et al., 2014). and largely restricted to small numbers of terminals and reticulation
events (Yu and Nakhleh, 2015). Introgression can also be assessed using

⁎ Address: Department of Herpetology, American Museum of Natural History, New York, NY, USA.
E-mail address: eddie.a.myers@gmail.com.

https://doi.org/10.1016/j.ympev.2021.107313
Received 30 December 2020; Received in revised form 28 August 2021; Accepted 14 September 2021
1055-7903/© 2021
E.A. Myers
site-based summary statistics of tree asymmetries across a known topol-
ogy (Durand et al., 2011; Green et al., 2010). These methods are useful
in identifying cases of introgression and are computationally efficient,
however, they are also less useful for identifying the number of intro-
gression events within groups that experience frequent gene flow
(Malinsky et al., 2018; Martin et al., 2015). Therefore, to understand di-
versification with gene flow, it is useful to combine these methods to in-
fer past introgression and reticulation across a phylogeny (Ferreira et
al., 2020).
Rattlesnakes (Crotalus and Sistrurus) are one of the most recogniz-
able and iconic snake groups. These snakes have long fascinated biolo-
gists and their life history, behavioral ecology, and venom are relatively
well-studied (Campbell and Lamar, 2004). Despite being the subject of
numerous phylogenetic analyses, many relationships within the genus
Crotalus remain unresolved (Reyes-Velasco et al., 2013). Additionally,
the species limits of many species complexes are poorly defined. For ex-
ample, phylogeographic and species delimitation studies of the C. viridis
species complex have resulted in strikingly different conclusions, rang-
ing from the existence of one species with nine subspecies to seven
species with the possibility of an additional two unnamed taxa (see
Ashton and de Queiroz, 2001; Davis et al., 2016; Douglas and Schuett,
2002; Pook et al., 2000). Genomic data suggest that there are additional
unnamed lineages within this group that do not correspond to the tradi-
tional subspecific taxonomy (Holding et al., 2021). Additionally, within
C. scutulatus up to four lineages have been detected with genomic data
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(e.g., Myers et al. 2017; Schield et al., 2018), but how much of this pop-
ulation structure is discrete versus the result of isolation-by-distance is
not clear (e.g., Myers et al., 2019; Watson et al., 2019). Substantial phe-
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notypic variation within species (Campbell and Lamar, 2004; Klauber,
1956) coupled with widespread gene flow among lineages likely cause
disagreements in delimiting and diagnosing species (Schield et al.,
2019). Furthermore, interspecific gene flow occurs between C. viridis
and C. scutulatus, and these two species maintain a narrow hybrid zone
in southwestern New Mexico (Glenn and Straight, 1990; Murphy and
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Ben Crabtree, 1988; Zancolli et al., 2016). Estimates of phylogenetic re-
lationships among the members of the western rattlesnake species
group (here defined as being composed of the C. viridis species complex,
C. mitchellii species complex, C. scutulatus, and C. tigris) also differ
among studies depending on the molecular marker and method of
analysis used (Blair and Sánchez-Ramírez, 2016; Castoe and Parkinson,
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2006; Meik et al., 2015; Murphy et al., 2002; Reyes-Velasco et al.,
2013). The young age of this species group (∼4.8 mya; Reyes-Velasco et
al., 2013) coupled with widespread intraspecific gene flow, suggests
both ILS and introgression must be accounted for when inferring phylo-
genetic relationships.
Here, I demonstrate that all species within the western rattlesnake
group have a history of introgression with at least one other taxon and
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that the MSC and MSNC approaches infer different topologies. This sug-
gests a slow accumulation of reproductive isolating barriers in rat-
tlesnakes. To do this, I generated a reduced representation genomic
dataset for multiple individuals per species within the group. Then I es-
timated a species tree and assessed the fit of these data to the MSC
model using posterior predictive simulations. I also used both MSNC
and site-base summary statistics to assess introgression and reticula-
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tion, then dated the timing of divergence within this group to incorpo-
rate all observed introgression events.
2. Methods
2.1. Sampling and taxon selection
I acquired tissue samples from 58 individuals representing the diver-
sity of species within the western rattlesnake species group from my
own fieldwork, museum loans, and colleagues who did not collect
voucher specimens (Supplemental Table 1). Additionally, I sampled
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Molecular Phylogenetics and Evolution xxx (xxxx) 107313
two individual Crotalus atrox for use as an outgroup taxon (following
Reyes-Velasco et al., 2013). My ingroup sampling included two C. cer-
berus, one C. mitchellii, seven C. oreganus, five C. pyrrhus, 25C. scutulatus,
one C. stephensi, seven C. tigris, and 10 C. viridis (see Fig. 1 for sampling
of specimens across the distributions of these species). The species lim-
its within some of these groups are unclear; however, I selected these
taxa as they are representative of these species complexes and are
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therefore useful to study historical relationships and lineage diversifica-
tion above the species level (Fig. 1).
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2.2. Data generation and bioinformatics
DNA was extracted using a Qiagen DNeasy kit, following manufac-
turer protocols. I submitted DNA to the University of Wisconsin-
Madison Biotechnology Center for genotyping-by-sequencing (GBS)
services using a dual enzyme DNA digest protocol. DNA concentration
was verified using the Quant-iT™ PicoGreen® dsDNA kit (Life Tech-
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nologies, Grand Island, NY). Libraries were prepared following Elshire
et al. (2011) with minimal modification; in short, 150 ng of DNA was
digested using PstI and MspI restriction enzymes (New England Biolabs,
Ipswich, MA) after which barcoded adapters amenable to Illumina se-
quencing were added by ligation with T4 ligase (New England Biolabs,
Ipswich, MA). Ninety-six adapter-ligated samples were pooled and am-
plified to provide library quantities amenable to sequencing, and
adapter dimers were removed by SPRI bead purification. Quality and
quantity of the finished libraries were assessed using the Agilent Bioan-
alyzer High Sensitivity Chip (Agilent Technologies, Inc., Santa Clara,
CA) and Qubit dsDNA HS Assay Kit (Life Technologies, Grand Island,
NY), respectively. Libraries were standardized to 2 nM. Sequencing was
performed using single read, 100 bp sequencing and HiSeq SBS Kit v4
(Illumina) on a HiSeq2500 sequencer; samples were multiplexed to a
total of 144 samples per lane.
I used ipyrad (Eaton and Overcast, 2020) to demultiplex Illumina
reads, de novo assemble loci, and call SNPs. Within ipyrad I allowed for
4 low quality base calls within a locus, a minimum depth of 6 for statis-
tical and majority rule base calling, a maximum clustering depth of
10,000 within samples, a 0.85 clustering threshold, zero mismatched
barcodes, a 50 base-pair minimum length of reads after trimming, a
maximum of five N’s in consensus loci, a maximum of 10 SNPs per lo-
cus, and a minimum of 36 individuals per locus (loci must con-
tain > 60% of all sequenced individuals).
2.3. Species tree estimation and fit to the MSC model
I used the multispecies coalescent (MSC) species-tree approach
SNAPP v1.5 (Bryant et al., 2012) in BEAST v2.6 (Bouckaert et al., 2014)
to estimate relationships among the western rattlesnake species group.
A total of 14 samples were used in this analysis: 2 individuals per lin-
eage with the exception of C. mitchellii and C. stephensi for which only
one individual was available. These samples were selected to minimize
missing data; missing data on a per individual basis ranged from 1.1 to
15.9%. A VCF file with these 14 samples was thinned to contain 1 SNP
per GBS locus to reduce linkage using vcftools (Danecek et al., 2011),
this was then converted to a NEXUS file using vcf2phylip v2.0 (Ortiz,
2019) keeping SNPs only if they were present in >10 samples, finally
the R package phrynomics (https://github.com/bbanbury/phrynomics)
was used to format the file to SNAPP input specifications. This final bi-
allelic SNP alignment contained 14,500 SNPs. In SNAPP, the mutation
rates u and v were set to 1.0, the lambda prior was set to a gamma dis-
tribution with alpha = 2.0 and beta = 200.0, the ‘snapprior’ was set
to default values. SNAPP was run for 4,500,000 iterations, sampling the
MCMC chain every 4,500 iteration. The first 10% of samples were dis-
carded as burn-in and convergence was assessed by examining trace
plots in TRACER v1.6 (Rambaut et al., 2014) ensuring that all parame-
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Fig. 1. Geographic distributions of all species, sampled individuals, and photos of representative species. (A) Distribution and sampling of Crotalus stephensi (ma-
roon), C. pyrrhus (blue), and C. mitchellii (yellow); (B) Distribution and sampling of C. oreganus (navy blue), C. viridis (orange), and C. cerberus (lavender); (C) C.
mitchellii; (D) Distribution and sampling of C. scutulatus; (E) Distribution and sampling of C. tigris; (F) C. scutulatus. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
ter ESS >200. A maximum clade credibility tree was generated from
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the post burn-in samples.
I then used posterior predictive simulations to test if these data are a
good fit to the multispecies coalescent model implemented in SNAPP. I
used the R package P2C2M.SNAPP (Duckett et al., 2020) to conduct
these simulations. This method uses a posterior distribution of species
trees produced from SNAPP run with empirical data to simulate poste-
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rior predictive datasets and then uses summary statistics to compare the
empirical data to this posterior predictive distribution to identify poten-
tial violations of the MSC model. This method requires a mutation rate
for simulations for which I used 7.62 × 10-9 substitutions/site/genera-
tion which has previously been used as a genome wide rate in Crotalus
(Harrington et al., 2017). P2C2M.SNAPP was used to simulate 100 pos-
terior predictive datasets that contained the same number of samples
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and sites as my empirical data. These simulated datasets were then run
in SNAPP v1.5. To compare the posterior distribution from the empiri-
cal data to the predictive distribution all three summary statistics im-
plemented in P2C2M.SNAPP were used, these are the Robinson-Foulds
tree distance (RF), the standard deviation of maximum likelihoods of
posterior trees, and pairwise FST outliers.
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2.4. Introgression and reticulation
To differentiate between hybridization and incomplete lineage sort-
ing, I used the D statistic (also referred to as the ABBA/BABA test;
Green et al., 2010). This analysis uses a 4-taxon statement, an outgroup
and three ingroup species, and assesses the number of shared bi-allelic
sites (labeled as A or B alleles) among the ingroup species. Within the
ingroup, species 1 and species 2 are most closely related and species 3
has a derived allele (allele B). Under a scenario of hybridization or ILS
there are then two possible configurations in which the derived allele is
shared between species 1 or 2 and species 3, (((A, B), B), A) or (((B, A),
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B), A). If ILS alone is responsible for the distribution of these ‘B’ alleles
across species, then the total number of both of these configurations is
expected to be equal. Deviations from such an expectation can be inter-
preted as evidence of introgression between species 3 and species 1
(more BABA configurations) or species 2 (more ABBA configurations;
Durand et al., 2011). I conducted a total of 21 comparisons between
species based on the SNAPP topology to test for gene flow between all
possible pairs. These tests were conducted using the R package admixr
(Petr et al., 2019) which interfaces with the ADMIXTOOLS (Patterson et
al., 2012) command-line programs. This method uses only bi-allelic
SNPs and allows for multiple individuals per taxon, therefore all indi-
viduals were used in this analysis (with the exception of ROM38478
and CAS228045 that were removed because of high levels of missing
data). ADMIXTOOLS outputs a z-score, the number of standard errors
that the D statistic deviates from 0, and I used an absolute z-score
of > 3 as evidence of a significant D statistic (Durand et al., 2011).
To assess whether the geographic location of specimens used in the
calculation of the D-statistic can influence the outcome of this analysis I
also conducted every pairwise comparison between C. viridis and C. scu-
tulatus as well as between C. cerberus and C. tigris. These two taxon pairs
were chosen for this analysis because they showed significant gene flow
from the above D-statistic test and have multiple individuals sampled
across geographic space. For the C. viridis and C. scutulatus analysis this
involved 250 tests with a tree topology of (((C. oreganus, C. viridis speci-
men), C. scutulatus specimen), C. atrox). The C. cerberus and C. tigris
comparisons involved 14 tests with a topology of (((C. scutulatus, C. cer-
berus specimen), C. tigris specimen), C. atrox). Geographic distance be-
tween sampled individuals was calculated using the earth.dist function
in the R package fossil (Vavrek, 2011). Using a linear model, I tested for
an association between D-statistic values and straight-line geographic
distances between sampled individuals.
I also used the multi-species network coalescent (MSNC) approach
SNaQ to simultaneously infer the topology of species relationships and
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historical reticulation events. This method which is implemented in the
Julia package PhyloNetworks (Solís-Lemus et al., 2017) is a maximum
pseudolikelihood approach that uses a coalescent model extended to in-
clude reticulation events (Solís-Lemus and Ané, 2016). Concordance
factors (CF) of 4-taxon statements are used to estimate a semi-directed
species network with estimated reticulation events and γ-values (inheri-
tance probabilities of ancestral contributions to hybridization events). I
converted the unlinked SNP PHYLIP file from ipyrad to a CF table using
the R package SNPs2CF (Olave and Meyer, 2020) using all sampled in-
dividuals and calculated CFs between species only for a total of 35,176
quartets. Phylogenetic networks with the number of hybridization
events (hmax) ranging from 0 to 5 were then reconstructed with the
snaq! function using this CF table, running 10 independent analyses for
each hmax value using the SNAPP tree as the starting topology. The
best number of reticulation events was inferred as the point where
adding an additional migration event did not improve the likelihood
score. With the best hmax, I then used the pseudoreplication approach
in SNPs2CF to create a CF with credibility intervals that was used with
the bootsnaq() command of PhyloNetworks to conduct 100 bootstrap
replications to estimate support for phylogenetic relationships and mi-
gration edges.
2.5. Divergence dating
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To estimate divergence times I used the full likelihood approach im-
plemented in G-PhoCS (Gronau et al., 2011). G-PhoCS uses the multi-
species coalescent to estimate divergence times and effective popula-
tion sizes (Ne) from multi-locus sequence data and models gene flow be-
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tween populations given user-defined migration events (Gronau et al.,
2011). This method uses gamma distributions to specify prior distribu-
tions on theta (θ = 4Neμ, where μ is the mutation per nucleotide site
per generation), τ (species divergence time, TDIV = τ/μ), and msx (the
proportion of individuals in population x that arrive via migration from
population s per generation). I used a random subset of 2,500 GBS loci
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for this analysis and up to five individuals per taxon depending on sam-
pling within species. I ran G-PhoCS analyses under two different priors
(τ – θ [1, 300] and [1, 30]) that represent recent and deeper diver-
gences as well as smaller and larger Ne (Oswald et al., 2017). Each of
these prior settings were run twice to ensure consistency among runs.
Two different topologies were fixed in these analyses, one following the
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MCC tree from SNAPP and the other given the best topology from
SNaQ. Migration bands were set in the SNAPP topology run based on
results from the ABBA/BABA and P2C2M.SNAPP analyses, whereas mi-
gration in the SNaQ topology run was based on ABBA/BABA and migra-
tion inferred in SNaQ. Each analysis was run for 5,000,000 generations,
sampling every 500 iterations after a burn-in of 5,000 iterations. Con-
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Fig. 2. Topologies inferred under the MSC and MSNC models, inferred introgression, and estimated branch lengths. (A) SNAPP topology with migration, numbers
at nodes represent posterior probabilities; (B) SNaQ with migration, numbers at nodes represent bootstrap support values. Red arrows indicate significant D-
statistic instances of introgression, green arrows are inferred migration from P2C2M.SNAPP, and blue arrows are reticulations from SNaQ (blue numbers are values
of γ). Placement of arrows on trees do not represent when introgression occurred, nor do they necessarily represent that introgression was bidirectional. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Molecular Phylogenetics and Evolution xxx (xxxx) 107313
vergence was checked by examining trace plots from the MCMC runs in
Tracer v1.6 (Rambaut et al., 2014). To convert τ into divergence time in
years before present I used a previously estimated divergence time be-
tween C. atrox and the ingroup species (6.1 mya; Reyes-Velasco et al.,
2013) to fix the root time of divergence in these analyses. This allowed
me to scale estimated divergence times within the western rattlesnake
species group across all G-PhoCS analyses.
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3. Results
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3.1. GBS data
I sequenced 60 individual samples (including all western rattlesnake
in-group specimens, plus two C. atrox samples) for GBS resulting in 168
million reads, with 2.79 million reads per specimen on average (0.425 –
5.42 million reads/specimen; see Supplemental Table 1). After process-
ing these data with ipyrad the final dataset contained 24,302 loci with
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150,947 variable sites and 94,015 parsimony informative sites. Demul-
tiplexed fastq files for all samples have been accessioned on NCBI’s SRA
(SRA data PRJNA756778; specimen accession numbers SAM-
N20925586 – SAMN20925645).
3.2. Species tree and model fit
The SNAPP species tree analysis resulted in a well-resolved phy-
logeny of the C. viridis species group with posterior probabilities equal
to 1.0 at all nodes (Fig. 2). While the relationships in this phylogeny are
well-supported, posterior predictive simulations in P2C2M.SNAPP
demonstrate that the GBS data are not a good fit to the assumptions of
the MSC model used in SNAPP. The RF p-value was 0.005
(P2C2M.SNAPP uses a p-value > 0.05 as an indication of model viola-
tion for this metric; Duckett et al., 2020), however, both the MLSD (p-
value = 0) and the pairwise FST metrics suggest MSC model violations.
The pairwise FST metric demonstrates a poor fit of this model with C.
stephensi and C. pyrrhus as outliers, suggesting that there has been or
that there currently is gene flow between these two taxa. These MSC
model violations indicate that a strictly branching phylogeny may be a
poor representation of the evolutionary history of these species.
3.3. Introgression and reticulation
Eleven of the total 21 ABBA/BABA tests recovered significant gene
flow among taxa (Fig. 3; Supplemental Table 2). Several of these tests
were set up with combinations of species such that multiple tests sug-
gest gene flow between the same two pairs. For example, the tests (((C.
stephensi, C. pyrrhus), C. oreganus), C. atrox) and (((C. stephensi, C.
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Fig. 3. Results from D-statistic analysis between all species level comparisons. Dots are calculated D statistic values, bars are confidence intervals based on the
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standard errors. Those highlighted in red are statistically significant, values in black are not (significance values are based on z scores). Positive D statistics indi-
cate introgression between species 1 and species 3, while negative values indicate introgression between species 2 and species 3. (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the web version of this article.)

mitchellii), C. oreganus), C. atrox) both suggest gene flow between C. high bootstrap support values at most nodes. The topological differ-
stephensi and C. oreganus (Fig. 3; Supplemental Table 2). In total these ences are with the placement of C. tigris and the relationships among
ABBA/BABA tests suggest there have been at least seven introgression the C. mitchellii species complex (Fig. 2). The highest support for the
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events during the diversification of this group and all species are impli-
cated in past hybridization (Fig. 3), providing overwhelming support
for introgression either between extant species pairs or between ances-
tral lineages.
Testing for an association between the signal of gene flow from the
D-statistic test and geographic distance between samples demonstrates
that there is an association such that higher values of the D-statistic are
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number of reticulation events was for hmax = 2 with historical gene
flow between (1) C. pyrrhus and C. stephensi and (2) between C. tigris
and the ancestral lineage leading to (C. oreganus, C. cerberus). The pro-
portion of loci inferred to have followed these hybridization edges, γ,
was inferred to be 49% from C. stephensi into C. pyrrhus and 32.8% from
C. tigris into the (C. oreganus, C. cerberus) lineage. The ABBA/BABA tests
do not detect the SNaQ inferred C. pyrrhus and C. stephensi reticulation

calculated between samples within closer geographic proximity (Fig.
4). A linear model for the comparison between C. scutulatus and C.
viridis resulted in an adjusted r2 = 0.1 (p-value < 0.05), and an ad-
justed r2 = 0.57 between C. tigris and C. cerberus (p-value < 0.05). All
pair-wise D-statistic comparisons resulted in statistically significant ab-
solute z-scores. Together these results suggest that the strength of the
signal of gene flow detected by the D-statistic is influenced by the geo-
graphic sampling of specimens; however, in these cases gene flow is al-
ways detected regardless of pairwise comparison.
The network approach implemented in SNaQ recovers similar phy-
logenetic relationships as found using the species tree approach with
event (note, however, that these two taxa were treated as sister in all
ABBA/BABA analyses and therefore introgression could not be tested),
however both analyses do infer past hybridization between C. tigris and
the (C. oreganus, C. cerberus) lineage.
3.4. Divergence dating
The MCMC runs in G-PhoCS reached stationarity as assessed in
Tracer so I combined replicate runs of the same prior combinations for
divergence time estimation. These divergence times were robust to the
two sets of priors used and I report the results of the τ – θ [1, 300] runs.

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Fig. 4. Individual specimen based pairwise D statistic values compared to geographic distance between sampling locality. More negative D statistic values indicate a
greater proportion of sites that suggest introgression has occurred. The comparison between all C. scutulatus and C. viridis specimens are in grey and comparisons be-
tween C. tigris and C. cerberus are in black. Associations in both cases are positive indicating fewer sites implicated in introgression as geographic distances increase;
however, all comparisons are significant indicating that introgression has occurred.

Fixing the divergence time between C. atrox and the ingroup taxa at 4. Discussion
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6.1 mya resulted in a root divergence time of 5.7 mya (95% CI: 2.2 –
9.3 mya) and 5.5 mya (95% CI: 2.1 – 8.9 mya) for the SNAPP and SNaQ
topologies respectively (Fig. 2). Divergence time estimates between
these two topologies and parameterizations of gene flow are similar,
however the timing of divergence is much younger within the C.
mitchellii species complex in the SNaQ topology (Fig. 2; Supplemental
Table 3). Despite these differences, the estimated divergence times have
wide confidence intervals that largely overlap between the SNAPP and
SNaQ topologies (Supplemental Table 3).
All species within the western rattlesnake group have a signature of
past hybridization and introgression with at least one other taxon. This
has several implications for estimating phylogenies and understanding
diversification. First, the signature of past introgression is found across
the geographic distributions of species and therefore detecting hy-
bridization within other groups may be possible by only sampling a few
specimens per species. Second, the topological differences between the
MSC and MSNC approaches are driven by taxa also implicated in past
hybridization, suggesting that gene flow will obscure phylogenetic rela-
tionships when not accounted for. Furthermore, mechanisms that cause
reproductive isolation are seemingly slow to evolve and rampant intro-

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gression likely causes difficulty in delimiting species in this group. Fu-
ture studies will need to incorporate reticulating phylogenies to better
understand trait evolution and biogeographic history within rat-
tlesnakes.
4.1. Biogeography of introgression
The phylogenetic relationships I estimated using genomic data and
the MSC model are similar to the relationships presented in several re-
cent studies that use a few Sanger generated loci (mtDNA and single
copy nuclear DNA genes; Alencar et al., 2016; Blair and Sánchez-
Ramírez, 2016; Reyes-Velasco et al., 2013). However, all species within
this radiation are implicated in at least one introgression event. Most of
the identified introgression events are between lineages that are in
close geographic proximity. This suggests that the geographic distribu-
tions of many of these rattlesnake taxa may have remained relatively
stable through time irrespective of the glacial cycles of the Pleistocene
that impacted the historical demographic dynamics of many North
American species (e.g., Comes and Kadereit, 1998; Hewitt, 2004).
There are, however, a few exceptions to this within the western rat-
tlesnake group, the most striking example being the detection of gene
flow between allopatric C. oreganus and C. tigris, two species separated
by > 185 km (based on the two closest specimens listed on VertNet).
Therefore, it is possible that the geographic distributions of these
D
species have shifted through time and may have repeatedly been in con-
tact with subsequent isolation. This scenario is possible given paleo-
projections of ecological niche models for several of these taxa that sug-
gest much smaller regions of suitable climate during the Last Glacial
TE
Maximum when compared to current geographic distributions (Schield
et al., 2019). Similarly, a recent study on montane rattlesnakes suggests
that past hybridization events likely only occurred during the Pleis-
tocene when the geographic distributions of these species were more
widespread and allowed for contact (Blair et al., 2019).
By examining the signal of past introgression using individual based
EC
pairwise estimates of the D-statistic I have demonstrated that the
strength of the signal of introgression declines with geographic dis-
tance, however, this signal is always detected. Therefore, regardless of
the geographic origin of specimens, a signal of past introgression can be
detected in the data and it may be sufficient to infer hybridization and
introgression between taxa by sampling genomic data from only a small
RR
number of individuals across a species’ range. This is further demon-
strated here where sampling was unbalanced among taxa, with only a
single specimen representing C. stephensi and two individuals sampled
from C. cerberus whereas 25C. scutulatus specimens were sequenced;
however both of these taxa represented by 1–2 specimens were found to
have a signature of interspecific introgression.
This pattern of detecting introgression regardless of geographic lo-
CO
cation of sampled specimens is in contrast to some previous findings.
For example, within live oaks the sampling location of specimens did
influence the ability to detect admixture between lineages, where re-
sults of significant admixture are restricted to samples that occur in
close geographic proximity of the hybridizing lineages (Eaton et al.,
2015). Furthermore, within the western rattlesnakes, introgression has
been suggested between C. oreganus and C. scutulatus based on a data
UN
from venom genes (Dowell et al., 2018), this potential introgression
event was not detected in the data presented here. Further sampling of
individuals across potential zones of sympatry or within communities
where multiple members of the western rattlesnake group co-occur is
necessary to address whether there are additional introgression events
not detected here or if signatures of admixture are intensified in these
geographic regions (Zancolli et al., 2016).
7
Molecular Phylogenetics and Evolution xxx (xxxx) 107313
4.2. Influence of gene flow on phylogenies
The differences in topology between the MSC tree and the MSNC
tree are between two taxa that have an inferred history of introgression
based on the SNaQ analysis (Fig. 2). For example, these analyses place
C. tigris in two different locations in the phylogeny with strong support
(PP = 1.0, BS = 97). However, this discordance likely reflects a his-
F
tory of introgression between this taxon and the C. viridis species com-
plex that is not accounted for in a strictly diverging phylogeny. Addi-
OO
tionally, the relationships recovered here within the C. mitchellii com-
plex differ from those inferred in a recent study of species limits within
this group (Meik et al., 2015). These authors generated a ddRAD
dataset, used similar MSC methods to those implemented here (e.g.,
SNAPP), and therefore should be comparable to the present study. This
observed discordance is likely the result of introgression among lin-
eages, as both the current study and Meik et al. (2015) demonstrate that
gene flow is an important process within this group. Because the MSC
PR
model is a poor fit to these genomic data, the relationships recovered
assuming no gene flow within SNAPP may be biased, necessitating the
use of these network approaches. However, previous studies in other
groups of organisms using both MSC and MSNC approaches have found
remarkably similar topologies between these models (e.g., Blair et al.,
2019). This may reflect the intensity of introgression between taxa,
where higher amounts of gene flow will lead to more discordant topolo-
gies inferred between MSC and MSNC methods. Yet we still do not un-
derstand how widespread divergence with gene flow is across many
taxonomic groups and how often these events result in reticulate phylo-
genies that necessitate the use of MSNC approaches. For example, gene
flow that occurred early in the radiation of an ancient group may not be
detectable and therefore is not necessary to model when reconstructing
evolutionary histories. This demonstrates the importance of using ap-
proaches that test the fit of a particular model to phylogenomic data
(Cai and Ané, 2020; Duckett et al., 2020).
Hybridization and introgression have likely resulted in poorly de-
fined species boundaries within this group of rattlesnakes. Gene flow
was detected among taxa within the youngest species complexes, for ex-
ample between C. pyhrrus and C. stephensi and between C. viridis and C.
oreganus. This demonstrates that there are porous genomic boundaries
between these species (e.g., Harrison and Larson, 2014), which is ex-
pected given that many of these taxa hybridize during the early stages
of divergence, suggesting that speciation with gene flow is common in
this group (e.g., Meik et al., 2015; Schield et al., 2019). Introgression re-
sulting in poorly defined species is best exemplified within the C. viridis
species complex where authors using similar data suggest that taxo-
nomic diversity ranges between one and nine species (Ashton and de
Queiroz, 2001; Douglas and Schuett, 2002; Pook et al., 2000). Addi-
tional phylogeographic data for these groups may resolve species limits,
however, it is likely that these taxa will be found to maintain gene flow
across contact zones. This is a common finding in many species delimi-
tation studies (Burbrink and Guiher, 2015; Melville et al., 2019;
Oliveira et al., 2015) and the width and nature of these contact zones
can provide important insights into the nature of species divergence
and maintenance of species boundaries (Burbrink et al., 2020). Strict
reproductive isolation is likely not necessary for the maintenance of
species boundaries; however, it is likely that genomic islands of specia-
tion underlying traits responsible for local adaptation are keeping these
nascent lineages distinct (Nosil and Feder, 2012).
Although there is rampant gene flow in the nuclear genome of these
taxa, previous studies suggest that there is little introgression of the
mtDNA genome among these species. For example, mtDNA introgres-
sion is only known to occur between C. oreganus and C. viridis in a nar-
row region of contact and seemingly only from C. viridis into C. oreganus
(Schield et al., 2019). Furthermore, within the C. mitchellii complex all
taxa are monophyletic in an mtDNA gene tree with no detected mtDNA
introgression (Meik et al., 2015).
E.A. Myers
4.3. Species diversification despite hybridization
There are multiple hypotheses to explain species richness within pit
vipers. Two that receive the most attention are the evolution of the lo-
real pit as a key innovation (Goris, 2011; Roelke and Childress, 2007)
and the invasion of the Western Hemisphere triggering rapid diversifi-
cation (Wüster et al., 2008). However, these hypotheses alone cannot
account for increased rates of speciation within this group (Alencar et
al., 2016). Recent work has suggested that a lack of reproductive isola-
tion and continued hybridization are important processes in promoting
diversification via a combinatorial mechanism within rapid radiations
(Marques et al., 2019). For example, a recent study has suggested that
salamander lineages with increased rates of diversification naturally
hybridize (Patton et al., 2020). Furthermore, gene flow among species
is a protracted process that can occur for millions of years post-
divergence (Barth et al., 2020; Pulido-Santacruz et al., 2020; Esquerré
et al. 2021) and rates of reproductive isolation and speciation are de-
coupled (Rabosky and Matute, 2013). This decoupling may be ex-
plained by the protracted period of gene flow between lineages and
would suggest that the accumulation of species over evolutionary
timescales is limited by processes other than strict reproductive isola-
tion.
A similar process may be occurring within the radiation of viperids,
where groups that have experienced increased diversification rates are
D
also those that have a signature of historical introgression and contem-
porary gene flow. Given this common theme of divergence with gene
flow, we might expect that there is a signature of introgression among
the earliest diverging branches as well as among older extant lineages.
TE
Within the western rattlesnake radiation, introgression occurs among
the most divergent lineages as there is evidence of gene flow between C.
stephensi and the C. oreganus/C. cerberus pair, even though these taxa
share a common ancestor >5.74 mya. From the estimated age of diver-
gence of C. stephensi this suggests that this introgression occurred
within the last ∼1.27 mya. It is possible that within this clade of rat-
EC
tlesnakes there is little association between strong hybrid incompatibil-
ities and macroevolutionary speciation rates. Incorporating historical
signals of introgression and gene flow into macroevolutionary studies
of diversification rates may illuminate whether lineages lacking strong
reproductive isolation barriers have higher rates of species diversifica-
tion.
RR
4.4. Trait evolution
Incorporating historical signals of gene flow in macroevolutionary
studies can also allow us to study how introgression influences trait
evolution. The western rattlesnake group exhibits extensive intra- and
interspecific venom variation, but how this variation has evolved is
CO
largely unknown. The phospholipase A2 encoded neurotoxin, often re-
ferred to as Mojave toxin (after the Mojave rattlesnake, Crotalus scutula-
tus) is present in many populations but is missing in others. Within C.
oreganus, populations in east-central Utah state and southern California
possess this neurotoxic venom (French et al., 2004; Glenn and Straight,
1977) where this species hybridizes with C. scutulatus (Dowell et al.,
2018). Similarly, C. viridis populations in southwest New Mexico and
UN
northern Arizona possess neurotoxins potentially because of hybridiza-
tion with C. scutulatus (Glenn and Straight, 1990; Werman, 2008), how-
ever this neurotoxin is only found in C. viridis populations within a nar-
row hybrid zone (Zancolli et al., 2016). Researchers have also hypothe-
sized that C. tigris obtained Mojave toxin genes from past introgression
with C. scutulatus (Werman, 2008). These hypotheses are feasible given
the high levels of hybridization and introgression within this group and
that populations with similar venom phenotypes are often sympatri-
cally distributed. Implementing recent developments in methods for an-
cestral state and biogeographic reconstruction that account for reticula-
tion (e.g., Karimi et al., 2020) is imperative for understanding trait evo-
8
Molecular Phylogenetics and Evolution xxx (xxxx) 107313
lution and the importance of introgression acting as a driver of pheno-
typic evolution within rattlesnakes.
5. Conclusions
Phylogenetic reticulation and introgression are a common compo-
nent of diversification within the western rattlesnake species group.
F
This suggests the accumulation of reproductive isolating barriers in rat-
tlesnakes are slow to accumulate. Topological differences in estimated
OO
phylogenies are the result of historical introgression and reticulation, a
pattern that is likely common in other groups. Furthermore, introgres-
sion may influence trait evolution and could help drive species diversi-
fication in this group, future studies should explore these mechanisms
in more detail.
Declaration of Competing Interest
PR
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
EAM was funded by the Gerstner Scholar/Theodore Roosevelt (at
AMNH) and Peter Buck/Walter Rathbone Bacon (at NMNH) fellow-
ships. I would like to thank the entire AMNH Herpetology department
for support and additional funding while conducting this research, and
C. Raxworthy for sponsoring my postdoctoral fellowship at the AMNH.
Fieldwork was conducted under AZ permit number SP760664 and a
Navajo Nation Department of Fish and Wildlife permit number 1043.
Portions of the computational analyses were conducted on the Smith-
sonian Institution High Performance Cluster (SI/HPC; https://doi.org/
10.25572/SIHPC). J. Servis provided exceptional editorial feedback
and suggestions. Thanks to M. Petr and M. Kweskin for assistance with
admixr, and to R. Bell who provided helpful suggestions on early drafts
of this manuscript. Tissue samples were provided by B. Murphy at the
Royal Ontario Museum, L. Scheinberg at the California Academy of Sci-
ences, D. DeNardo, J. Badman, M. Goode, P. Rosen, and T. Reeder. B.
Reid was a huge help in the field and C. J. Cole and C. R. Townsend
truly facilitated fieldwork in Arizona.
Funding
EAM was funded by the Gerstner Scholar/Theodore Roosevelt (at
AMNH) and Peter Buck/Walter Rathbone Bacon (at NMNH) fellow-
ships.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ympev.2021.107313.
References
Alencar, L.R.V., Quental, T.B., Grazziotin, F.G., Alfaro, M.L., Martins, M., Venzon, M.,
Zaher, H., 2016. Diversification in vipers: phylogenetic relationships, time of divergence
and shifts in speciation rates. Mol. Phylogenet. Evol. 105, 50–62.
Ashton, K.G., de Queiroz, A., 2001. Molecular systematics of the western rattlesnake,
Crotalus viridis (Viperidae), with comments on the utility of the D-loop in phylogenetic
studies of snakes. Mol. Phylogenet. Evol. 21, 176–189.
Barth, J.M.I., Gubili, C., Matschiner, M., Tørresen, O.K., Watanabe, S., Egger, B., Han, Y.-
S., Feunteun, E., Sommaruga, R., Jehle, R., 2020. Stable species boundaries despite ten
million years of hybridization in tropical eels. Nat. Commun. 11, 1–13.
Blair, C., Bryson, Jr., R.W., Linkem, C.W., Lazcano, D., Klicka, J., McCormack, J.E., 2019.
Cryptic diversity in the Mexican highlands: thousands of UCE loci help illuminate
phylogenetic relationships, species limits and divergence times of montane rattlesnakes
(Viperidae: Crotalus). Mol. Ecol. Resour. 19, 349–365.
Blair, C., Sánchez-Ramírez, S., 2016. Diversity-dependent cladogenesis throughout
western Mexico: evolutionary biogeography of rattlesnakes (Viperidae: Crotalinae:
E.A. Myers
Crotalus and Sistrurus). Mol. Phylogenet. Evol. 97, 145–154.
Bouckaert, R., Heled, J., Kühnert, D., Vaughan, T., Wu, C.H., Xie, D., Suchard, M.A.,
Rambaut, A., Drummond, A.J., 2014. BEAST 2: A software platform for bayesian
evolutionary analysis. PLoS Comput. Biol. 10. https://doi.org/10.1371/
journal.pcbi.1003537.
Bryant, D., Bouckaert, R., Felsenstein, J., Rosenberg, N.A., RoyChoudhury, A., 2012.
Inferring species trees directly from biallelic genetic markers: bypassing gene trees in a
full coalescent analysis. Mol. Biol. Evol.. https://doi.org/10.1093/molbev/mss086.
Burbrink, F., Gehara, M., McKelvy, A.D., Myers, E.A., 2020. Resolving spatial complexities
of hybridization in the context of the gray zone of speciation in North American
ratsnakes (Pantherophis obsoletus complex). Evolution 75, 260–277.
Burbrink, F.T., Guiher, T.J., 2015. Considering gene flow when using coalescent methods
to delimit lineages of North American pitvipers of the genus Agkistrodon. Zool. J. Linn.
Soc. 173, 505–526. https://doi.org/10.1111/zoj.12211.
Cai, R., Ané, C., 2020. Assessing the fit of the multi-species network coalescent to multi-
locus data. Bioinformatics.
Campbell, J.A., Lamar, W.W., 2004. The Venomous Reptiles of the Western Hemisphere.
Cornell University Press, Ithaca, NY.
Castoe, T.A., Parkinson, C.L., 2006. Bayesian mixed models and the phylogeny of pitvipers
(Viperidae: Serpentes). Mol. Phylogenet. Evol. 39, 91–110.
Comes, H.P., Kadereit, J.W., 1998. The effect of Quaternary climatic changes on plant
distribution and evolution. Trends Plant Sci. 3, 432–438.
Danecek, P., Auton, A., Abecasis, G., Albers, C.A., Banks, E., DePristo, M.A., Handsaker,
R.E., Lunter, G., Marth, G.T., Sherry, S.T., McVean, G., Durbin, R., 2011. The variant call
format and VCFtools. Bioinformatics 27, 2156–2158. https://doi.org/10.1093/
bioinformatics/btr330.
Davis, M.A., Douglas, M.R., Collyer, M.L., Douglas, M.E., 2016. Deconstructing a species-
complex: geometric morphometric and molecular analyses define species in the Western
Rattlesnake (Crotalus viridis). PLoS One 11, e0146166.
Douglas, M.E., Schuett, G.W., 2002. Phylogeography of the western rattlesnake (Crotalus
viridis) complex, with emphasis on the Colorado Plateau. Eagle Mountain Pub..
Dowell, N.L., Giorgianni, M.W., Griffin, S., Kassner, V.A., Selegue, J.E., Sanchez, E.E.,
D
Carroll, S.B., 2018. Extremely divergent haplotypes in two toxin gene complexes encode
alternative venom types within rattlesnake species. Curr. Biol. 28, 1016–1026.
Duckett, D.J., Pelletier, T.A., Carstens, B.C., 2020. Identifying model violations under the
multispecies coalescent model using P2C2M. SNAPP. PeerJ 8, e8271.
Durand, E.Y., Patterson, N., Reich, D., Slatkin, M., 2011. Testing for ancient admixture
TE
between closely related populations. Mol. Biol. Evol. 28, 2239–2252.
Eaton, D.A.R., Hipp, A.L., González-Rodríguez, A., Cavender-Bares, J., 2015. Historical
introgression among the American live oaks and the comparative nature of tests for
introgression. Evolution 69, 2587–2601.
Eaton, D.A.R., Overcast, I., 2020. ipyrad: Interactive assembly and analysis of RADseq
datasets. Bioinformatics 36, 2592–2594.
Edwards, S., 2009. Is a new and general theory of molecular systematics emerging?.
EC
Evolution 63, 1–19.
Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler, E.S., Mitchell,
S.E., 2011. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity
species. PLoS One 6. https://doi.org/10.1371/journal.pone.0019379.
Esquerré, D., Keogh, J.S., Demangel, D., Morando, M., Avila, L.J., Sites, J.W., Leaché,
A.D., 2021. Rapid radiation and rampant reticulation: Phylogenomics of South American
Liolaemus lizards. Syst. Biol..
Ferreira, M.S., Jones, M.R., Callahan, C.M., Farelo, L., Tolesa, Z., Suchentrunk, F., Boursot,
P., Mills, L.S., Alves, P.C., Good, J.M., Melo-Ferreira, J., 2020. The Legacy of Recurrent
RR
Introgression during the Radiation of Hares. Syst. Biol.. https://doi.org/10.1093/sysbio/
syaa088.
French, W.J., Hayes, W.K., Bush, S.P., Cardwell, M.D., Bader, J.O., Rael, E.D., 2004.
Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and
geographic characterization. Toxicon 44, 781–791.
Glenn, J.L., Straight, R., 1977. The midget faded rattlesnake (Crotalus viridis concolor)
venom: lethal toxicity and individual variability. Toxicon 15, 129–132.
Glenn, J.L., Straight, R.C., 1990. Venom characteristics as an indicator of hybridization
CO
between Crotalus viridis viridis and Crotalus scutulatus scutulatus in New Mexico. Toxicon
28, 857–862.
Goris, R.C., 2011. Infrared organs of snakes: an integral part of vision. J. Herpetol. 45,
2–14.
Green, R.E., Krause, J., Briggs, A.W., Maricic, T., Stenzel, U., Kircher, M., Patterson, N., Li,
H., Zhai, W., Fritz, M.-H.-Y., 2010. A draft sequence of the Neandertal genome. Science
328, 710–722.
Gronau, I., Hubisz, M.J., Gulko, B., Danko, C.G., Siepel, A., 2011. Bayesian inference of
ancient human demography from individual genome sequences. Nat. Genet. 43,
UN
1031–1035. https://doi.org/10.1038/ng.937.
Harrington, S.M., Hollingsworth, B.D., Higham, T.E., Reeder, T.W., 2017. Pleistocene
climatic fluctuations drive isolation and secondary contact in the red diamond
rattlesnake (Crotalus ruber) in Baja California. J. Biogeogr.. https://doi.org/10.1111/
jbi.13114.
Harrison, R.G., Larson, E.L., 2014. Hybridization, introgression, and the nature of species
boundaries. J. Hered. 105, 795–809.
Hejase, H.A., VandePol, N., Bonito, G.M., Liu, K.J., 2018. Fastnet: fast and accurate
statistical inference of phylogenetic networks using large-scale genomic sequence data.
In: RECOMB International Conference on Comparative Genomics. Springer, pp.
242–259.
Hewitt, G.M., 2004. Genetic consequences of climatic oscillations in the Quaternary.
Philos. Trans. R. Soc. Lond. B. Biol. Sci. 359, 183–195. https://doi.org/10.1098/
rstb.2003.1388.
Holding, M.L., Sovic, M.G., Colston, T.J., Gibbs, H.L., 2021. The scales of coevolution:
comparative phylogeography and genetic demography of a locally adapted venomous
9
Molecular Phylogenetics and Evolution xxx (xxxx) 107313
predator and its prey. Biol. J. Linn. Soc. 132, 297–317.
Jónsson, H., Schubert, M., Seguin-Orlando, A., Ginolhac, A., Petersen, L., Fumagalli, M.,
Albrechtsen, A., Petersen, B., Korneliussen, T.S., Vilstrup, J.T., Lear, T., Myka, J.L.,
Lundquist, J., Miller, D.C., Alfarhan, A.H., Alquraishi, S.A., Al-Rasheid, K.A.S.,
Stagegaard, J., Strauss, G., Bertelsen, M.F., Sicheritz-Ponten, T., Antczak, D.F., Bailey, E.,
Nielsen, R., Willerslev, E., Orlando, L., 2014. Speciation with gene flow in equids despite
extensive chromosomal plasticity. Proc. Natl. Acad. Sci. U. S. A. 111, 18655–18660.
https://doi.org/10.1073/pnas.1412627111.
Karimi, N., Grover, C.E., Gallagher, J.P., Wendel, J.F., Ané, C., Baum, D.A., 2020.
F
Reticulate evolution helps explain apparent homoplasy in floral biology and pollination
in baobabs (Adansonia; Bombacoideae; Malvaceae). Syst. Biol. 69, 462–478.
Kautt, A.F., Kratochwil, C.F., Nater, A., Machado-Schiaffino, G., Olave, M., Henning, F.,
OO
Torres-Dowdall, J., Härer, A., Hulsey, C.D., Franchini, P., 2020. Contrasting signatures of
genomic divergence during sympatric speciation. Nature 588, 106–111.
Klauber, L.M., 1956. Rattlesnakes. Their Habits, Life Histories, and Influence on Mankind.
University of California Press, Los Angeles and Berkeley.
Malinsky, M., Svardal, H., Tyers, A.M., Miska, E.A., Genner, M.J., Turner, G.F., Durbin, R.,
2018. Whole-genome sequences of Malawi cichlids reveal multiple radiations
interconnected by gene flow. Nat. Ecol. Evol. 2, 1940–1955.
Mallet, J., Besansky, N., Hahn, M.W., 2016. How reticulated are species?. BioEssays 38,
140–149.
Marques, D.A., Meier, J.I., Seehausen, O., 2019. A combinatorial view on speciation and
PR
adaptive radiation. Trends Ecol. Evol. 34, 531–544.
Martin, S.H., Davey, J.W., Jiggins, C.D., 2015. Evaluating the use of ABBA–BABA statistics
to locate introgressed loci. Mol. Biol. Evol. 32, 244–257.
Mayr, E., 1963. Animal Species and Evolution. Belknap Press of Harvard University Press,
Cambridge, MA.
Meik, J.M., Streicher, J.W., Lawing, A.M., Flores-Villela, O., Fujita, M.K., 2015.
Limitations of climatic data for inferring species boundaries: insights from speckled
rattlesnakes. PLoS One 10, e0131435.
Melville, J., Chaplin, K., Hipsley, C.A., Sarre, S.D., Sumner, J., Hutchinson, M., 2019.
Integrating phylogeography and high-resolution X-ray CT reveals five new cryptic
species and multiple hybrid zones among Australian earless dragons. R. Soc. open Sci. 6,
191166.
Murphy, R.W., Ben Crabtree, C., 1988. Genetic identification of a natural hybrid
rattlesnake: Crotalus scutulatus scutulatus × C. viridis viridis. Herpetologica 44, 119–123.
Murphy, R.W., Fu, J., Lathrop, A., Feltham, A., Kovac, V., 2002. Phylogeny of the
rattlesnakes (Crotalus and Sistrurus) inferred from sequences of five mitochondiral
genes, in: Schuett, G.W., Hoggren, M., Douglas, M.E., Greene, H.W. (Eds.), Biology of the
Vipers. Eagle Mountain Pub., pp. 69–92.
Myers, E.A., Hickerson, M.J., Burbrink, F.T., 2017. Asynchronous diversification of snakes
in the North American warm deserts. J. Biogeogr. 44, 461–474.
Myers, E.A., Xue, A.T., Gehara, M., Cox, C.L., Davis Rabosky, A.R., Lemos-Espinal, J.,
Martínez-Gómez, J.E., Burbrink, F.T., 2019. Environmental heterogeneity and not
vicariant biogeographic barriers generate community-wide population structure in
desert-adapted snakes. Mol. Ecol. 28, 4535–4548.
Nosil, P., 2008. Speciation with gene flow could be common. Mol. Ecol. 17, 2103–2106.
Nosil, P., Feder, J.L., 2012. Genomic divergence during speciation: causes and
consequences. Phil. Transc. Royal Soc. B. 367, 332–342.
Olave, M., Meyer, A., 2020. Implementing large genomic single nucleotide polymorphism
data sets in phylogenetic network reconstructions: a case study of particularly rapid
radiations of cichlid fish. Syst. Biol. 69, 848–862.
Oliveira, E.F., Gehara, M., São-Pedro, V.A., Chen, X., Myers, E.A., Burbrink, F.T.,
Mesquita, D.O., Garda, A.A., Colli, G.R., Rodrigues, M.T., 2015. Speciation with gene
flow in whiptail lizards from a Neotropical xeric biome. Mol. Ecol. 24, 5957–5975.
Ortiz, E.M., 2019. vcf2phylip v2. 0: convert a VCF matrix into several matrix formats for
phylogenetic analysis. URL https://doi.org/10.5281/zenodo 2540861.
Oswald, J.A., Overcast, I., Mauck, W.M., Andersen, M.J., Smith, B.T., 2017. Isolation with
asymmetric gene flow during the nonsynchronous divergence of dry forest birds. Mol.
Ecol.. https://doi.org/10.1111/mec.14013.
Patterson, N., Moorjani, P., Luo, Y., Mallick, S., Rohland, N., Zhan, Y., Genschoreck, T.,
Webster, T., Reich, D., 2012. Ancient admixture in human history. Genetics 192,
1065–1093.
Patton, A.H., Margres, M.J., Epstein, B., Eastman, J., Harmon, L.J., Storfer, A., 2020.
Hybridizing salamanders experience accelerated diversification. Sci. Rep. 10, 1–12.
Petr, M., Vernot, B., Kelso, J., 2019. admixr—R package for reproducible analyses using
ADMIXTOOLS. Bioinformatics 35, 3194–3195.
Pook, C.E., Wüster, W., Thorpe, R.S., 2000. Historical biogeography of the western
rattlesnake (Serpentes: Viperidae: Crotalus viridis), inferred from mitochondrial DNA
sequence information. Mol. Phylogenet. Evol. 15, 269–282.
Pulido-Santacruz, P., Aleixo, A., Weir, J.T., 2020. Genomic data reveal a protracted
window of introgression during the diversification of a Neotropical woodcreeper
radiation. Evolution 74, 842–858.
Rabosky, D.L., Matute, D.R., 2013. Macroevolutionary speciation rates are decoupled
from the evolution of intrinsic reproductive isolation in Drosophila and birds. Proc. Natl.
Acad. Sci. 110, 15354–15359.
Rambaut, A., Suchard, M.A., Xie, D., Drummond, A.J., 2014. Tracer v1. 6. Comput. Progr.
Doc. Distrib. by author, website http://beast.bio.ed.ac.uk/Tracer.
Reyes-Velasco, J., Meik, J.M., Smith, E.N., Castoe, T.A., 2013. Phylogenetic relationships
of the enigmatic longtailed rattlesnakes (Crotalus ericsmithi, C. lannomi, and C. stejnegeri).
Mol. Phylogenet. Evol. 69, 524–534.
Roelke, C.E., Childress, M.J., 2007. Defensive and infrared reception responses of true
vipers, pitvipers, Azemiops and colubrids. J. Zool. 273, 421–425.
Roux, C., Fraïsse, C., Romiguier, J., Anciaux, Y., Galtier, N., Bierne, N., 2016. Shedding
light on the grey zone of speciation along a continuum of genomic divergence. PLoS Biol.
14, e2000234. https://doi.org/10.1371/journal.pbio.2000234.
E.A. Myers
Schield, D.R., Adams, R.H., Card, D.C., Corbin, A.B., Jezkova, T., Hales, N.R., Meik, J.M.,
Perry, B.W., Spencer, C.L., Smith, L.L., 2018. Cryptic genetic diversity, population
structure, and gene flow in the Mojave Rattlesnake (Crotalus scutulatus). Mol.
Phylogenet. Evol..
Schield, D.R., Perry, B.W., Adams, R.H., Card, D.C., Jezkova, T., Pasquesi, G.I.M.,
Nikolakis, Z.L., Row, K., Meik, J.M., Smith, C.F., 2019. Allopatric divergence and
secondary contact with gene flow: a recurring theme in rattlesnake speciation. Biol. J.
Linn. Soc. 128, 149–169.
Singhal, S., Moritz, C., 2013. Reproductive isolation between phylogeographic lineages
scales with divergence. Proc. R. Soc. B Biol. Sci. 280. https://doi.org/10.1098/
rspb.2013.2246.
Solís-Lemus, C., Ané, C., 2016. Inferring phylogenetic networks with maximum
pseudolikelihood under incomplete lineage sorting. PLoS Genet. 12. https://doi.org/
10.1371/journal.pgen.1005896.
Solís-Lemus, C., Bastide, P., Ané, C., 2017. PhyloNetworks: a package for phylogenetic
networks. Mol. Biol. Evol. 34, 3292–3298.
Vavrek, M.J., 2011. Fossil: palaeoecological and palaeogeographical analysis tools.
Palaeontol. Electron. 14, 1T.
Watson, J.A., Spencer, C.L., Schield, D.R., Butler, B.O., Smith, L.L., Flores-Villela, O.,
Campbell, J.A., Mackessy, S.P., Castoe, T.A., Meik, J.M., 2019. Geographic variation in
morphology in the Mohave Rattlesnake (Crotalus scutulatus Kennicott 1861)(Serpentes:
D
TE
EC
RR
CO
UN
10
Molecular Phylogenetics and Evolution xxx (xxxx) 107313
Viperidae): implications for species boundaries. Zootaxa 4683, 129–143.
Wen, D., Yu, Y., Zhu, J., Nakhleh, L., 2018. Inferring phylogenetic networks using
PhyloNet. Syst. Biol. 67, 735–740.
Werman, S.D., 2008. Phylogeny and evolution of B-neurotoxic phospholipases A2 (PLA2)
in the venomos of rattlesnakes, Crotalus and Sistrurus (Serpentes: Viperidae), in: Hayes,
W.K., Beaumont, M.A., Cardwell, M.D., Bush, S.P. (Eds.), The Biology of Rattlesnakes.
Loma Linda University Press, Loma Linda, cA, pp. 511–536.
Wüster, W., Peppin, L., Pook, C.E., Walker, D.E., 2008. A nesting of vipers: phylogeny and
historical biogeography of the Viperidae (Squamata: Serpentes). Mol. Phylogenet. Evol.
F
49, 445–459.
Yu, Y., Nakhleh, L., 2015. A maximum pseudo-likelihood approach for phylogenetic
networks. BMC Genomics 16, S10.
OO
Zancolli, G., Baker, T.G., Barlow, A., Bradley, R.K., Calvete, J.J., Carter, K.C., De Jager, K.,
Owens, J.B., Price, J.F., Sanz, L., 2016. Is hybridization a source of adaptive venom
variation in rattlesnakes? A test, using a Crotalus scutulatus × viridis hybrid zone in
southwestern New Mexico. Toxins (Basel) 8, 188.
Zhang, C., Ogilvie, H.A., Drummond, A.J., Stadler, T., 2018. Bayesian inference of species
networks from multilocus sequence data. Mol. Biol. Evol. 35, 504–517.
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