Journal of Biogeography (J. Biogeogr.
) (2011) 38, 697–710
Evolutionary drivers of phylogeographical diversity in the highlands of Mexico: a case study of the Crotalus triseriatus species group of montane rattlesnakes
Robert W. Bryson Jr1*, Robert W. Murphy2,3, Amy Lathrop2 and David Lazcano-Villareal4
School of Life Sciences, University of Nevada, Las Vegas, Las Vegas, NV, USA, 2Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, Toronto, ON, Canada, 3 State 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
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 speciesspeciﬁc responses in co-distributed Mexican highland taxa. The high degree of
*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: firstname.lastname@example.org
ª 2010 Blackwell Publishing Ltd
These high levels of genetic divergence suggest that endemism in the Mexican highlands may be vastly underestimated. 1993. Gene ﬂow would have been affected by these events. for example. ¨ Navarro-Siguenza et al. Mittermeier et al.. remain poorly documented. ravus. Several morphologically cryptic maternal lineages occur within the Transvolcanic Belt.. Brattstrom.. 2005. Those species currently allied with the Crotalus triseriatus species group (C. 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. 1940. W. 1990. 2000.
INTRODUCTION The geographical location. Becerra. 1964.. Finally. Furthermore. pine–oak forest.. 2007). This development may have had a signiﬁcant impact on the diversiﬁcation of highland taxa. Dorcas.. 2008).. 1979. 2004) and is found in all of the major mountainous regions of Mexico. Jaeger et al.R. and Quaternary climate change have been the postulated drivers of evolutionary diversiﬁcation in western North America (e. phylogeography. mitochondrial DNA (mtDNA) differentiation is high 698
and allopatric populations are generally monophyletic (Sulli´ van et al. 2008).. 2005). largely due to orogenesis.. LeonPaniagua et al. Harris et al. 2005). The evolutionary drivers of this diversity. McDonald. triseriatus species group. Viperidae. Leon´ Paniagua et al.
genetic differentiation recovered in our study and others suggests that the Mexican highlands may contain considerably more diversity than currently recognized. are contentious. few studies explore the historical diversiﬁcation of Mexican highland taxa. 2008. In small mammals.. 2006) are especially difﬁcult to classify. This large group includes 40% of the total number of currently recognized species of rattlesnakes (Campbell & Lamar. however. 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. and isolation in high elevation refugia during the ˜ interglacials (Moreno-Letelier & Pinero. 2007. 2008b). Ferrusquıa-Villafranca & Gon´ ´ zalez-Guzman. Puebla-Olivares et al. 1946. however. 2000. 1992. diversiﬁcation rates. triseriatus. 2002. dating back to Dunn (1931). We also estimate dates of lineage divergences based on a relaxed molecular clock to provide a probabilistic temporal calibration for the phylogeny. complex topography. Klauber. Smith. C.g. divergence dating.... 2005. Murphy et al. Mexico.. 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.. several authors suggest that the Mexican highlands may harbour one or more cryptic species within the C. The phylogenetic relationships among these rattlesnakes. C. 2002). The content of this species group varies despite more than 65 years of intensive systematic effort (Gloyd. 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. aquilus. 1993. 1993). lepidus. 2002). 1997. Our study addresses several questions relating to the evolutionary history of the C. Molecular studies of montane Mexican taxa often discover complex phylogeographical patterns. the Transvolcanic Belt of central Mexico ´ was formed during the Neogene (Ferrusquıa-Villafranca. Riddle & Hafner. 1972. 697–710 ª 2010 Blackwell Publishing Ltd
.. Harris et al. Subsequent post-glacial fragmentation of Mexican pine–oak woodlands (Van Devender. 1957. The relatively small-bodied montane rattlesnakes (Viperidae) inhabiting the pine–oak forests of mainland Mexico represent an ideal model system for investigating historical patterns of diversiﬁcation in the Mexican highlands. Castoe & Parkinson. pusillus and C. Murphy et al.. Murphy et al. we look for phylogeographical patterns
Journal of Biogeography 38. C. and linked previously isolated highland biotas (AnduchoReyes et al. Bryson Jr et al. and dynamic tectonic and climatic history of the Mexican highlands provide a matrix for the evolution of a spectacularly diverse biota. McCormack et al. because the uplift created new geographical barriers and montane habitats. Sierra Madre Oriental and Sierra Madre del Sur (small mammals: Sullivan ´ et al. 2005.g. 1997. 2005) and a level of biotic endemism scarcely rivalled elsewhere (Peterson et al. Van Devender.. 1993). Although most of the major mountain ranges in Mexico are relatively ancient ´ ´ (Ferrusquıa-Villafranca. The Mexican highlands harbour a signiﬁcant amount of western North America’s biodiversity (Ramamoorthy et al. Neogene vicariance. 1952. Keywords Biogeography. 2008a). birds: Garcıa-Moreno et al. Transvolcanic Belt. triseriatus species group (Armstrong & Murphy. 2009). 1993. Arellano et al. 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. 2006). reptiles. This displacement could have resulted in population and range expansions in highland species during glacial periods. Hafner et al.. 2004. 1990) may have caused fragmentation of these isolated refugial populations (e. Despite early broad-scale inferences about the biogeographical history of Mexico...
. Asterisks denote multiple samples obtained from the same locality. Ct197 and Ct201. Castoe & Parkinson.Phylogeography of the Crotalus triseriatus group in the C.5) in a lysis buffer (100 mm Tris.
Journal of Biogeography 38. and ATPase subunits 8 and 6 (ATPase 8. Total genomic DNA was extracted from liver. Four samples were collected from a morphologically distinct. 1).’). These gene regions have been shown to be informative at different levels of divergence within rattle¨ snakes (Pook et al. Symbols indicate major mitochondrial DNA (mtDNA) lineages inferred in this study. Murphy et al. 2004) for the Crotalus triseriatus species group. we used Sistrurus catenatus and S. Wuster et al. 2008). 2000. Shed skins often required 2–3 days to fully
46 59 36 112
41 99 55 163
R21 7 222
226 8 29 140* 161 20 252 162* 12* 237 142 118* 32* 160 215 127
116* 227* 143* 135 233* 209* 254 18* 259 2* 201
26* 223 10 31 9 197 R55 30 3
Major mtDNA lineages lepidus morulus aquilus triseriatus armstrongi pusillus Crotalus sp. Wuster et al. pusillus (herein referred to as ‘Crotalus sp.2% SDS) and incubated at 37 °C.. 2002. and numbers refer to speciﬁc sample numbers (see Appendix S1). ravus
266 126 15* 27 14 33* 155 122 255 238* 261* 157 6 R57 262* 1* 21 230* 172
Figure 1 Map of Mexico and the southwestern United States depicting the sample localities and distribution (adapted from Campbell & Lamar. NADH dehydrogenase subunit 4 and ﬂanking tRNAs (ND4). Based on recent phylogenetic analyses (Murphy ¨ et al. 0. lepidus and C. 5 mm Na2EDTA. 697–710 ª 2010 Blackwell Publishing Ltd
208 R42 144* 17 121* 267* 150
199 225 139 204 152
Crotalus sp.. including 12S and 16S ribosomal RNA genes. 2006... triseriatus group that are shared with other co-distributed highland taxa. undescribed species closely related to C. aquilus (Ct160. or ventral scale clips using proteinase K (22 mg mL)1 in 10 mm Tris-HCl. 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. The preﬁx ‘Ct’ was omitted from the sample numbers for clarity. 2002. and three samples were from specimens intermediate between C. shed skins. lepidus x aquilus’). 2005. miliarus as outgroup taxa. ATPase 6). We sequenced relatively slowly evolving and more quickly evolving regions of the mitochondrial genome. 200 mm NaCl. herein referred to as ‘C. triseriatus group (Fig... Douglas et al. pH 7.
a mean branch length exponential prior of 100. Output parameters were visualized using the program Tracer v1. 48–50 °C for 45 s. Samples were analysed with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). contortrix in the Late Miocene (Late Hemphillian. Felsenstein. and an additional 12.. Foster City.8 (Drummond & Rambaut. For ampliﬁcation of ND4 we modiﬁed one of the forward primers ´ of Arevalo et al. (2002. clear bands were excised from the gel and placed in a ﬁlter tip (Sorenson.7 mL Eppendorf tube after centrifuging the DNA through the ﬁlter tip for 10 min at 16. followed by 39 cycles of: 94 °C for 30 s. 1. We used 4 lL of the cleaned PCR product in one-quarter reaction volume of that recommended by ABI (Applied Biosystems). 2004) was used to select a best-ﬁt model of evolution. for each partition.8 lL deoxynucleoside triphosphates (dNTPs) (10 mm). 2007) with the posterior probability limit set to 0. Fisherbrand. ND4. 1994). The GTRGAMMA model was used.1 (Nylander. 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). DNA was collected in a 1. we summarized parameter values of the samples from the posterior on the maximum clade credibility tree using TreeAnnotator 1.5 and mean node heights summarized.R. Additionally. We sequenced in both directions using the ampliﬁcation primers and Big Dye Terminator v. Results were displayed in Tracer to conﬁrm acceptable mixing and likelihood stationarity of the Markov chain Monte Carlo (MCMC) analyses. 2008). Identical sequences for samples from the same locality were collapsed into one haplotype.4 (Drummond & Rambaut. All gene regions were ampliﬁed via polymerase chain reaction (PCR) in a 25 lL reaction volume containing 0. the ﬁnal analyses were run on an unpartitioned dataset. a variable rate prior. appropriate burn-in. 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. Forward and reverse sequences for each individual were edited and manually aligned using BioEdit 5. we conservatively discarded all samples obtained during the ﬁrst one million (25%) generations as burn-in.1 rcf. 2). and (2) the earliest record of A. 697–710 ª 2010 Blackwell Publishing Ltd
. 12S.0 lL template DNA. 1999). We considered those nodes with ‡95% Bayesian posterior probability and ‡70% bootstrap support as strongly supported (Hillis & Bull. ML analyses were conducted using RAxML 7.000 generations of each run. DNA was denatured at 94 °C for 2 min. combined tRNAs. CA.3 (Stamatakis.0. USA). digest. 2004). We further assessed convergence by evaluating posterior probability clade-support values post burn-in using the on-line application Are We There Yet (AWTY. which included one or two individuals from each geographically structured maternal group within each lineage (Fig. Phylogenetic analyses We analysed our sequence data using Bayesian inference (BI) and maximum likelihood (ML) phylogenetic methods. 2. Analyses on a partitioned-by-gene dataset resulted in effective sample sizes below 50 for several parameters. Analyses were run for 40 million generations. Previously published primer sequences are given in Murphy et al. Divergence dating We estimated divergence dates using a Bayesian relaxed molecular clock as implemented in beast v. We also included sequences from several other North American pitvipers to calibrate the tree (Appendix S2).5. Bryson Jr et al. USA). 12S. with samples retained every 1000 generations.75 U Taq DNA polymerase (Fisherbrand). W.5 lL 1· PCR buffer (1. 72 °C for 45 s. ATPase 6. Holman. and with a Yule tree prior. BI analyses were conducted using MrBayes 3.1 (Ronquist & Huelsenbeck.02. Sharp.1. 2007). 2000). Pittsburgh. Parmley & Holman. 16S). Best-ﬁt models of evolution were estimated from the new dataset using MrModeltest.3. 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¢).9 (Hall. 19. 1993. we inferred divergence estimates for a reduced dataset. 2007). The entire 25 lL reaction was visualized on a 1% agarose gel containing ethidium bromide. we used the estimated age of divergence between
Journal of Biogeography 38. After determining chain convergence.1 cycle sequencing kit (Applied Biosystems. Wilgenbusch et al. and 16S).5 lL of proteinase K was added every 24 h. 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. and heating temperature of 0. which occurred during the ﬁrst 500.0 lL double-distilled water. (1994) (12931L: 5¢-CTA CCA AAA GCT CAT ´ GTA GAA GC-3¢) and used the LEU reverse primer (Arevalo et al. Therefore. MrModeltest 2. 2003) on the combined mtDNA dataset.4.. 0.4 (Rambaut & Drummond. 2007) to ascertain stationarity and whether or 700 not the duplicated runs had converged on the same mean likelihood. and 1000 nonparametric bootstrap replicates were performed to assess nodal support. PA. 2006) with the same partitioning scheme used for the BI analyses. After discarding the ﬁrst 4 million generations (10%) 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. and 1. 2004). 75-30550T). A ﬁnal extension phase of 72 °C for 7 min terminated the protocol. Because of potential problems associated with model parameter variance across heterogeneous datasets (Guiher & Burbrink.0 lL each primer (10 pm). implementing separate models for each gene region (ATPase 8. and adequate effective sample sizes (>200 for each estimated parameter). We implemented an uncorrelated lognormal clock and node constraints obtained from the fossil and geological record with lognormal distributions to estimate divergence dates throughout the tree.5 mm MgCl2. Initially. MrBayes settings included random starting trees.0. based on the Akaike information criterion (AIC).
aquilus ´ from north-eastern Queretaro (Ct164) and C. black dots represent strongly supported nodes (‡ 95% posterior probability. Ct267 Crotalus sp. The branch drawn in grey is not to scale. MEX Ct268 Crotalus sp. White dots additionally indicate 12 strongly supported (with one exception) geographically cohesive subgroups clustered within major phylogroups and used in diversiﬁcation rate analyses.221 klauberi DUR Ct35 lepidus TX Ct140. maximum likelihood bootstrap below). The single divergent samples of C. Numbers at nodes indicate support values (Bayesian posterior probability above.05 sub. Roman numerals indicate major phylogroups nested within major lineages.
.34 aquilus HID Ct27 aquilus HID Ct122 aquilus HID Ct283 maculosus NAY I Ct118 klauberi ZAC Ct119 klauberi ZAC Ct116 klauberi AGS Ct201 lepidus x aquilus AGS Ct117 klauberi AGS II Ct227 klauberi ZAC Ct228 klauberi ZAC Ct143 klauberi JAL Ct195 klauberi JAL Ct12.147 pusillus MICH I Ct145 pusillus MICH Ct208 pusillus JAL pusillus R42 pusillus MICH II Ct17 pusillus MICH Ct18 armstrongi MICH Ct172 triseriatus PUE Ct155 triseriatus PUE Ct157 triseriatus PUE I Ct238 triseratus VER Ct242 triseratus VER Ct255 triseriatus MICH Ct262 triseriatus MICH triseriatus Ct263 triseriatus MICH Ct230 triseriatus PUE Ct245 triseratius PUE II Ct1 triseriatus MEX R57 triseriatus DF Ct6 triseriatus MEX Ct4 triseriatus MEX Ct21 triseriatus DF
Ct216 morulus TAM Ct217 morulus TAM Ct223 morulus TAM I Ct31 morulus TAM Ct9 morulus TAM Ct10 morulus TAM Ct127 morulus NL morulus Ct26 morulus NL Ct133 morulus NL Ct215 morulus NL II Ct252 morulus COAH Ct20 morulus NL Ct11 morulus NL Ct25 morulus NL Ct164 aquilus QTO I Ct126 aquilus QTO Ct261 aquilus MEX Ct269 aquilus MEX Ct124 aquilus QTO Ct2 16 aquilus AGS II Ct239 aquilus GTO Ct241 aquilus GTO Ct209 aquilus MICH Ct224 aquilus MICH Ct3 aquilus QTO aquilus Ct160 lepidus x aquilus SLP Ct165 aquilus SLP III R55 aquilus SLP Ct30 aquilus SLP Ct197 lepidus x aquilus SLP Ct15 aquilus HID Ct266 aquilus HID Ct123 aquilus HID Ct14 aquilus HID IV Ct33.158 Crotalus sp.Journal of Biogeography 38. 70% bootstrap). MICH Ct264 Crotalus sp. miliarius Ct150 exiguus GRO Ct149 exiguus GRO I Ct151 exiguus GRO Ct152 brunneus OAX II Ct168 brunneus OAX Ct153 ravus MEX ravus Ct225 ravus PUE Ct204 ravus PUE Ct139 ravus PUE III R44 ravus PUE Ct199 ravus TLAX Ct154 ravus PUE Ct211 ravus VER Ct135 armstrongi JAL I Ct137. miliarus were used as outgroup taxa.138 armstrongi COL Ct259 armstrongi MICH Ct254 armstrongi MICH II Ct257 armstrongi MICH armstrongi Ct136 armstrongi JAL Ct193 armstrongi JAL III Ct233 armstrongi JAL Ct256 armstrongi JAL Ct121. catenatus
0. lepidus maculosus from Nayarit (Ct238) are labelled as phylogroups for simplicity. Sistrurus catenatus and S. MICH Crotalus sp. 697–710 ª 2010 Blackwell Publishing Ltd
S. MEX Ct144.13 maculosus DUR Ct142 maculosus DUR III Ct237 maculosus SIN Ct32 klauberi DUR IV Ct220.141 lepidus COAH Ct24 lepidus TX V Ct112 lepidus TX lepidus Ct59 lepidus NM Ct161 klauberi DUR Ct162 klauberi DUR Ct250 klauberi DUR Ct29 klauberi CHIH 88 R21 klauberi CHIH Ct8 klauberi CHIH 92 Ct222 klauberi CHIH Ct226 klauberi SON Ct41 klauberi AZ Ct55 klauberi AZ VI Ct99 klauberi AZ Ct36 klauberi TX Ct39 klauberi NM Ct61 klauberi NM Ct53 klauberi NM Ct46 klauberi NM Ct7 klauberi CHIH Ct163 klauberi CHIH Ct23 klauberi CHIH
Phylogeography of the Crotalus triseriatus group
Figure 2 Phylogenetic estimate for the Crotalus triseriatus species group based on mixed-model Bayesian inference (tree shown) and maximum likelihood analyses of mitochondrial DNA sequence data. / site
Wuster et al. Two lineages represented the subspecies C. we used two sets of dates estimated in beast for diversiﬁcation-rate analyses.. C.. Because underestimates of genetic diversity potentially bias inferred rates of diversiﬁcation (Esselstyn et al.5 Ma (Holman.R. 2006b). 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. W.01. The second set included estimated diversiﬁcation dates between an additional 12 geographically cohesive monophyletic subgroups clustered within several of the major phylogroups. The node representing the most recent common ancestor (MRCA) of A. and references therein). 2002. Strong phylogeographical structure was present within most of these taxa (Fig. ruber-atrox clade was given a lognormal mean of 1. 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.5 Ma (Carreno & Helenes. and references therein). The node representing the MRCA of the C. 1989. a lognormal mean of 0. These lognormal distributions with hard lower bounds best reﬂect the prediction. 2002. The ﬁrst set consisted of estimated divergence dates between the major phylogroups (Fig. and this was a more liberal approach. Murphy et al.76. 2009). and a lognormal standard deviation of 0. pusillus triseriatus aquilus morulus lepidus
Murphy et al. RESULTS Sequence characteristics and phylogenetic estimate The ﬁnal dataset used for phylogenetic inference consisted of 2408 aligned nucleotide positions. All sequences were deposited in GenBank (Appendix S2). and a lognormal standard deviation of 0. 2009). 2000). ATPase 6. Model ﬁt was measured using AIC scores. 2000). atrox (Castoe et al. 2006
Figure 3 Comparison of phylogenetic relationships for the Crotalus triseriatus species group based on previous molecular studies (a. the geographical distribution of subclades was consistent with recognized subspecies (C. 3.3 (Rabosky. 2004). 2009) due to the Pliocene ´ ˜ marine incursion of the Sea of Cortes (Carreno & Helenes. C. b) and this study (c). ravus. Bryson Jr et al. in previous studies. r. C. The stem of Sistrurus (Guiher & ¨ Burbrink. which we considered to be a conservative approach.. armstrongi and C. Differences are in part due to new lineages inferred from our expanded taxonomic and geographical sampling (dotted lines). Relationships among lineages were generally congruent with those of previous molecular studies (Fig. contortrix was given a zero offset of 6 Ma. ruber and C.42. ravus. These smaller subgroups represented the ﬁnest division of phylogeographical diversity that could be reasonably inferred from our study. 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. based on the high likelihood of fossil nonpreservation.
Journal of Biogeography 38. t. Temporal patterns of diversiﬁcation We analysed temporal shifts in diversiﬁcation rates using MLbased diversiﬁcation-rate analysis (Rabosky. lepidus (Campbell & Lamar. that any true divergence date will probably be older than the oldest known fossil. producing a median age of 7 Ma and a 95% PCI extending to the start of the Late Hemphillian at 8 Ma (Holman. l.. 2). 2008. 2006a). ravus. ﬁve of which corresponded to the species C.. 2). 2002
Castoe & Parkinson. C. 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 purebirth model. triseriatus. C. One lineage represented an undescribed taxon.. brunneus and C. In C. a lognormal mean of 0. 2008) was constrained with a zero offset (hard upper bound) of 8 million years ago (Ma).1 and a lognormal standard deviation of 0.
ravus other Crotalus pusillus lepidus triseriatus aquilus triseriatus (LG) *
ravus pusillus triseriatus lepidus aquilus triseriatus (LG) *
ravus armstrongi Crotalus sp. and two-rate purebirth) was computed with laser 2. triseriatus (labelled with an asterisk). of a mislabelled sample of C. 2009. ND4. triseriatus group (Figs 2 & 3). r. and a 95% PCI extending to the beginning of the development ´ ˜ of the Sea of Cortes at 5. aquilus and C. and the use. 12S) and HKY+I+G (tRNAs and 16S).01. We identiﬁed eight major maternal lineages within the C.37. 697–710 ª 2010 Blackwell Publishing Ltd
. exiguus). 2002. morulus. and birth–death) and three variable rates (exponential and logistic density-dependent. rather than younger (Ho & Phillips. Castoe & Parkinson. Kelly et al. 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. r. Models of sequence evolution selected for the mtDNA partitions were GTR+I+G (ATPase 8. pusillus. The ﬁt of different birth–death models implementing two constant rates (pure-birth. No zero offset was used.
09 diversiﬁcation events per million years.1) for the liberal dataset. Arrows denote the placement of fossil calibrations detailed in the text. piscivorous A.53 events per million years.45 diversiﬁcation events per million years. Values at nodes represent mean divergence dates between major lineages. Phylogeographical structure within these lineages appeared to have been generated primarily during the Pleistocene.16 aquilus AGS Ct30 aquilus SLP Ct3 aquilus QTO Ct122 aquilus HID Ct15 aquilus HID Ct283 maculosus NAY Ct195 klauberi JAL Ct118 klauberi ZAC Ct12. The LTT plots suggested either a constant rate of diversiﬁcation or a decline in the Pleistocene (Fig. ruber Ct150 exiguus GRO Ct149 exiguus GRO Ct168 brunneus OAX Ct152 brunneus OAX Ct153 ravus MEX Ct204 ravus PUE Ct135 armstrongi JAL Ct137.Phylogeography of the Crotalus triseriatus group 2006).4
3. the null hypothesis of rate-constancy was rejected (P = 0. suggesting the rate-variable yule2rate model provided a better ﬁt. Dating estimates suggested that diversiﬁcation in the C.3
4.13 maculosus DUR Ct32 klauberi DUR Ct140.1
3.02 Ma.9 2.04). contortrix S. A change in net diversiﬁcation rate took place 1. 5). with bars indicating 95% highest posterior densities. Thus. According to the scenario suggested by this model. the null hypothesis of rate-constancy was not rejected (P = 0. when the rate shifted dramatically to 0. P-values for the change in AIC scores between the two models differed between datasets. MICH Ct268 Crotalus sp. Divergence times and tempo of diversiﬁcation The GTR+I+G model of sequence evolution was selected for the beast analyses.3
5. 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.147 pusillus MICH Ct145 pusillus MICH Ct17 pusillus MICH Ct208 pusillus JAL Ct155 triseriatus PUE Ct242 triseratus VER Ct245 triseratius PUE Ct262 triseriatus MICH Ct216 morulus TAM Ct9 morulus TAM Ct11 morulus NL Ct26 morulus NL Ct164 aquilus QTO Ct261 aquilus MEX Ct2. depending on the scale of phylogeographical diversity recognized by each dataset. the C. The ML estimate of the diversiﬁcation rate under the best rate-constant pureBirth model was 0.138 armstrongi COL Ct259 armstrongi MICH Ct254 armstrongi MICH Ct233 armstrongi JAL Ct193 armstrongi JAL Ct264 Crotalus sp. I II I II triseriatus pusillus armstrongi ravus
II I morulus
III IV I II III IV V VI lepidus aquilus
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. For the conserved dataset. triseriatus group had an initial net diversiﬁcation rate of 0. miliarius C.
6. and the prior use of a misidentiﬁed sample (see below). atrox C. 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).4
Gloydius A. MEX Ct144. 4).1 4. and divergences between most major lineages occurred in the Late Miocene and Pliocene (Fig. 697–710 ª 2010 Blackwell Publishing Ltd
. catenatus S. Differences were largely attributable to our expanded taxonomic and geographic sampling. In contrast.
Journal of Biogeography 38. a strong signal of diversiﬁcation rate change was not indicated after accounting for ﬁne-scaled phylogeographical diversity.141 lepidus COAH Ct59 lepidus NM Ct161 klauberi DUR Ct29 klauberi CHIH Ct46 klauberi NM Ct226 klauberi SON Ct55 klauberi AZ
I II III I II III Crotalus sp. However. triseriatus group probably began in the Late Miocene.
. The divergence of C. 2009. 1993.5
3.02 million years ago. C. low elevation breaks (Fig.
Log-Lineages Through Time
3. insects: Masta. such as the Rio Panuco basin (AnduchoReyes et al. Both Neogene vicariance and Quaternary climate change had comparably strong effects on driving diversiﬁcation rates.0 2. t. or desert-grassland. 1997. RodrıguezBanderas et al.. l. Five of the eight major lineages (C.. 1990. aquilus and C.0 r1 = 0. C. and estimated divergence dates between these lineages fell within the orogenic timeframe ´ for the area (Ferrusquıa-Villafranca. Paternally mediated gene ﬂow may occur between seemingly isolated populations. 2008.5
2. st = time of diversiﬁcation rate shift from yule2rate model estimates. glacial climatic cycles probably contributed to the fragmentation of Mexican pine–oak forests and may have driven divergences. Anducho-Reyes et al.5 r2 = 0. 6). unless the dispersed male is sampled. 2005. t. aquilus from C. mesquite-. This inference was broadly congruent with diversiﬁcation patterns seen in several Middle American highland pitvipers (Castoe et al. Uplift of the Central Mexican Plateau coupled with the subsequent aridiﬁcation and ﬁnal Pliocene development of the Chihuahuan Desert (Jaeger et al. Bryson Jr et al. Leon-Paniagua et al.45
Figure 5 Lineage through time plots derived from Bayesian relaxed clock estimates of divergence dates within the Crotalus triseriatus species group.. Male rattlesnakes can disperse relatively long distances between populations during the breeding season (e.. Table 1)..5
Log Lineages 2. 2009) and other montane ´ ˜ taxa (pines: Moreno-Letelier & Pinero. 2008). 2004.. 2008. l. Whereas some phylogroups appear to be separated by well deﬁned. ´ 2007.
Journal of Biogeography 38. Early divergences were temporally and geographically congruent with the formation of the Transvolcanic Belt. r = diversiﬁcation rate. triseriatus group occurred over the last six million years. morulus to the east from western populations of C.) were distributed on or near the Transvolcanic Belt. might not reﬂect isolated gene ﬂow. King & Duvall. small mammals: Sullivan ´ et al. morulus). McCormack et al. Puebla-Olivares et al. Becerra. 2009.0
1. l. Our results revealed strong geographical partitioning of genetic diversity within nearly all lineages. 2008). suggesting that the development of this mountain range played a critical role in early diversiﬁcation of this widespread highland group. Thus. lizards: Zaldivar-Riveron et al. triseriatus group Our analyses indicated that a progressive diversiﬁcation of the C.09 3. Observed phylogeographical patterns in the C. 2000. Edwards & Bradley. 2005). triseriatus. armstrongi. aquilus).53 2. 1977. 697–710 ª 2010 Blackwell Publishing Ltd
.g. 2008) and Cerritos-Arista and Saladan Filter Barriers (Morafka. however. Most of this structure appeared to have developed during the Quaternary. W. Clark et al. and male-biased dispersal cannot be accounted for in a mtDNA gene tree..5 st = 1.. lepidus)... C.0
1. The remaining. lepidus. 2007) (Fig.0
r1 = 0.. and references therein) may have separated C. 6. morulus along the Sierra Madre Oriental may have been facilitated by the development of any one of several hypoth704
´ esized ﬁlter barriers.0
1. triseriatus group. (a) Diversiﬁcation rates for inferred lineages and major phylogroups suggest a diversiﬁcation rate shift 1. 2005.R. more northerly three lineages occupied the Central Mexican Plateau and southern Sierra Madre Oriental (C. birds: Garcıa-Moreno et al.5
6 0 Time From Basal Divergence (million years ago)
1.02 3. pusillus and Crotalus sp. northern Sierra Madre Oriental (C. ravus.
DISCUSSION Evolutionary drivers of diversity within the C. (b) Diversiﬁcation rates for inferred lineages and all possible phylogroups suggest a constant diversiﬁcation rate through time. Smith & Farrell. Bryson et al.. 2008b. 2005. Tennessen & ´ Zamudio. and Sierra Madre Occidental (western C. others may be marginally separated by midelevation swathes of oak-. 2002.
(a) Generalized areas of major mountain ranges in Mexico: SMOcc. C.
Journal of Biogeography 38. Sierra Madre Oriental. (10) Colima and Tepic-Zocoalco Grabens (rattlesnakes in this study). Future testing with nuclear gene markers is needed to elucidate patterns of gene ﬂow. (b) Biogeographical barriers to highland Mexican taxa (see Table 2): (1) Balsas Basin (including the Tepalcatepec Depression). Leon-Paniagua et al. 2008a] and ´ (12) unnamed barrier [spiny mice (Habromys). armstrongi I/II+III C. C. l. (6) Rio Mezquital ´ basin. Sierra Madre Occidental. and their thickness corresponds to the number of taxa exhibiting genetic break (thick = two or more taxa. ravus II/III C. Numbered lines span approximate locations of barriers. Rio Papaloapan basin (Campbell & Armstrong. triseriatus I/II l. t. C. 697–710 ª 2010 Blackwell Publishing Ltd
Phylogeographical diversity in the Mexican highlands Despite an overall paucity of phylogeographical studies of Mexican highland taxa. (2) Rio Verde basin. The Central Mexican Plateau (CMP) is also shown. C. C. C.
Phylogroup division C.
Table 1 Potential isolating barriers between major phylogroups within the Crotalus triseriatus species group inferred in this study. SMOr. Transvolcanic Belt. TVB. Jalisco to Jalisco/Colima border) Colima and Tepic-Zocoalco Grabens Rio Ahuijullo basin. (5) Lerma-Santiago Basin. Geographical barriers are shown in Fig. C. C. SMdS. Sierra Madre del Sur.g. morulus I/II aquilus I/II+III+IV aquilus II/III+IV aquilus III/IV lepidus I/II lepidus I+II/III+IV+V+VI lepidus III+IV+V/VI lepidus III/IV+V lepidus IV/V Potential isolating barriers Balsas Basin. (11) Aguascalientes Graben [Mexican jays (Aphelocoma ultramarina). lepidus. C. (8) Rio Panuco basin. C. (9) Rio Ahuijullo basin. Tepalcatepec Depression (Campbell & Lamar. ravus I/II+III C. morulus) are not obvious. t. emerging patterns suggest mixed 705
. C. (3) Rio Papaloapan basin. (7) Cerritos-Arista and Saladan Filter Barriers. 2004) of the Balsas Basin Oriental Basin Unknown ´ Unknown (potentially mid-elevations <1800 m surrounding the isolated ‘sky island’ in north-eastern Queretaro ´ and adjacent San Luis Potosı) Unknown ´ Rio Panuco basin Tributaries of the Rio Santiago basin Rio Mezquital basin Unknown Unknown (potentially high elevation >2700 m ridges along crest of the Sierra Madre Occidental) Unknown
Boundaries within some northern phylogroups (e. 2007]. Phylogroups follow those in Fig.Phylogeography of the Crotalus triseriatus group
Figure 6 Map of Mexico depicting pine–oak forests above 1800 m elevation. Rio Verde basin Balsas Basin.. thin = one taxon).. 2. 6. armstrongi II/III pusillus I/II t. C. McCormack et al. (4) Oriental Basin. 1979) Unknown (potentially mid-elevations <1800 m extending south-southeast of Ameca. western C.
In southern Mexico. Mexican jays (Aphelocoma ultramarina)10 Montane rattlesnakes (Crotalus triseriatus group)1. some geographical barriers. in part. 2000. Table 2). deer mice (Peromyscus aztecus species group)*2.. bark beetle (Dendroctonus mexicanus)3 Montane rattlesnakes (Crotalus triseriatus group)1. The Rio Verde additionally divides the Sierra Madre del Sur in western Oaxaca. (2002). ravus in the C. Rio Tepalcatepec.. 2008) are also broadly attributable to the formation of the Transvolcanic Belt. Rio Atoyac) that form the Balsas Basin. Mexican kingsnakes (Lampropeltis mexicana species group)11 Montane rattlesnakes (Crotalus triseriatus group)1. Van Devender. Matrilineal genealogy of the C. bark beetle (Dendroctonus mexicanus)3 Montane rattlesnakes (Crotalus triseriatus species group)1. However. The ancient development of most of the major mountains in Mexico probably pre-dates diversiﬁcation of the extant highland species. Demastes et al. 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. Neotropical brush-ﬁnches (Buarremon)*4. These largely overlooked barriers are probably responsible for disrupting Pleistocene pine–oak forest corridors (Demastes et al. ´ ´ ˜ 2008. 10McCormack et al. 2Sullivan et al.. deer mice (Peromyscus aztecus species group)*2.. ¨ Navarro-Siguenza et al.
Table 2 Biographical barriers shared by co-distributed Mexican highland taxa. 2004.
Journal of Biogeography 38. triseriatus group. 5Edwards & Bradley. 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. Bryson Jr et al. In the C. spiny mice (Habromys)*8 Montane rattlesnakes (Crotalus triseriatus species group)1. The Rio Ahuijullo isolates these areas to the west. such as between phylogroups of C.. triseriatus group. may have served as ﬁlter barriers. 1997. aquilus. 6. Murphy et al. we inferred several novel historical relationships for the C. W. 2002. the Rio ´ Panuco bisects the Sierra Madre Oriental. southwestern white pine (Pinus strobiformis)9. Further west. 1990). birds: McCormack et al. the Sierra Madre del Sur is isolated from the Transvolcanic Belt to the north by the Rio Balsas and its associated tributaries (e. the Rios Lerma and Rio Santiago ﬂank the southern Sierra Madre Occidental and northern Transvolcanic Belt and separate these two highlands. 2008. 2007.. deer mice (Peromyscus aztecus species group)2. further explain inferred historical disjunctions between highland taxa distributed across the Central Mexican Plateau.g. 9Moreno-Letelier & Pinero. ravus (McCranie. Plesiomorphic morphological attributes shared with Sistrurus have seemingly confounded the phylogenetic placement of C. However. The Tepalcatepec Depression was included as a western branch of the Balsas Basin. In concert. 2008.. 2008a.. 7Garcıa-Moreno et al. Further north. and references therein).R.. 4Navarro-Siguenza et al. Paniagua & Morrone. emerald toucanet (Aulacorhynchus prasinus species group)*6. Mexican woodrats (Neotoma mexicana species group)*5 Montane rattlesnakes (Crotalus triseriatus species group)1. 2002) and could. Mexican woodrats (Neotoma mexicana species group)*5. Historical ﬁlter barriers across the Central Mexican Plateau may also include large interconnected palaeo-lakes ´ (Mejıa-Madrid et al. 2007.. Mexican jays (Aphelocoma ultramarina)*10. bark beetle (Dendroctonus mexicanus)3. as evidenced by shared genetic breaks in co-distributed taxa (Fig. ´ Leon-Paniagua et al. 8Leon-Paniagua et al. 2008a. Neotropical brush-ﬁnches (Buarremon)*4. and several phylogroups are embedded within this mountain range.. 3Anducho-Reyes et al.. initial diversiﬁcation appears to be tightly linked to the uplifting of the Transvolcanic Belt. 6Puebla-Olivares et al.. The topology strongly supported the placement of 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. bark beetle (Dendroctonus mexicanus)3 Bark beetle (Dendroctonus mexicanus)3. common bush-tanager (Chlorospingus ophthalmicus)*7 Montane rattlesnakes (Crotalus triseriatus species group)1. emerald toucanet (Aulacorhynchus prasinus species group)6. To the north. 2009. 697–710 ª 2010 Blackwell Publishing Ltd
. 2007. 2007..
Biogeographical barrier (1) Balsas Basin Taxon Montane rattlesnakes (Crotalus triseriatus species group)1. responses to past geological and climatic events despite a presumed shared history in the same region (Sullivan et al. 1997. and not directly stated in the original study. Some pine–oak expansions probably resulted in ephemerally contiguous highland biotas in the Mexican sierras (Toledo. 1988. such as major river drainages. Neotropical brush-ﬁnches (Buarremon)*4
(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
¨ This study. Further. Numbers correspond to barriers shown in Fig. Asterisks denote barriers inferred from the phylogeny or delineated on maps. 1982. 2002. the Sierra Madre del Sur is separated from the Transvolcanic Belt and Sierra Madre Oriental by the Rio Papaloapan basin across northern Oaxaca.. 11Bryson et al. 2002). the Rio Mezquital bisects the Sierra Madre Occidental. Edwards & Bradley. triseriatus group Utilizing expanded taxonomic and geographical sampling and phylogenetic mixed-model analyses of mtDNA. 6. the Neogene formation of the Transvolcanic Belt is undoubtedly a major force driving the evolutionary history of these taxa.. 2002. 2009). Deep divergences and high phylogeographical diversity in other co-distributed highland taxa (small mammals: Sullivan et al. common bush-tanager (Chlorospingus ophthalmicus)*7.
armstrongi and C. lepidus. but divergences within this group appeared to be relatively recent (Fig. l. J. aquilus and C. D. 49.R. F. 707
. l. J. Mueller. Strong support was obtained for a sister relationship between C. B. KS. Jr (1994) Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosome races of the Sceloporus grammicus complex (Phrynosomatidae) in central Mexico. R. 43. ´ Queen. U. 4)..F. 1–3. triseriatus LG’ (ROM 18114. l. l. Franklin and E. curators and institutions for providing or assisting with tissue samples: D. (2009) Funds enough. maculosus) together formed a monophyletic group. 1992). 2002. M. Armstrong. A.W.K. & Whitlock.R. AF259087. However. which we determined to be C. Torocco.. Setser and G. Collecting was conducted under permits granted by SEMARNAT to R. t. Peterson. M. lepidus in the west (C. lepidus (Dorcas. MacCulloch (Royal Ontario Museum).W. D.. and funded by the Natural Sciences and Engineering Research Council of Canada Discovery Grant A3148. E. F. 10919–10923.. 529–540. Nieto-Montes (Universidad Nacional ´ ´ Autonoma de Mexico). G.O. Romero. lepidus. Swinford.M. lepidus. & Zink. aquilus to other C. t. labelled as ‘C.R. We further thank M. Two anonymous referees and S. l. Michoacan).. l. triseriatus group except for C. REFERENCES Anducho-Reyes. Several studies suggest that multiple. Systematic Biology.Phylogeography of the Crotalus triseriatus group We inferred a novel placement for C. Molecular Phylogenetics and Evolution. Proches provided ¸ ¸ comments that greatly improved the ﬁnal version of this manuscript.D. 729–744. Crotalus aquilus has oscillated between being considered as a subspecies of C. K.. Becerra. R. While phylogenetic inferences based on one marker (e. (2005) Timing the origin and expansion of the Mexican tropical dry forests. t. 2008. J.J. morulus. P. ´ Arellano. Price. Samples of Crotalus obtained along the central portion of the Transvolcanic Belt (Figs 1 & 2) provide clear support for this prediction.T. 2004) yet it is much more likely to detect geographical limits and cryptic species than studies based on nuclear DNA gene sequences (Moore. 2004).P. It formed the sister clade to all other species of the C.. Zink & Barrowclough. Barrowclough & Zink.. We thank the following people. S.A.T. Quintero-Dıaz. & Murphy. klauberi and C. ¨ C. We thank the numerous people who assisted in the ﬁeld. this association was based on a misidentiﬁed sample. Narvaez and J. Mackessy. D. L. B. AF259161. Ulises de la Rosa-Lozano. Morafka and F. & Sites.L. 1979. 13. Jones. Molecular Phylogenetics and Evolution. Campbell.W. J. Flores-Villela and A.V. Cognato.. aquilus.R.X. Frost (American Museum of Natural History).B. aquilus than to other lineages of C. Smith and A. 2002. G. Gonzalez-Cozatl. 5 of the Museum of Natural History University of Kansas. 2003. Hudson & Coyne. Mendoza-Quijano. J. Beltran-Sanchez. lepidus was supported. (2004) The incomplete natural history of mitochondria.L. 387–418. Davis.L. morulus may be more closely related to C. M. Dixon. 18. Garcıa-Padilla ´ and E. Special Publication No. R. Canseco-Marquez. maternally inherited data can lead to signiﬁcant biogeographical discoveries (e. triseriatus (Campbell & Lamar. this study would not have been possible. Ballard & Whitlock. triseriatus (Campbell.g. 102. Recent molecular studies (Murphy et al. J. G.M. G. A. B. 1992). Riddle et al. R.A. Mendoza-Paz.C. Although historically considered to be C.. geographically isolated species may be represented under the name C. 1997. armstrongi near the base of the tree and distant from C. mtDNA) should be interpreted with caution. nuDNA and the discovery of divergence. Egan assisted with use of laser and R. Hartman. E. t.A. (2008) Phylogeography of the bark beetle Dendroctonus mexicanus Hopkins (Coleoptera: Curculionidae: Scolytinae). Molecular Ecology. Garcıa-Vazquez. C. J. triseriatus group taxa have been contentious (see Gutberlet & Harvey. including S. Alvarado-Dıaz and ˜ A. ACKNOWLEDGEMENTS We dedicate this study to the late Fernando Mendoza-Quijano.S. Other subspecies of C. M. & Zuniga. t.B. Graham. in contrast to strong morphological support for a monophyletic C. Future studies using unlinked nuclear loci can test whether or not the genealogy also reﬂects the macroevolutionary species phylogeny. & Rogers. J. J. 930–940. A. aquilus and C. Molecular Ecology. (2005) Molecular systematics of Middle American harvest mice Reithrodontomys (Muridae). 2009). J. 697–710 ª 2010 Blackwell Publishing Ltd
years. AF259124. morulus sister relationship suggested that C.A. (1979) The natural history of Mexican rattlesnakes. 2004). C. E. Campbell for other assistance that greatly improved this project. Kansas. Ponce-Campos. O.W. Kardon (San Antonio ´ ´ Zoo). Hayes. J. and this clade was sister to all other C. All sequencing work was conducted in the laboratory of R. 2002. lepidus.N. Barrowclough. Murphy & Aguirre´ Leon. l. The phylogenetic afﬁnities of C. Only the monophyly of C. and time: mtDNA. Gutberlet & Harvey. D. R. AF259199). 2004). 37. Lawrence.. Proceedings of the National Academy of Sciences USA. these genetically distinctsnakesarelikelytorepresentanundescribedcrypticspecies. Dorcas. Castoe & Parkinson. Ballard. Without his enthusiasm and unfailing support through the
Journal of Biogeography 38. An mtDNA-only approach has several limitations (Funk & Omland.I.X.. Mocino-Deloya.O. ´ Smith (University of Texas at Arlington). 2002). Upton & Murphy. ravus. triseriatus. GenBank numbers AF259231. ´ Arevalo. triseriatus (as originally described) and a distinct species closely related to C. Lemos˜ Espinal. Smith. Our genealogical inferences serve as a robust hypothesis of matrilineal relationships within the C.g.W. 1995. 1979. estimated from mitochondrial cytochrome b gene sequences. C. Our ﬁnding of a C. E. Banda-Leal. triseriatus species group. J. Harrison. Quijada-Mascarenas (Universidad Michoacana de San ´ ´ Nicolas de Hidalgo. This ﬁnding contradicted previous suggestions based on morphological evidence of a close relationship between C. 2000. 2006) proposed a sister relationship between C. triseriatus.M. ´ K. triseriatus (Armstrong & Murphy. Gruenwald. Murphy et al. aquilus–C.
(2004) The evolution of New World venomous snakes. & Demastes. L. F.E.M. & Bull.T. pp.R.W. (2005) Northern Mexico’s landscape. Molecular Phylogenetics and Evolution. (2006) Evolution of rattlesnakes (Viperidae: Crotalus) in warm deserts of western North America shaped by Neogene vicariance and Quaternary climate change.. by T. Timm. Fa). Castoe. (2006) Bayesian mixed models and the phylogeny of pitvipers (Serpentes: Viperidae). Cornell University Press.. F. (1979) A new rattlesnake (Reptilia. Hafner. Navarro-Siguenza. (2004) Genetic variation coincides with geographic structure in the common bush-tanager (Chlorospingus ophthalmicus) complex from Mexico. Spradling. & Armstrong.A.V. J. 185–268.M.. 697–710 ª 2010 Blackwell Publishing Ltd
. T.. Drummond. 43. 106–119.W. & Gonzalez-Guzman. T. Biological diversity of Mexico: origins and distribution (ed. 1557–1565. J. C. 86. D. & Zamudio. (1979) Geographic variation in the Mexican pygmy rattlesnake. 36.T. 144–154. M. 2595–2610. & Reed.F. (2002) Mathematical consequences of the genealogical species concept. M. (1964) Evolution of the pit vipers. Lot and J. Ceballos and R. Pastorini. Castoe.. (2007) BEAST: Bayesian evolutionary analysis by sampling trees.-L. W. R. J. Nucleic Acids Symposium Series. Transactions of the Kansas Academy of Science. Annual Review of Ecology and Systematics.E. A. B.. Douglas. (2007) A phylogeny of the Lampropeltis mexicana complex (Serpentes: Colubridae) based on mitochondrial DNA sequences suggests evidence for species-level polyphyly within Lampropeltis. Sistrurus ravus. & Burbrink. 95–98. 1931. M. Herpetologica. 63. causes. & Coyne.T. (1993) An empirical test of bootstrapping as a method for assessing conﬁdence in phylogenetic analysis. R. T. ˜ Carreno. Case. 365–370. (1940) The rattlesnakes. NY. Ithaca. ´ ¨ Garcıa-Moreno.L. part II: the biotic setting across time. D. & Bradley. Chippindale. P. Hall. A study in zoogeography and evolution. 4.K.A. D. and consequences. U. & Omland. and conservation in northern Mexico (ed. Brant. Brown. D. K. 22. Systematic Biology. Cornell University Press. 304–317. Molecular Phylogenetics and Evolution. Evolution. 56. Journal of Mammalogy. Molecular Phylogenetics and Evolution. (2000) Phylogeography of Peromyscus furvus (Rodentia. Oxford University Press. J. by T.J.M. (2009) Comparative phylogeography of pitvipers suggests a consensus of ancient Middle American highland biogeography. Jr & Harvey. J.I. by J. Edwards.D..A. Brattstrom. (2003) Species-level paraphyly: frequency. pp.L.W.S.L. C. genera Sistrurus and Crotalus.A.W. 39. B. Indianapolis. Campbell. J. & Phillips.A.J. L. 34.J. A new island biogeography of the Sea of Cortes (ed. E.A. Ramamoorthy. 71–88. pp. Molecular Ecology. Mexico. IN. L. A. D. Molecular Phylogenetics and Evolution.J.W. I. 543–553. Riddle.T. (2002) Systematics and phylogeography of pocket gophers in the genera Cratogeomys and Pappogeomys. & Forstner. by J. J. Viperidae) from Jalisco..H. A. Hafner.L. 20–30. Funk. Sasa. J. T.. 17. Felger). Holman.. & Porras. J. Dunn. & Parkinson. 1–270. evolution. Dorcas. Clark. J.Y. R. G. (2002) Molecular systematics and historical phylobiogeography of the Neotoma mexicana species group. Burbrink.N.L. M. with insights from animal mitochondrial DNA. T. Douglas. New York.. A. Lamar). ´ ´ ´ Ferrusquıa-Villafranca. W. T. Bryson. M. ecosystems. Stechert.J. Molecular Phylogenetics and Evolution.D. NY. Oxford University Press.. 39–51.A. S. K. J. & ´ ´ Sanchez-Gonzalez. Gloyd. 35. by J. Brodie Jr). Selva. 83.W.W. Campbell.A.M. Demastes. & Helenes. & Lamar.W. W. H. Campbell and W. Molecular Ecology. paleoecology. R. Copeia. 674–684. J. G.. Ho. MA. Ithaca. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. 367–380. Campbell and E. A. Tyler. 15. pp. (2004) Inferring phylogenies..B. J.. 81.E. (2008) Integrating individual behaviour and landscape genetics: the population structure of timber rattlesnake hibernacula.. Sunderland. NY. Special Publication of the Chicago Academy of Sciences. ´ Ferrusquıa-Villafranca. 58. 48. Sinauer Associates. (2009) Accounting for calibration uncertainty in phylogenetic estimation of evolutionary divergence times. B. M. Oxford. (1992) Relationships among montane populations of Crotalus lepidus and Crotalus triseriatus. & Bradford. TX.A.. 91–110. Transactions of the San Diego Society of Natural History. R. The venomous reptiles of the Western Hemisphere (ed. Spradling. Felsenstein. distribution.W. Jaeger. 13. (2009) Do geological or climatic processes drive speciation in dynamic 708 archipelagos? The tempo and mode of diversiﬁcation in SE Asian shrews.R. D. Peterson. 41. I. Oxford University Press. & Lamar. (1993) Geology of Mexico: a synopsis. (1931) The herpetological fauna of the Americas. Harris. with the description of a new subspecies. Cornell University Press. 186–196. Systematic Biology.R.J. Daza.M.R. Hudson.A. (2008) Demographic and phylogeographic histories of two venomous North American snakes of the genus Agkistrodon. C. & Rambaut. 88–103.. J. Cartron. 3–107.A.G. Esselstyn. 33.. & Parkinson. S. 214. New York. Molecular Ecology. Journal of Biogeography. Hafner. Indiana University Press. W. BMC Evolutionary Biology. Biology of the pitvipers (ed. Light.E. 7. J. Ezcurra). & Brown. Cratogeomys merriami.R. (2004) Venomous reptiles of the Western Hemisphere. M. (2005) Cryptic species in the Mexican pocket gopher. J. R. 14–40. R. M. 2129–2135. D.. (2005) Cryptic Neogene vicariance and Quaternary dispersal of the
Journal of Biogeography 38.R. 397–423.. 3353–3374. J. & Sullivan.A. Muridae) based on cytochrome b sequence data. D. J.L.A.E.R. Evolution. 719–730.R.. Cody and E.J. M. (2000) Fossil snakes of North America: origin. Kuch..S. Hafner. (1989) The venomous reptiles of Latin America. Bye. Serpentes.A. 9. 182–192. Bryson Jr et al. pp. A.. M.. Campbell. 42.A.J. Campbell. Ithaca.A. Hillis.L. Smith. Schuett.S. Rogers. J.S.S. Biodiversity. Gutberlet.P. Guiher. (2002) Geology and ages of the ´ islands. 1095–1108. R. 634–682. E. R.A. Journal of Mammalogy.J. Campbell.
across the Chihuahuan Desert ﬁlter-barrier. N.ed. 47. A.B. V. & Holman. Peterson.. Biogeographica. Flores-Villela. Molecular Ecology. 2nd edn. L. Douglas and H. P. 21– 35. R. 39. Biological diversity in Mexico: origins and distribution (ed.. Garcıa´ Deras... 33–38. 1699–1711. Available at: http://beast. Uppsala. Molecular Ecology.T. 3033–3048. Puebla-Olivares. & Perez-Ponce de ´n. A.M. (2008a) Speciation in the highlands of Mexico: genetic and phenotypic divergence in the Mexican jay (Aphelocoma ultramarina). Journal of Herpetology.A. Bonaccorso. Evolution. M.. M. R. Moore.M. (2000) Phylogeography of the jumping spider Habronattus pugillis (Araneae: Salticidae): recent vicariance of sky island populations? Evolution.. Klauber. J. J. 125. 787–801. Nyari. Ecology. Bye.A. J.S. C. 2.. D.. Rabosky. Navarro-Siguenza. 49.R. 69–92.P. Brooks. (2008) Speciation in the emerald toucanet (Aulacorhynchus prasinus) complex.uk/Tracer.E. 709
.. Case. M. (2006b) LASER: a maximum likelihood toolkit for detecting temporal shifts in diversiﬁcation rates from molecular phylogenies. Ramamoorthy. & Morrone. (1990) Prairie rattlesnake seasonal migrations: episodes of movement. A. (2002) Phylogeny of the rattlesnakes (Crotalus and Sistrurus) inferred from sequences of ﬁve mitochondrial DNA genes. 123–126. A. G. E. 17.Phylogeography of the Crotalus triseriatus group red-spotted toad (Bufo punctatus): insights on the evolution of North American warm desert biotas.G. (2008b) Integrating paleoecology and genetics of bird populations in two sky island archipelagos. (1952) Taxonomic studies of the rattlesnakes of mainland Mexico. M. D. Ramamoorthy. pp.A.T. B..ac. Berkeley and Los Angeles.J. & Pinero. 15. & Drummond. 697–710 ª 2010 Blackwell Publishing Ltd
˜ Moreno-Letelier. A. & Fa..S. Rabosky. 54.. A. Luis-Martinez. Emberizidae) in Mesoamerica.. (2007) Tracer v1.. L. 257–260. Llorente-Bousquets. (2009) Phylogeographic structure of Pinus strombiformis Engelm.W. (1993) Phytogeography and history of the alpine–subalpine ﬂora of northeastern Mexico. DC. Oxford. Leo G. ´ ´ ˜ ¨ Leon-Paniagua. Gil.. J. J. McCormack. G.J. (1988) Description of the hemipenis of Sistrurus ravus (Serpentes: Viperidae). J. & Aguirre-Leon. 9.P. Washington. Klauber.H. L. (2002) The non-avian reptiles: origins and evolution. J..E. & Vargas-Fernandez. Evolution. I. 44. A new island biogeography of ´ the Sea of Cortes (ed. 38–63. (2004) MrModeltest v2.J. 6. J. Animal Behaviour. by T.S. T. Bonaccorso. 26. Kelly. Mittermeier.B. Oxford University Press.
Journal of Biogeography 38. Bulletin of the Zoological Society of San Diego. Lathrop. BMC Biology. Molecular Phylogenetics and Evolution. Peterson. (2006a) Likelihood methods for inferring temporal shifts in diversiﬁcation rates. J. M. Rambaut. R.E. 1939–1944. J. Vazquez-Domınguez. Ezcurra). B. Schuett. 1–313. Bowen. Espinosa de los Monteros. 60. 39–50.J. 181–220. New York. life histories and inﬂuence on mankind.B. F.L.L. T. 36. Torres-Chavez. 1.. Hoggren. D. J. 1152– 1164.G. & Smith. C. Hoffman.B.. W. Masta.E.W.H.. 41. ¨ Pook.. Bye. & Broadley. D. Fa). Journal of Biogeography. A.bio.. Leon-Paniagua. A. King. A.E. Journal of Biogeography. J.R. Eagle Mountain Publishing. Paniagua. (2009) Do the Oaxacan Highlands represent a natural biotic unit? A cladistic biogeographical test based on vertebrate taxa. R.. pp. ´ Peterson. Hernandez-Banos. CA. Cody and E. B.E. J. Uppsala University. 269–282.S. 924–935. (2009) Phylogeny. M.S. R. University of California Press. Omland. (2008) Phylogeography of the Buarremon brush-ﬁnch complex (Aves. M. T. A. Biodiversity Letters.W. & Harrell.G. (1977) A biogeographical analysis of the Chihuahuan desert through its herpetofauna. (2005) Hotspots revisited: Earth’s biologically richest and most endangered terrestrial ecoregions. Murphy. Eagle Mountain. Evolutionary Bioinformatics Online.E. 34. M. Molecular Phylogenetics and Evolution. & da Fonseca. 681–703. 468–480.. D. Peterson. A. & Duvall. Cladistics..E. Lot and J. (2007) Diversiﬁcation of the arboreal mice of the genus Habromys (Rodentia: Cricetidae: Neotominae) in the Mesoamerican highlands.W. 25. A.A. Parmley. Morafka. ´ Murphy. Conservation International.. & Thorpe. UT. 42. K. L. & Morales.M. Villet. 653–664.G. Herpetologica. H. Barker. (1993) Biological diversity of Mexico: origins and distribution. & ¨ Navarro-Siguenza. Wuster. J. J. ˆ Biology of the vipers (ed. 1–143.R. Feltham. Evolutionary Biology Centre. Mittermeier. biogeography and classiﬁcation of the snake superfamily Elapoidea: a rapid radiation in the late Eocene.. E. P.A.G. A. Llorente-Bousquets. McDonald. O.G. L. (2000) Historical biogeography of the western rattlesnake (Serpentes: Viperidae: Crotalus viridis) inferred from mitochondrial DNA sequence information. 36.T. Nylander. D.E. G. 141–144. Martin. S.. & Smith. Oxford University Press. 28.. & Kovak. NavarroSiguenza. McCranie. (1993) Conservation priorities in northern Middle America: moving up in the world. 718–726. New York.T. The Auk. McCormack.A. T. Fu. D. Greene). Lot.L. 38. ´ ´ ´ ´ Mejıa-Madrid. (2007) Phylogeography and freshwater basins in central Mexico: recent history as revealed by the ﬁsh parasite Rhabdochona lichtenfelsi (Nematoda). by T.4. & Garcıa-Moreno. vernal foraging and sex differences. A. 14. Pilgrim. A. 2505–2521. R. E. W.. (2007) Earliest fossil record of a pigmy rattlesnake (Viperidae: Sistrurus Garman).. Molecular Phylogenetics and Evolution.E... J. C. ´ ¨ Navarro-Siguenza.V. Journal of Biogeography.C. Lamoreux. (1995) Inferring phylogenies from mtDNA variation: mitochondrial gene trees versus nuclear gene trees. pp. (1972) Rattlesnakes: their habits. Oxford University Press.L.A..G. (1957) The Pleistocene history of temperate biotas in Mexico and eastern United States. Program distributed by the author..A. by G. A. 121–131.
C. Toledo. & Laclette. pp. 145–160. building on their shared interests in rattlesnake systematics and biogeography of Mexico.fsu. & Zamudio. 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).M.J. analysed the data. D.E. Journal of Zoological Systematics and Evolutionary Research.
Editor: Serban Proches ¸ ¸
Journal of Biogeography 38. C. Molecular Ecology. Betancourt. & Thorpe.. (1946) Preliminary notes and speculations on the ´ Triseriatus group of rattlesnakes in Mexico. Sceloporus virgatus (Squamata: Phrynosomatidae). Buonamici.I. 435–461. Upton.J. Pook. Molecular Phylogenetics and Evolution. & Farrell. 426–440. and A. D. Packrat middens. 31. A.F. 93–111. (2005) Range expansions in the ﬂightless longhorn cactus beetles. & Vendramin.R. Arellano. Bryson Jr et al. 8. Martin).L. Vargas-Mendoza.
BIOSKETCHES Robert W. (2008) Genetic differentiation among mountain island populations of the striped plateau lizard. Journal of Biogeography. 2688–2690.G. and R. Tennessen.W. Sullivan. 66. D. collected the data. 75–101.B. & Swofford. T. C. 17. Tucson. but are not copy-edited or typeset. G. New York. ´ Zaldivar-Riveron. was a Master’s student at Sul Ross State University.T. 14.L. Bryson Jr and Robert W.A.R. Pook. K. 46.C. D. M.B. & Rogers. (1997) Phylogeny of the sideblotched lizards (Phrynosomatidae: Uta) based on mtDNA sequences: support for a midpeninsular seaway in Baja California. A. 155. D. V. Ronquist. R. D..E. Molecular Phylogenetics and Evolution. & Huelsenbeck. (2000) Phylogeography and systematics of the Peromyscus eremicus species group and the historical biogeography of North American warm regional deserts. Available at: http://ceb..W. in response to Pleistocene climate changes.) studies. B. (1990) Late Quaternary vegetation and climate of the Chihuahuan Desert.W.P.) and ecological (D. Murphy began this collaborative research while R.R.S. Zink. L. Moneilema gigas and Moneilema armatum..M.W. (2000) Comparative phylogeography of Mesoamerican highland rodents: concerted versus independent response to past climatic ﬂuctuations. United States and Mexico. Author contributions: R. this journal provides supporting information supplied by the authors. J. 2008.. (2006) RAxML-VI-HPC: Maximum likelihoodbased phylogenetic analyses with thousands of taxa and mixed models. R. J.W. led the writing.. Such materials are peer-reviewed and may be re-organized for online delivery. Technical support issues arising from supporting information (other than missing ﬁles) should be addressed to the authors. 49. D. Molecular Ecology. 755–768. 558–564. Van Devender and P.. 1025–1044. The last 40.W. 43.L.csit. A. & Murphy.W. Salomao. As a service to our authors and readers. 22.E. (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. (1982) Pleistocene changes of vegetation in tropical Mexico. W. Van Devender. R. & Walker. & Kilpatrick. Riddle.W.B. All authors are broadly interested in better understanding the biodiversity of Mexico through evolutionary (R. and R. Markert. Smith.L. Bioinformatics. J.L. Biological diversiﬁcation in the tropics (ed. C. Quijana-Mascarenas.B. ˜ ¨ Wuster. R. Systematic Biology. (2008) Mitochondrial DNA under siege in avian phylogeography. 36.G. J. J. J.E.F. D. 697–710 ª 2010 Blackwell Publishing Ltd
.. R.. J.W.M.L.. Appendix S2 GenBank accession numbers for genetic samples used in this study. Nieto-Montes de Oca..R. Hafner. ˜ C.V. The American Naturalist. R. (2005) Tracing an invasion: landbridges. Warren. (2009) Genetic diversity and phylogeographic analysis of Pinus leiophylla: a post-glacial range expansion. University of Arizona Press. A. F.M. B.000 years of biotic change (ed. and R. 1095–1108. W..R. W. (2005) Phylogeny and evolution of dorsal pattern in the Mexican endemic lizard genus Barisia (Anguidae: Gerrhonotinae). AZ. Columbia University Press..L. (2006) A step-wise approach to integrating phylogeographic and phylogenetic biogeographic perspectives on the history of a core North American warm deserts biota.M.L. A. 243–257. conceived the ideas. Copeia. & Alexander. & Hafner. University of Kansas Science Bulletin. J. 1572–1574. ¨ Wuster. E.B.. 2107–2121. ´ Rodrıguez-Banderas. Peppin.W. refugia and the phylogeography of the Neotropical rattlesnake (Serpentes: Viperidae: Crotalus durissus). B. Prance). 14.D. G. 17. (1997) Phylogeography and molecular systematics of the Peromyscus aztecus group (Rodentia: Muridae) inferred using parsimony and likelihood. Wilgenbusch. Bioinformatics. (2008) A nesting of vipers: phylogeny and historical biogeography of the Viperidae (Squamata: Serpentes)..W.M. A. Riddle.edu/awty. by J.E.M. 104–113.W. by G. J.V. Molecular Ecology. Molecular Phylogenetics and Evolution.A.R. Ferguson. Journal of Arid Environments. L.S. 104–133.. Stamatakis. 445–459.B.P.S. and A. 19.A. & Barrowclough. 1807–1820. (2004) AWTY: a system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference. Sullivan. T. H. Smith.F. pp.. R.