2011 Bryson Et Al C Triseriatus Group

ORIGINALARTICLE

Evolutionary drivers ofphylogeographical diversity in thehighlands of Mexico: a case study of theCrotalus triseriatus species group ofmontane rattlesnakes

Robert W. Bryson Jr1*, Robert W. Murphy2,3, Amy Lathrop2 and

David Lazcano-Villareal4

1School of Life Sciences, University of Nevada,

Las Vegas, Las Vegas, NV, USA, 2Centre for

Biodiversity and Conservation Biology, Royal

Ontario Museum, Toronto, ON, Canada,3State Key Laboratory of Genetic Resources and

Evolution, Kunming Institute of Zoology,

The Chinese Academy of Sciences, Kunming,

China, 4Laboratorio de Herpetologıa,

Universidad Autonoma de Nuevo Leon, San

Nicolas de los Garza, Nuevo Leon, Mexico

*Correspondence: Robert W. Bryson Jr, School

of Life Sciences, University of Nevada, Las

Vegas, 4505 Maryland Parkway, Las Vegas, NV

89154-4004, USA.

E-mail: [email protected]

ABSTRACT

Aim To assess the genealogical relationships of widespread montane rattlesnakes

in the Crotalus triseriatus species group and to clarify the role of Late Neogene

mountain building and Pleistocene pine–oak forest fragmentation in driving the

diversification of Mexican highland taxa.

Location Highlands of mainland Mexico and the south-western United States

(Texas, New Mexico, and Arizona).

Methods A synthesis of inferences was used to address several associated

questions about the biogeography of the Mexican highlands and the

evolutionary drivers of phylogeographical diversity in co-distributed taxa. We

combined extensive range-wide sampling (130 individuals representing five

putative species) and mixed-model phylogenetic analyses of 2408 base pairs of

mitochondrial DNA to estimate genealogical relationships and divergence times

within the C. triseriatus species group. We then assessed the tempo of

diversification using a maximum likelihood framework based on the birth–

death process. Estimated times of divergences provided a probabilistic temporal

component and questioned whether diversification rates have remained

constant or varied over time. Finally, we looked for phylogeographical

patterns in other co-distributed taxa.

Results We identified eight major lineages within the C. triseriatus group, and

inferred strong correspondence between maternal and geographic history within

most lineages. At least one cryptic species was detected. Relationships among

lineages were generally congruent with previous molecular studies, with

differences largely attributable to our expanded taxonomic and geographic

sampling. Estimated divergences between most major lineages occurred in the

Late Miocene and Pliocene. Phylogeographical structure within each lineage

appeared to have been generated primarily during the Pleistocene. Although the

scale of genetic diversity recognized affected estimated rates of diversification,

rates appeared to have been constant through time.

Main conclusions The biogeographical history of the C. triseriatus group

implies a dynamic history for the highlands of Mexico. The Neogene formation of

the Transvolcanic Belt appears responsible for structuring geographic diversity

among major lineages. Pleistocene glacial–interglacial climatic cycles and

resultant expansions and contractions of the Mexican pine–oak forest appear

to have driven widespread divergences within lineages. Climatic change, paired

with the complex topography of Mexico, probably produced a myriad of species-

specific responses in co-distributed Mexican highland taxa. The high degree of

Journal of Biogeography (J. Biogeogr.) (2011) 38, 697–710

ª 2010 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/jbi 697doi:10.1111/j.1365-2699.2010.02431.x

INTRODUCTION

The geographical location, complex topography, and dynamic

tectonic and climatic history of the Mexican highlands provide

a matrix for the evolution of a spectacularly diverse biota. The

Mexican highlands harbour a significant amount of western

North America’s biodiversity (Ramamoorthy et al., 1993;

Mittermeier et al., 2005) and a level of biotic endemism

scarcely rivalled elsewhere (Peterson et al., 1993). The evolu-

tionary drivers of this diversity, however, remain poorly

documented. Despite early broad-scale inferences about the

biogeographical history of Mexico, dating back to Dunn (1931),

few studies explore the historical diversification of Mexican

highland taxa. This impedes the ability of researchers to identify

fine-scale biogeographical patterns and the extent to which

these apply to co-distributed taxa (McCormack et al., 2008a).

Neogene vicariance, largely due to orogenesis, and Quater-

nary climate change have been the postulated drivers of

evolutionary diversification in western North America (e.g.

Jaeger et al., 2005; Riddle & Hafner, 2006). Although most of

the major mountain ranges in Mexico are relatively ancient

(Ferrusquıa-Villafranca, 1993; Ferrusquıa-Villafranca & Gon-

zalez-Guzman, 2005), the Transvolcanic Belt of central Mexico

was formed during the Neogene (Ferrusquıa-Villafranca, 1993;

Becerra, 2005). This development may have had a significant

impact on the diversification of highland taxa, because the

uplift created new geographical barriers and montane habitats,

and linked previously isolated highland biotas (Anducho-

Reyes et al., 2008). Historical diversification of highland taxa

may also have been influenced by dramatic habitat fluctuations

during the Pleistocene that resulted in the cyclical downward

displacement and retraction of Mexican pine–oak woodlands

(Martin & Harrell, 1957; Van Devender, 1990; McDonald,

1993). This displacement could have resulted in population

and range expansions in highland species during glacial

periods, and isolation in high elevation refugia during the

interglacials (Moreno-Letelier & Pinero, 2009). Subsequent

post-glacial fragmentation of Mexican pine–oak woodlands

(Van Devender, 1990) may have caused fragmentation of these

isolated refugial populations (e.g. McCormack et al., 2008b).

Gene flow would have been affected by these events.

Molecular studies of montane Mexican taxa often discover

complex phylogeographical patterns. In small mammals, for

example, mitochondrial DNA (mtDNA) differentiation is high

and allopatric populations are generally monophyletic (Sulli-

van et al., 1997; Harris et al., 2000; Hafner et al., 2005; Leon-

Paniagua et al., 2007). Several morphologically cryptic mater-

nal lineages occur within the Transvolcanic Belt, Sierra Madre

Oriental and Sierra Madre del Sur (small mammals: Sullivan

et al., 1997; Harris et al., 2000; Arellano et al., 2005; Leon-

Paniagua et al., 2007; birds: Garcıa-Moreno et al., 2004;

Navarro-Siguenza et al., 2008; Puebla-Olivares et al., 2008).

These high levels of genetic divergence suggest that endemism

in the Mexican highlands may be vastly underestimated.

Studies of genetic structuring in other co-distributed taxa are

needed in order to develop a more complete understanding of

the evolutionary drivers of diversification.

The relatively small-bodied montane rattlesnakes (Viperi-

dae) inhabiting the pine–oak forests of mainland Mexico

represent an ideal model system for investigating historical

patterns of diversification in the Mexican highlands. This large

group includes 40% of the total number of currently recog-

nized species of rattlesnakes (Campbell & Lamar, 2004) and is

found in all of the major mountainous regions of Mexico. The

phylogenetic relationships among these rattlesnakes, however,

are contentious. Those species currently allied with the

Crotalus triseriatus species group (C. triseriatus, C. aquilus,

C. lepidus, C. pusillus and C. ravus; Murphy et al., 2002; Castoe

& Parkinson, 2006) are especially difficult to classify. The

content of this species group varies despite more than 65 years

of intensive systematic effort (Gloyd, 1940; Smith, 1946;

Klauber, 1952, 1972; Brattstrom, 1964; Dorcas, 1992; Murphy

et al., 2002). Furthermore, several authors suggest that the

Mexican highlands may harbour one or more cryptic species

within the C. triseriatus species group (Armstrong & Murphy,

1979; Murphy et al., 2002).

Our study addresses several questions relating to the

evolutionary history of the C. triseriatus species group. We

combine extensive range-wide sampling and mixed-model

phylogenetic analyses to formulate a robust hypothesis of

phylogenetic relationships and to address long-standing

uncertainties about cryptic diversity. We also estimate dates

of lineage divergences based on a relaxed molecular clock to

provide a probabilistic temporal calibration for the phylogeny.

We model the temporal distribution of divergence events to

assess the potential effects of Late Neogene mountain building

and Pleistocene pine–oak forest fragmentation on the tempo of

diversification. Finally, we look for phylogeographical patterns

genetic differentiation recovered in our study and others suggests that the

Mexican highlands may contain considerably more diversity than currently

recognized.

Keywords

Biogeography, divergence dating, diversification rates, Mexico, phylogeography,

pine–oak forest, reptiles, Transvolcanic Belt, Viperidae.

R. W. Bryson Jr et al.

698 Journal of Biogeography 38, 697–710ª 2010 Blackwell Publishing Ltd

in the C. triseriatus group that are shared with other

co-distributed highland taxa.

MATERIALS AND METHODS

Taxon sampling and laboratory methods

Between 1999 and 2009 we collected 130 samples (see

Appendix S1 in the Supporting Information) from throughout

the distribution of all putative taxa in the C. triseriatus group

(Fig. 1). Four samples were collected from a morphologically

distinct, undescribed species closely related to C. pusillus

(herein referred to as ‘Crotalus sp.’), and three samples were

from specimens intermediate between C. lepidus and C. aquilus

(Ct160, Ct197 and Ct201; herein referred to as ‘C. lepidus x

aquilus’). Based on recent phylogenetic analyses (Murphy

et al., 2002; Castoe & Parkinson, 2006; Wuster et al., 2008), we

used Sistrurus catenatus and S. miliarus as outgroup taxa.

We sequenced relatively slowly evolving and more quickly

evolving regions of the mitochondrial genome, including 12S

and 16S ribosomal RNA genes, NADH dehydrogenase subunit

4 and flanking tRNAs (ND4), and ATPase subunits 8 and 6

(ATPase 8, ATPase 6). These gene regions have been shown to

be informative at different levels of divergence within rattle-

snakes (Pook et al., 2000; Murphy et al., 2002; Wuster et al.,

2005; Douglas et al., 2006). Total genomic DNA was extracted

from liver, shed skins, or ventral scale clips using proteinase K

(22 mg mL)1 in 10 mm Tris-HCl, pH 7.5) in a lysis buffer

(100 mm Tris, 5 mm Na2EDTA, 200 mm NaCl, 0.2% SDS) and

incubated at 37 �C. Shed skins often required 2–3 days to fully

16º

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pusillus

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Major mtDNA lineages

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aquilus

ravus

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armstrongi

Figure 1 Map of Mexico and the south-

western United States depicting the sample

localities and distribution (adapted from

Campbell & Lamar, 2004) for the Crotalus

triseriatus species group. Symbols indicate

major mitochondrial DNA (mtDNA) lin-

eages inferred in this study, and numbers

refer to specific sample numbers (see

Appendix S1). Asterisks denote multiple

samples obtained from the same locality. The

prefix ‘Ct’ was omitted from the sample

numbers for clarity.

Phylogeography of the Crotalus triseriatus group

Journal of Biogeography 38, 697–710 699ª 2010 Blackwell Publishing Ltd

digest, and an additional 12.5 lL of proteinase K was added

every 24 h. Samples were cleaned using two washes of

phenol:chloroform:isoamyl alcohol (25:24:1) followed by a

final wash of chloroform:isoamyl alcohol (24:1).

All gene regions were amplified via polymerase chain

reaction (PCR) in a 25 lL reaction volume containing

0.8 lL deoxynucleoside triphosphates (dNTPs) (10 mm),

19.0 lL double-distilled water, 1.0 lL each primer (10 pm),

2.5 lL 1· PCR buffer (1.5 mm MgCl2; Fisherbrand, Pitts-

burgh, PA, USA), 0.75 U Taq DNA polymerase (Fisherbrand),

and 1.0 lL template DNA. Previously published primer

sequences are given in Murphy et al. (2002; 12S, 16S). For

amplification of ND4 we modified one of the forward primers

of Arevalo et al. (1994) (12931L: 5¢-CTA CCA AAA GCT CAT

GTA GAA GC-3¢) and used the LEU reverse primer (Arevalo

et al., 1994). Primers for ATPase were designed specifically for

this project: (9974L: 5¢-AGC ACT AGC CTT TTA AGY T-3¢and 10830H: 5¢-AGA AAC CCT ATT TTT AGT ACT AG-3¢).

Initially, DNA was denatured at 94 �C for 2 min, followed by

39 cycles of: 94 �C for 30 s, 48–50 �C for 45 s, 72 �C for 45 s.

A final extension phase of 72 �C for 7 min terminated the

protocol. The entire 25 lL reaction was visualized on a 1%

agarose gel containing ethidium bromide. Sharp, clear bands

were excised from the gel and placed in a filter tip (Sorenson;

75-30550T). DNA was collected in a 1.7 mL Eppendorf tube

after centrifuging the DNA through the filter tip for 10 min at

16.1 rcf.

We sequenced in both directions using the amplification

primers and Big Dye Terminator v.3.1 cycle sequencing kit

(Applied Biosystems, Foster City, CA, USA). We used 4 lL of

the cleaned PCR product in one-quarter reaction volume of

that recommended by ABI (Applied Biosystems). Samples were

analysed with an ABI Prism 3100 Genetic Analyzer (Applied

Biosystems). Forward and reverse sequences for each individ-

ual were edited and manually aligned using BioEdit 5.0.9

(Hall, 1999). Identical sequences for samples from the same

locality were collapsed into one haplotype.

Phylogenetic analyses

We analysed our sequence data using Bayesian inference (BI)

and maximum likelihood (ML) phylogenetic methods. BI

analyses were conducted using MrBayes 3.1 (Ronquist &

Huelsenbeck, 2003) on the combined mtDNA dataset, imple-

menting separate models for each gene region (ATPase 8,

ATPase 6, ND4, combined tRNAs, 12S, and 16S). MrModel-

test 2.1 (Nylander, 2004) was used to select a best-fit model of

evolution, based on the Akaike information criterion (AIC),

for each partition. MrBayes settings included random starting

trees, a variable rate prior, a mean branch length exponential

prior of 100, and heating temperature of 0.02. Analyses

consisted of four runs (n runs = 4) each conducted with three

heated and one cold Markov chain while sampling every 100

generations for 4 million generations. Output parameters were

visualized using the program Tracer v1.4 (Rambaut &

Drummond, 2007) to ascertain stationarity and whether or

not the duplicated runs had converged on the same mean

likelihood. We further assessed convergence by evaluating

posterior probability clade-support values post burn-in using

the on-line application Are We There Yet (AWTY; Wil-

genbusch et al., 2004). After determining chain convergence,

which occurred during the first 500,000 generations of each

run, we conservatively discarded all samples obtained during

the first one million (25%) generations as burn-in. A 50%

majority-rule consensus phylogram with nodal posterior

probability (PP) support was estimated from the combination

of the four runs post-burn-in. ML analyses were conducted

using RAxML 7.0.3 (Stamatakis, 2006) with the same parti-

tioning scheme used for the BI analyses. The GTRGAMMA

model was used, and 1000 nonparametric bootstrap replicates

were performed to assess nodal support. We considered those

nodes with ‡95% Bayesian posterior probability and ‡70%

bootstrap support as strongly supported (Hillis & Bull, 1993;

Felsenstein, 2004).

Divergence dating

We estimated divergence dates using a Bayesian relaxed

molecular clock as implemented in beast v.1.5.4 (Drummond

& Rambaut, 2007). Because of potential problems associated

with model parameter variance across heterogeneous datasets

(Guiher & Burbrink, 2008), we inferred divergence estimates

for a reduced dataset, which included one or two individuals

from each geographically structured maternal group within

each lineage (Fig. 2). We also included sequences from several

other North American pitvipers to calibrate the tree (Appen-

dix S2). Best-fit models of evolution were estimated from the

new dataset using MrModeltest. We implemented an

uncorrelated lognormal clock and node constraints obtained

from the fossil and geological record with lognormal distri-

butions to estimate divergence dates throughout the tree.

Analyses were run for 40 million generations, with samples

retained every 1000 generations, and with a Yule tree prior.

Results were displayed in Tracer to confirm acceptable

mixing and likelihood stationarity of the Markov chain Monte

Carlo (MCMC) analyses, appropriate burn-in, and adequate

effective sample sizes (>200 for each estimated parameter).

Analyses on a partitioned-by-gene dataset resulted in effective

sample sizes below 50 for several parameters. Therefore, the

final analyses were run on an unpartitioned dataset. After

discarding the first 4 million generations (10%) as burn-in, we

summarized parameter values of the samples from the

posterior on the maximum clade credibility tree using

TreeAnnotator 1.4.8 (Drummond & Rambaut, 2007) with

the posterior probability limit set to 0.5 and mean node

heights summarized.

Two fossil calibrations for the tree were obtained for North

American pitvipers: (1) the oldest fossil from the genus

Sistrurus from the Late Miocene (Clarendonian; Parmley &

Holman, 2007), and (2) the earliest record of A. contortrix in

the Late Miocene (Late Hemphillian; Holman, 2000). Addi-

tionally, we used the estimated age of divergence between



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C. ruber and C. atrox (Castoe et al., 2009) due to the Pliocene

marine incursion of the Sea of Cortes (Carreno & Helenes,

2002; and references therein). The stem of Sistrurus (Guiher &

Burbrink, 2008; Wuster et al., 2008) was constrained with a

zero offset (hard upper bound) of 8 million years ago (Ma), a

lognormal mean of 0.01, and a lognormal standard deviation

of 0.76. This produced a median age centred at 9 Ma and a

95% prior credible interval (PCI) extending to the beginning

of the Clarendonian at 11.5 Ma (Holman, 2000). The node

representing the most recent common ancestor (MRCA) of

A. contortrix was given a zero offset of 6 Ma, a lognormal mean

of 0.01, and a lognormal standard deviation of 0.42, producing

a median age of 7 Ma and a 95% PCI extending to the start of

the Late Hemphillian at 8 Ma (Holman, 2000). These lognor-

mal distributions with hard lower bounds best reflect the

prediction, based on the high likelihood of fossil non-

preservation, that any true divergence date will probably be

older than the oldest known fossil, rather than younger (Ho &

Phillips, 2009; Kelly et al., 2009). The node representing the

MRCA of the C. ruber-atrox clade was given a lognormal mean

of 1.1 and a lognormal standard deviation of 0.37, resulting in

a median age centred at the climax of the formation of the Sea

of Cortes and development of the Bouse embayment at 3 Ma,

and a 95% PCI extending to the beginning of the development

of the Sea of Cortes at 5.5 Ma (Carreno & Helenes, 2002, and

references therein). No zero offset was used.

Temporal patterns of diversification

We analysed temporal shifts in diversification rates using ML-

based diversification-rate analysis (Rabosky, 2006a). The fit of

different birth–death models implementing two constant rates

(pure-birth, and birth–death) and three variable rates (expo-

nential and logistic density-dependent, and two-rate pure-

birth) was computed with laser 2.3 (Rabosky, 2006b). Model

fit was measured using AIC scores. Significance of the change

in AIC scores (DAICrc) between the best rate-constant and

best rate-variable model was determined by creating a null

distribution for DAICrc. This was done by simulating 1000

trees using yuleSim in laser with the same number of nodes

and the same speciation rate as that estimated under the pure-

birth model. We additionally generated a lineage-through-time

(LTT) plot using the plotLtt function in laser to visualise the

pattern of accumulation of log-lineages over time.

Because underestimates of genetic diversity potentially bias

inferred rates of diversification (Esselstyn et al., 2009), we used

two sets of dates estimated in beast for diversification-rate

analyses. The first set consisted of estimated divergence dates

between the major phylogroups (Fig. 2), which we considered

to be a conservative approach. The second set included

estimated diversification dates between an additional 12

geographically cohesive monophyletic subgroups clustered

within several of the major phylogroups, and this was a more

liberal approach. These smaller subgroups represented the

finest division of phylogeographical diversity that could be

reasonably inferred from our study.

RESULTS

Sequence characteristics and phylogenetic estimate

The final dataset used for phylogenetic inference consisted of

2408 aligned nucleotide positions. Models of sequence evolu-

tion selected for the mtDNA partitions were GTR+I+G

(ATPase 8, ATPase 6, ND4, 12S) and HKY+I+G (tRNAs and

16S). All sequences were deposited in GenBank (Appendix S2).

We identified eight major maternal lineages within the

C. triseriatus group (Figs 2 & 3), five of which corresponded to

the species C. ravus, C. pusillus, C. triseriatus, C. aquilus and

C. lepidus (Campbell & Lamar, 1989, 2004). Two lineages

represented the subspecies C. t. armstrongi and C. l. morulus.

One lineage represented an undescribed taxon. Strong phylo-

geographical structure was present within most of these taxa

(Fig. 2). In C. ravus, the geographical distribution of subclades

was consistent with recognized subspecies (C. r. ravus,

C. r. brunneus and C. r. exiguus). Relationships among lineages

were generally congruent with those of previous molecular

studies (Fig. 3; Murphy et al., 2002; Castoe & Parkinson,

ravus

pusillus

lepidus

aquilus

triseriatus (LG) *

triseriatus

other Crotalus

ravus

pusillus

lepidus

aquilus

triseriatus (LG) *

triseriatus

ravus

pusillus

lepidus

aquilus

morulus

triseriatus

armstrongi

Crotalus sp.

(a) Murphy et al., 2002 (b) Castoe & Parkinson, 2006 (c) This study

Figure 3 Comparison of phylogenetic relationships for the Crotalus triseriatus species group based on previous molecular studies (a, b) and

this study (c). Differences are in part due to new lineages inferred from our expanded taxonomic and geographical sampling (dotted lines),

and the use, in previous studies, of a mislabelled sample of C. triseriatus (labelled with an asterisk).



2006). Differences were largely attributable to our expanded

taxonomic and geographic sampling, and the prior use of a

misidentified sample (see below).

Divergence times and tempo of diversification

The GTR+I+G model of sequence evolution was selected for

the beast analyses. Dating estimates suggested that diversifi-

cation in the C. triseriatus group probably began in the Late

Miocene, and divergences between most major lineages

occurred in the Late Miocene and Pliocene (Fig. 4). Phylo-

geographical structure within these lineages appeared to have

been generated primarily during the Pleistocene.

The LTT plots suggested either a constant rate of diversifi-

cation or a decline in the Pleistocene (Fig. 5), depending on the

scale of phylogeographical diversity recognized by each dataset.

The birth–death likelihood analyses chose the pureBirth and

yule2rate models as the best rate-constant and best rate-variable

models for both the ‘conserved’ and the ‘liberal’ datasets.

However, P-values for the change in AIC scores between the

two models differed between datasets. For the conserved

dataset, the null hypothesis of rate-constancy was rejected

(P = 0.04), suggesting the rate-variable yule2rate model pro-

vided a better fit. According to the scenario suggested by this

model, the C. triseriatus group had an initial net diversification

rate of 0.53 events per million years. A change in net

diversification rate took place 1.02 Ma, when the rate shifted

dramatically to 0.09 diversification events per million years. In

contrast, the null hypothesis of rate-constancy was not rejected

(P = 0.1) for the liberal dataset. The ML estimate of the

diversification rate under the best rate-constant pureBirth

model was 0.45 diversification events per million years. Thus, a

strong signal of diversification rate change was not indicated

after accounting for fine-scaled phylogeographical diversity.

I

II

III

I

II

I

II

III

III

IV

I

II

ravus

armstrongi

Crotalus sp.

pusillus

triseriatus

morulus

aquilus

Ct259 armstrongi MICH

Ct2,16 aquilus AGS

Ct137,138 armstrongi COL

S. miliarius

Ct152 brunneus OAX

Ct144,147 pusillus MICH

Ct242 triseratus VER

S. catenatus

Ct261 aquilus MEX

Ct26 morulus NL

Ct153 ravus MEX

Ct268 Crotalus sp. MEX

C. atrox

Ct262 triseriatus MICH

Ct164 aquilus QTO

Ct155 triseriatus PUE

Ct135 armstrongi JAL

Ct168 brunneus OAX

Ct30 aquilus SLP

Ct145 pusillus MICH

Ct11 morulus NL

Ct150 exiguus GRO

Ct9 morulus TAM

A. contortrix

Ct233 armstrongi JALCt254 armstrongi MICH

Ct264 Crotalus sp. MICH

Ct3 aquilus QTO

Ct193 armstrongi JAL

Ct245 triseratius PUE

A. piscivorous

Ct15 aquilus HIDCt122 aquilus HID

Gloydius

Ct17 pusillus MICH

Ct149 exiguus GRO

Ct216 morulus TAM

C. ruber

Ct208 pusillus JAL

Ct204 ravus PUEI

II

III

PleistocenePlioceneMiocene

15 10 5 0

II

IV

VI

V lepidus

Ct226 klauberi SON

Ct32 klauberi DUR

Ct161 klauberi DUR

Ct140,141 lepidus COAH

Ct46 klauberi NMCt29 klauberi CHIH

Ct59 lepidus NM

Ct118 klauberi ZACCt195 klauberi JAL

Ct12,13 maculosus DUR

Ct55 klauberi AZ

III

6.3

5.4

4.9

4.1

3.4

3.3

2.1

Ct283 maculosus NAY I

Figure 4 Chronogram with estimated divergence times for major lineages and phylogroups within the Crotalus triseriatus species

group inferred using Bayesian relaxed clock phylogenetic analyses. Arrows denote the placement of fossil calibrations detailed in the

text. Values at nodes represent mean divergence dates between major lineages, with bars indicating 95% highest posterior densities.

Maximum likelihood-based diversification-rate analyses utilized estimated divergences between lineages and major phylogroups (nodes

indicated with black dots) or lineages and all possible phylogroups (black plus white dots).



DISCUSSION

Evolutionary drivers of diversity within the

C. triseriatus group

Our analyses indicated that a progressive diversification of the

C. triseriatus group occurred over the last six million years.

Both Neogene vicariance and Quaternary climate change had

comparably strong effects on driving diversification rates. Early

divergences were temporally and geographically congruent

with the formation of the Transvolcanic Belt, suggesting that

the development of this mountain range played a critical role

in early diversification of this widespread highland group. Five

of the eight major lineages (C. ravus, C. t. triseriatus,

C. t. armstrongi, C. pusillus and Crotalus sp.) were distributed

on or near the Transvolcanic Belt, and estimated divergence

dates between these lineages fell within the orogenic timeframe

for the area (Ferrusquıa-Villafranca, 1993; Becerra, 2005). The

remaining, more northerly three lineages occupied the Central

Mexican Plateau and southern Sierra Madre Oriental

(C. aquilus), northern Sierra Madre Oriental (C. l. morulus),

and Sierra Madre Occidental (western C. lepidus). Uplift of the

Central Mexican Plateau coupled with the subsequent aridi-

fication and final Pliocene development of the Chihuahuan

Desert (Jaeger et al., 2005, and references therein) may have

separated C. aquilus and C. l. morulus to the east from western

populations of C. lepidus. The divergence of C. aquilus from

C. l. morulus along the Sierra Madre Oriental may have been

facilitated by the development of any one of several hypoth-

esized filter barriers, such as the Rio Panuco basin (Anducho-

Reyes et al., 2008) and Cerritos-Arista and Saladan Filter

Barriers (Morafka, 1977; Bryson et al., 2007) (Fig. 6).

Our results revealed strong geographical partitioning of

genetic diversity within nearly all lineages. Most of this

structure appeared to have developed during the Quaternary.

Thus, glacial climatic cycles probably contributed to the

fragmentation of Mexican pine–oak forests and may have

driven divergences. This inference was broadly congruent with

diversification patterns seen in several Middle American

highland pitvipers (Castoe et al., 2009) and other montane

taxa (pines: Moreno-Letelier & Pinero, 2009; Rodrıguez-

Banderas et al., 2009; insects: Masta, 2000; Smith & Farrell,

2005; Anducho-Reyes et al., 2008; small mammals: Sullivan

et al., 1997; Edwards & Bradley, 2002; Leon-Paniagua et al.,

2007; lizards: Zaldivar-Riveron et al., 2005; Tennessen &

Zamudio, 2008; birds: Garcıa-Moreno et al., 2004; McCor-

mack et al., 2008b; Puebla-Olivares et al., 2008).

Observed phylogeographical patterns in the C. triseriatus

group, however, might not reflect isolated gene flow. Male

rattlesnakes can disperse relatively long distances between

populations during the breeding season (e.g. King & Duvall,

1990; Clark et al., 2008), and male-biased dispersal cannot be

accounted for in a mtDNA gene tree, unless the dispersed male

is sampled. Paternally mediated gene flow may occur between

seemingly isolated populations. Whereas some phylogroups

appear to be separated by well defined, low elevation breaks

(Fig. 6, Table 1), others may be marginally separated by mid-

elevation swathes of oak-, mesquite-, or desert-grassland.

0 1 2 3 4 5 6

1.0

1.5

2.0

2.5

3.0

3.5

Log-Lineages Through Time

Time From Basal Divergence(million years ago)

Log

Line

ages

0 1 2 3 4 5 6

1.0

1.5

2.0

2.5

3.0

3.5

st = 1.02

r1 = 0.53

r2 = 0.09

r1 = 0.45

(a) (b)

Figure 5 Lineage through time plots derived from Bayesian relaxed clock estimates of divergence dates within the Crotalus triseriatus

species group. (a) Diversification rates for inferred lineages and major phylogroups suggest a diversification rate shift 1.02 million years ago.

(b) Diversification rates for inferred lineages and all possible phylogroups suggest a constant diversification rate through time. st = time of

diversification rate shift from yule2rate model estimates. r = diversification rate.



Boundaries within some northern phylogroups (e.g. western

C. lepidus, C. l. morulus) are not obvious. Future testing with

nuclear gene markers is needed to elucidate patterns of gene

flow.

Phylogeographical diversity in the Mexican highlands

Despite an overall paucity of phylogeographical studies of

Mexican highland taxa, emerging patterns suggest mixed

(a) (b)

Figure 6 Map of Mexico depicting pine–oak forests above 1800 m elevation. (a) Generalized areas of major mountain ranges in Mexico:

SMOcc, Sierra Madre Occidental; SMOr, Sierra Madre Oriental; TVB, Transvolcanic Belt; SMdS, Sierra Madre del Sur. The Central Mexican

Plateau (CMP) is also shown. (b) Biogeographical barriers to highland Mexican taxa (see Table 2): (1) Balsas Basin (including the

Tepalcatepec Depression), (2) Rio Verde basin, (3) Rio Papaloapan basin, (4) Oriental Basin, (5) Lerma-Santiago Basin, (6) Rio Mezquital

basin, (7) Cerritos-Arista and Saladan Filter Barriers, (8) Rio Panuco basin, (9) Rio Ahuijullo basin, (10) Colima and Tepic-Zocoalco

Grabens (rattlesnakes in this study), (11) Aguascalientes Graben [Mexican jays (Aphelocoma ultramarina), McCormack et al., 2008a] and

(12) unnamed barrier [spiny mice (Habromys), Leon-Paniagua et al., 2007]. Numbered lines span approximate locations of barriers, and

their thickness corresponds to the number of taxa exhibiting genetic break (thick = two or more taxa; thin = one taxon).

Table 1 Potential isolating barriers between major phylogroups within the Crotalus triseriatus species group inferred in this study.

Phylogroups follow those in Fig. 2. Geographical barriers are shown in Fig. 6.

Phylogroup division Potential isolating barriers

C. ravus I/II+III Balsas Basin, Rio Verde basin

C. ravus II/III Balsas Basin, Rio Papaloapan basin (Campbell & Armstrong, 1979)

C. t. armstrongi I/II+III Unknown (potentially mid-elevations <1800 m extending south-southeast of Ameca, Jalisco to Jalisco/Colima

border)

C. t. armstrongi II/III Colima and Tepic-Zocoalco Grabens

C. pusillus I/II Rio Ahuijullo basin, Tepalcatepec Depression (Campbell & Lamar, 2004) of the Balsas Basin

C. t. triseriatus I/II Oriental Basin

C. l. morulus I/II Unknown

C. aquilus I/II+III+IV Unknown (potentially mid-elevations <1800 m surrounding the isolated ‘sky island’ in north-eastern Queretaro

and adjacent San Luis Potosı)

C. aquilus II/III+IV Unknown

C. aquilus III/IV Rio Panuco basin

C. lepidus I/II Tributaries of the Rio Santiago basin

C. lepidus I+II/III+IV+V+VI Rio Mezquital basin

C. lepidus III+IV+V/VI Unknown

C. lepidus III/IV+V Unknown (potentially high elevation >2700 m ridges along crest of the Sierra Madre Occidental)

C. lepidus IV/V Unknown



responses to past geological and climatic events despite a

presumed shared history in the same region (Sullivan et al.,

2000; Paniagua & Morrone, 2009). The ancient development of

most of the major mountains in Mexico probably pre-dates

diversification of the extant highland species. However, the

Neogene formation of the Transvolcanic Belt is undoubtedly a

major force driving the evolutionary history of these taxa. In

the C. triseriatus group, initial diversification appears to be

tightly linked to the uplifting of the Transvolcanic Belt, and

several phylogroups are embedded within this mountain range.

Deep divergences and high phylogeographical diversity in

other co-distributed highland taxa (small mammals: Sullivan

et al., 1997; Demastes et al., 2002; Edwards & Bradley, 2002;

Leon-Paniagua et al., 2007; birds: McCormack et al., 2008a;

Navarro-Siguenza et al., 2008) are also broadly attributable to

the formation of the Transvolcanic Belt.

In concert, Quaternary glacial–interglacial climatic cycles

and the complex topography of Mexico had the potential to

produce a myriad of intraspecific phylogeographical patterns

in highland taxa. Some pine–oak expansions probably resulted

in ephemerally contiguous highland biotas in the Mexican

sierras (Toledo, 1982; Van Devender, 1990). However, some

geographical barriers, such as major river drainages, may have

served as filter barriers, as evidenced by shared genetic breaks

in co-distributed taxa (Fig. 6, Table 2). In southern Mexico,

the Sierra Madre del Sur is isolated from the Transvolcanic Belt

to the north by the Rio Balsas and its associated tributaries

(e.g. Rio Tepalcatepec, Rio Atoyac) that form the Balsas Basin.

The Rio Ahuijullo isolates these areas to the west. Further, the

Sierra Madre del Sur is separated from the Transvolcanic Belt

and Sierra Madre Oriental by the Rio Papaloapan basin across

northern Oaxaca. The Rio Verde additionally divides the Sierra

Madre del Sur in western Oaxaca. To the north, the Rio

Panuco bisects the Sierra Madre Oriental, and the xeric

interior of the Oriental Basin in Puebla and Veracruz separates

the highlands of the Sierra Madre Oriental from those of the

Transvolcanic Belt. Further west, the Rios Lerma and Rio

Santiago flank the southern Sierra Madre Occidental and

northern Transvolcanic Belt and separate these two highlands.

Further north, the Rio Mezquital bisects the Sierra Madre

Occidental. Historical filter barriers across the Central Mexican

Plateau may also include large interconnected palaeo-lakes

(Mejıa-Madrid et al., 2007, and references therein). These

largely overlooked barriers are probably responsible for

disrupting Pleistocene pine–oak forest corridors (Demastes

et al., 2002) and could, in part, further explain inferred

historical disjunctions between highland taxa distributed

across the Central Mexican Plateau, such as between phylo-

groups of C. aquilus.

Matrilineal genealogy of the C. triseriatus group

Utilizing expanded taxonomic and geographical sampling and

phylogenetic mixed-model analyses of mtDNA, we inferred

several novel historical relationships for the C. triseriatus

group. The topology strongly supported the placement of

C. ravus in the C. triseriatus group despite over 100 years of

inclusion in the genus Sistrurus and a recent transfer into

Crotalus as the sister species to all other Crotalus (Murphy

et al., 2002). Plesiomorphic morphological attributes shared

with Sistrurus have seemingly confounded the phylogenetic

placement of C. ravus (McCranie, 1988; Murphy et al. (2002).

Table 2 Biographical barriers shared by co-distributed Mexican highland taxa. Numbers correspond to barriers shown in Fig. 6. Asterisks

denote barriers inferred from the phylogeny or delineated on maps, and not directly stated in the original study. The Tepalcatepec

Depression was included as a western branch of the Balsas Basin.

Biogeographical barrier Taxon

(1) Balsas Basin Montane rattlesnakes (Crotalus triseriatus species group)1, deer mice (Peromyscus aztecus species group)2,

bark beetle (Dendroctonus mexicanus)3, Neotropical brush-finches (Buarremon)*4, Mexican woodrats

(Neotoma mexicana species group)*5

(2) Rio Verde basin Montane rattlesnakes (Crotalus triseriatus species group)1, Neotropical brush-finches (Buarremon)*4, Mexican

woodrats (Neotoma mexicana species group)*5, emerald toucanet (Aulacorhynchus prasinus species group)6,

common bush-tanager (Chlorospingus ophthalmicus)*7

(3) Rio Papaloapan basin Montane rattlesnakes (Crotalus triseriatus species group)1, deer mice (Peromyscus aztecus species group)*2,

emerald toucanet (Aulacorhynchus prasinus species group)*6, common bush-tanager (Chlorospingus

ophthalmicus)*7, spiny mice (Habromys)*8

(4) Oriental Basin Montane rattlesnakes (Crotalus triseriatus species group)1, deer mice (Peromyscus aztecus species group)*2,

bark beetle (Dendroctonus mexicanus)3

(5) Lerma-Santiago Basin Bark beetle (Dendroctonus mexicanus)3, southwestern white pine (Pinus strobiformis)9, Mexican jays

(Aphelocoma ultramarina)10

(6) Rio Mezquital basin Montane rattlesnakes (Crotalus triseriatus group)1, bark beetle (Dendroctonus mexicanus)3

(7) Cerritos-Arista and Saladan

Filter Barriers

Montane rattlesnakes (Crotalus triseriatus group)1, Mexican jays (Aphelocoma ultramarina)*10, Mexican

kingsnakes (Lampropeltis mexicana species group)11

(8) Rio Panuco basin Montane rattlesnakes (Crotalus triseriatus group)1, bark beetle (Dendroctonus mexicanus)3

(9) Rio Ahuijullo basin Montane rattlesnakes (Crotalus triseriatus species group)1, Neotropical brush-finches (Buarremon)*4

1This study; 2Sullivan et al., 1997; 3Anducho-Reyes et al., 2008; 4Navarro-Siguenza et al., 2008; 5Edwards & Bradley, 2002; 6Puebla-Olivares et al.,

2008; 7Garcıa-Moreno et al., 2004; 8Leon-Paniagua et al., 2007; 9Moreno-Letelier & Pinero, 2009; 10McCormack et al., 2008a; 11Bryson et al., 2007.



We inferred a novel placement for C. t. armstrongi near the

base of the tree and distant from C. t. triseriatus. It formed the

sister clade to all other species of the C. triseriatus group except

for C. ravus. This finding contradicted previous suggestions

based on morphological evidence of a close relationship

between C. t. armstrongi and C. t. triseriatus (Campbell, 1979;

Dorcas, 1992).

Strong support was obtained for a sister relationship

between C. aquilus and C. l. morulus, and this clade was sister

to all other C. lepidus. The phylogenetic affinities of C. aquilus

to other C. triseriatus group taxa have been contentious (see

Gutberlet & Harvey, 2004). Crotalus aquilus has oscillated

between being considered as a subspecies of C. triseriatus (as

originally described) and a distinct species closely related to

C. lepidus. Recent molecular studies (Murphy et al., 2002;

Castoe & Parkinson, 2006) proposed a sister relationship

between C. aquilus and C. triseriatus. However, this association

was based on a misidentified sample, labelled as ‘C. triseriatus

LG’ (ROM 18114; GenBank numbers AF259231, AF259087,

AF259124, AF259161, AF259199), which we determined to be

C. aquilus. Our finding of a C. aquilus–C. l. morulus sister

relationship suggested that C. l. morulus may be more closely

related to C. aquilus than to other lineages of C. lepidus, in

contrast to strong morphological support for a monophyletic

C. lepidus (Dorcas, 1992). Other subspecies of C. lepidus in the

west (C. l. lepidus, C. l. klauberi and C. l. maculosus) together

formed a monophyletic group, but divergences within this

group appeared to be relatively recent (Fig. 4). Only the

monophyly of C. l. lepidus was supported.

Several studies suggest that multiple, geographically isolated

species may be represented under the name C. triseriatus

(Armstrong & Murphy, 1979; Murphy et al., 2002; Gutberlet &

Harvey, 2004). Samples of Crotalus obtained along the central

portion of the Transvolcanic Belt (Figs 1 & 2) provide clear

support for this prediction. Although historically considered to

be C. t. triseriatus (Campbell & Lamar, 2004), these genetically

distinctsnakesarelikelytorepresentanundescribedcrypticspecies.

While phylogenetic inferences based on one marker (e.g.

mtDNA) should be interpreted with caution, maternally inher-

ited data can lead to significant biogeographical discoveries (e.g.

Upton & Murphy, 1997; Riddle et al., 2000; Murphy & Aguirre-

Leon, 2002). An mtDNA-only approach has several limitations

(Funk & Omland, 2003; Ballard & Whitlock, 2004) yet it is much

more likely to detect geographical limits and cryptic species than

studies based on nuclear DNA gene sequences (Moore, 1995;

Hudson & Coyne, 2002; Zink & Barrowclough, 2008; Bar-

rowclough & Zink, 2009). Our genealogical inferences serve as a

robust hypothesis of matrilineal relationships within the

C. triseriatus species group. Future studies using unlinked

nuclear loci can test whether or not the genealogy also reflects

the macroevolutionary species phylogeny.

ACKNOWLEDGEMENTS

We dedicate this study to the late Fernando Mendoza-Quijano.

Without his enthusiasm and unfailing support through the

years, this study would not have been possible. We thank the

following people, curators and institutions for providing or

assisting with tissue samples: D.R. Frost (American Museum of

Natural History), R.D. MacCulloch (Royal Ontario Museum),

O. Flores-Villela and A. Nieto-Montes (Universidad Nacional

Autonoma de Mexico), J.A. Campbell, C. Franklin and E.N.

Smith (University of Texas at Arlington), J. Alvarado-Dıaz and

A. Quijada-Mascarenas (Universidad Michoacana de San

Nicolas de Hidalgo, Michoacan), A. Kardon (San Antonio

Zoo), P. Ponce-Campos, U.O. Garcıa-Vazquez, J. Lemos-

Espinal, L. Canseco-Marquez, G. Swinford, E. Mocino-Deloya,

K. Setser and G. Quintero-Dıaz. We thank the numerous

people who assisted in the field, including S.P. Mackessy,

M. Torocco, F.R. Mendoza-Paz, B.T. Smith, J. Banda-Leal,

G. Ulises de la Rosa-Lozano, R. Romero, D. Hartman, R.

Queen, K. Peterson, M. Price, C. Harrison, E. Garcıa-Padilla

and E. Beltran-Sanchez. B.T. Smith and A. Egan assisted with

use of laser and R. We further thank M.R. Graham, J. Jones,

C. Gruenwald, J.A. Mueller, J.R. Dixon, A. Narvaez and

J.A. Campbell for other assistance that greatly improved this

project. Two anonymous referees and S. Proches provided

comments that greatly improved the final version of this

manuscript. Collecting was conducted under permits granted

by SEMARNAT to R.W.B., R.W.M., D.L.V., D.J. Morafka and

F. Mendoza-Quijano. All sequencing work was conducted in

the laboratory of R.W.M., and funded by the Natural Sciences

and Engineering Research Council of Canada Discovery Grant

A3148.

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(2008) Phylogeography of the bark beetle Dendroctonus

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Appendix S1 Collection and voucher data for genetic

samples used in this study and deposited in the Royal Ontario

Museum (ROM).

Appendix S2 GenBank accession numbers for genetic

samples used in this study.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such mate-

rials are peer-reviewed and may be re-organized for online

delivery, but are not copy-edited or typeset. Technical support

issues arising from supporting information (other than

missing files) should be addressed to the authors.

BIOSKETCHES

Robert W. Bryson Jr and Robert W. Murphy began this

collaborative research while R.W.B. was a Master’s student at

Sul Ross State University, building on their shared interests in

rattlesnake systematics and biogeography of Mexico. All

authors are broadly interested in better understanding the

biodiversity of Mexico through evolutionary (R.W.B., R.W.M.,

A.L.) and ecological (D.L.V.) studies.

Author contributions: R.W.B. and R.W.M. conceived the

ideas; R.W.B., D.L.V. and A.L. collected the data; R.W.B.,

R.W.M. and A.L. analysed the data; and R.W.B. and R.W.M.

led the writing.

Editor: Serban Proches



2011 Bryson Et Al C Triseriatus Group

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