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

ORIGINAL Evolutionary drivers of ARTICLE phylogeographical diversity in the highlands of : a case study of the triseriatus group of montane rattlesnakes Robert W. Bryson Jr1*, Robert W. Murphy2,3, Amy Lathrop2 and David Lazcano-Villareal4

1School of Life Sciences, University of Nevada, ABSTRACT Las Vegas, Las Vegas, NV, USA, 2Centre for Aim To assess the genealogical relationships of widespread montane rattlesnakes Biodiversity and Conservation Biology, Royal Ontario Museum, Toronto, ON, Canada, in the Crotalus triseriatus species group and to clarify the role of Late Neogene 3State Key Laboratory of Genetic Resources and mountain building and Pleistocene pine–oak forest fragmentation in driving the Evolution, Kunming Institute of Zoology, diversification of Mexican highland taxa. The Chinese Academy of Sciences, Kunming, Location Highlands of mainland Mexico and the south-western United States 4 China, Laboratorio de Herpetologı´a, (, , and ). Universidad Auto´noma de Nuevo Leo´n, San Nicolas de los Garza, Nuevo Leo´n, Me´xico 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 *Correspondence: Robert W. Bryson Jr, School of Life Sciences, University of Nevada, Las to have driven widespread divergences within lineages. Climatic change, paired Vegas, 4505 Maryland Parkway, Las Vegas, NV with the complex topography of Mexico, probably produced a myriad of species- 89154-4004, USA. specific responses in co-distributed Mexican highland taxa. The high degree of E-mail: [email protected]

ª 2010 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/jbi 697 doi:10.1111/j.1365-2699.2010.02431.x R. W. Bryson Jr et al.

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, , Transvolcanic Belt, .

and allopatric populations are generally monophyletic (Sulli- INTRODUCTION van et al., 1997; Harris et al., 2000; Hafner et al., 2005; Leo´n- The geographical location, complex topography, and dynamic Paniagua et al., 2007). Several morphologically cryptic mater- tectonic and climatic history of the Mexican highlands provide nal lineages occur within the Transvolcanic Belt, Sierra Madre a matrix for the evolution of a spectacularly diverse biota. The Oriental and Sierra Madre del Sur (small : Sullivan Mexican highlands harbour a significant amount of western et al., 1997; Harris et al., 2000; Arellano et al., 2005; Leo´n- North America’s biodiversity (Ramamoorthy et al., 1993; Paniagua et al., 2007; birds: Garcı´a-Moreno et al., 2004; Mittermeier et al., 2005) and a level of biotic endemism Navarro-Sigu¨enza et al., 2008; -Olivares et al., 2008). scarcely rivalled elsewhere (Peterson et al., 1993). The evolu- These high levels of genetic divergence suggest that endemism tionary drivers of this diversity, however, remain poorly in the Mexican highlands may be vastly underestimated. documented. Despite early broad-scale inferences about the Studies of genetic structuring in other co-distributed taxa are biogeographical history of Mexico, dating back to Dunn (1931), needed in order to develop a more complete understanding of few studies explore the historical diversification of Mexican the evolutionary drivers of diversification. highland taxa. This impedes the ability of researchers to identify The relatively small-bodied montane rattlesnakes (Viperi- fine-scale biogeographical patterns and the extent to which dae) inhabiting the pine–oak forests of mainland Mexico these apply to co-distributed taxa (McCormack et al., 2008a). represent an ideal model system for investigating historical Neogene vicariance, largely due to orogenesis, and Quater- patterns of diversification in the Mexican highlands. This large nary climate change have been the postulated drivers of group includes 40% of the total number of currently recog- evolutionary diversification in western North America (e.g. nized species of rattlesnakes (Campbell & Lamar, 2004) and is Jaeger et al., 2005; Riddle & Hafner, 2006). Although most of found in all of the major mountainous regions of Mexico. The the major mountain ranges in Mexico are relatively ancient phylogenetic relationships among these rattlesnakes, however, (Ferrusquı´a-Villafranca, 1993; Ferrusquı´a-Villafranca & Gon- are contentious. Those species currently allied with the za´lez-Guzma´n, 2005), the Transvolcanic Belt of central Mexico Crotalus triseriatus species group (C. triseriatus, C. aquilus, was formed during the Neogene (Ferrusquı´a-Villafranca, 1993; C. lepidus, C. pusillus and C. ravus; Murphy et al., 2002; Castoe Becerra, 2005). This development may have had a significant & Parkinson, 2006) are especially difficult to classify. The impact on the diversification of highland taxa, because the content of this species group varies despite more than 65 years uplift created new geographical barriers and montane habitats, of intensive systematic effort (Gloyd, 1940; Smith, 1946; and linked previously isolated highland biotas (Anducho- Klauber, 1952, 1972; Brattstrom, 1964; Dorcas, 1992; Murphy Reyes et al., 2008). Historical diversification of highland taxa et al., 2002). Furthermore, several authors suggest that the may also have been influenced by dramatic habitat fluctuations Mexican highlands may harbour one or more cryptic species during the Pleistocene that resulted in the cyclical downward within the C. triseriatus species group (Armstrong & Murphy, displacement and retraction of Mexican pine–oak woodlands 1979; Murphy et al., 2002). (Martin & Harrell, 1957; Van Devender, 1990; McDonald, Our study addresses several questions relating to the 1993). This displacement could have resulted in population evolutionary history of the C. triseriatus species group. We and range expansions in highland species during glacial combine extensive range-wide sampling and mixed-model periods, and isolation in high elevation refugia during the phylogenetic analyses to formulate a robust hypothesis of interglacials (Moreno-Letelier & Pin˜ero, 2009). Subsequent phylogenetic relationships and to address long-standing post-glacial fragmentation of Mexican pine–oak woodlands uncertainties about cryptic diversity. We also estimate dates (Van Devender, 1990) may have caused fragmentation of these of lineage divergences based on a relaxed molecular clock to isolated refugial populations (e.g. McCormack et al., 2008b). provide a probabilistic temporal calibration for the phylogeny. Gene flow would have been affected by these events. We model the temporal distribution of divergence events to Molecular studies of montane Mexican taxa often discover assess the potential effects of Late Neogene mountain building complex phylogeographical patterns. In small mammals, for and Pleistocene pine–oak forest fragmentation on the tempo of example, mitochondrial DNA (mtDNA) differentiation is high diversification. Finally, we look for phylogeographical patterns

698 Journal of Biogeography 38, 697–710 ª 2010 Blackwell Publishing Ltd Phylogeography of the Crotalus triseriatus group in the C. triseriatus group that are shared with other aquilus’). Based on recent phylogenetic analyses (Murphy co-distributed highland taxa. et al., 2002; Castoe & Parkinson, 2006; Wu¨ster et al., 2008), we used Sistrurus catenatus and S. miliarus as outgroup taxa. We sequenced relatively slowly evolving and more quickly MATERIALS AND METHODS evolving regions of the mitochondrial genome, including 12S and 16S ribosomal RNA genes, NADH dehydrogenase subunit Taxon sampling and laboratory methods 4 and flanking tRNAs (ND4), and ATPase subunits 8 and 6 Between 1999 and 2009 we collected 130 samples (see (ATPase 8, ATPase 6). These gene regions have been shown to Appendix S1 in the Supporting Information) from throughout be informative at different levels of divergence within rattle- the distribution of all putative taxa in the C. triseriatus group (Pook et al., 2000; Murphy et al., 2002; Wu¨ster et al., (Fig. 1). Four samples were collected from a morphologically 2005; Douglas et al., 2006). Total genomic DNA was extracted distinct, undescribed species closely related to C. pusillus from liver, shed skins, or ventral scale clips using proteinase K ) (herein referred to as ‘Crotalus sp.’), and three samples were (22 mg mL 1 in 10 mm Tris-HCl, pH 7.5) in a lysis buffer from specimens intermediate between C. lepidus and C. aquilus (100 mm Tris, 5 mm Na2EDTA, 200 mm NaCl, 0.2% SDS) and (Ct160, Ct197 and Ct201; herein referred to as ‘C. lepidus x incubated at 37 C. Shed skins often required 2–3 days to fully

61 46

53 39 59 41 36 55 99 112 163 lepidus

klauberi 35 R21 24 7

222 226 8

29

140* 161 20 252 111* 162* 215 127 12* 32* morulus 26* 237 142 160 223 10 118* 31 9 216* maculosus 197 116* 283 165 R55 Major mtDNA lineages 30 aquilus 201 227* 2* 164 lepidus 3 239* 124 266 morulus 143* 126 15* 27 135 209* 14 aquilus 233* 33* 155 254 255 122 triseriatus 193 261* 157 238* armstrongi 137* R57 armstrongi 136 18* 262* 6 259 triseriatus pusillus 1* 21 230* Crotalus sp. 172 ravus

Figure 1 Map of Mexico and the south- western United States depicting the sample localities and distribution (adapted from

Campbell & Lamar, 2004) for the R44 211 153 200 triseriatus species group. Symbols indicate 199 pusillus 225 major mitochondrial DNA (mtDNA) lin- 154 139 121* 204 208 17 eages inferred in this study, and numbers R42 150 152 refer to specific sample numbers (see 267* 144* Crotalus sp. 149* Appendix S1). Asterisks denote multiple exiguus 168168 brunneus samples obtained from the same locality. The 16º prefix ‘Ct’ was omitted from the sample numbers for clarity.

Journal of Biogeography 38, 697–710 699 ª 2010 Blackwell Publishing Ltd R. W. Bryson Jr et al. digest, and an additional 12.5 lL of proteinase K was added not the duplicated runs had converged on the same mean every 24 h. Samples were cleaned using two washes of likelihood. We further assessed convergence by evaluating phenol:chloroform:isoamyl alcohol (25:24:1) followed by a posterior probability clade-support values post burn-in using final wash of chloroform:isoamyl alcohol (24:1). the on-line application Are We There Yet (AWTY; Wil- All gene regions were amplified via polymerase chain genbusch et al., 2004). After determining chain convergence, reaction (PCR) in a 25 lL reaction volume containing which occurred during the first 500,000 generations of each 0.8 lL deoxynucleoside triphosphates (dNTPs) (10 mm), run, we conservatively discarded all samples obtained during 19.0 lL double-distilled water, 1.0 lL each primer (10 pm), the first one million (25%) generations as burn-in. A 50%

2.5 lL1· PCR buffer (1.5 mm MgCl2; Fisherbrand, Pitts- majority-rule consensus phylogram with nodal posterior burgh, PA, USA), 0.75 U Taq DNA polymerase (Fisherbrand), probability (PP) support was estimated from the combination and 1.0 lL template DNA. Previously published primer of the four runs post-burn-in. ML analyses were conducted sequences are given in Murphy et al. (2002; 12S, 16S). For using RAxML 7.0.3 (Stamatakis, 2006) with the same parti- amplification of ND4 we modified one of the forward primers tioning scheme used for the BI analyses. The GTRGAMMA of Are´valo et al. (1994) (12931L: 5¢-CTA CCA AAA GCT CAT model was used, and 1000 nonparametric bootstrap replicates GTA GAA GC-3¢) and used the LEU reverse primer (Are´valo were performed to assess nodal support. We considered those et al., 1994). Primers for ATPase were designed specifically for nodes with ‡95% Bayesian posterior probability and ‡70% this project: (9974L: 5¢-AGC ACT AGC CTT TTA AGY T-3¢ bootstrap support as strongly supported (Hillis & Bull, 1993; and 10830H: 5¢-AGA AAC CCT ATT TTT AGT ACT AG-3¢). Felsenstein, 2004). 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. Divergence dating A final extension phase of 72 C for 7 min terminated the protocol. The entire 25 lL reaction was visualized on a 1% We estimated divergence dates using a Bayesian relaxed agarose gel containing ethidium bromide. Sharp, clear bands molecular clock as implemented in beast v.1.5.4 (Drummond were excised from the gel and placed in a filter tip (Sorenson; & Rambaut, 2007). Because of potential problems associated 75-30550T). DNA was collected in a 1.7 mL Eppendorf tube with model parameter variance across heterogeneous datasets after centrifuging the DNA through the filter tip for 10 min at (Guiher & Burbrink, 2008), we inferred divergence estimates 16.1 rcf. for a reduced dataset, which included one or two individuals We sequenced in both directions using the amplification from each geographically structured maternal group within primers and Big Dye Terminator v.3.1 cycle sequencing kit each lineage (Fig. 2). We also included sequences from several (Applied Biosystems, Foster City, CA, USA). We used 4 lLof other North American pitvipers to calibrate the tree (Appen- the cleaned PCR product in one-quarter reaction volume of dix S2). Best-fit models of evolution were estimated from the that recommended by ABI (Applied Biosystems). Samples were new dataset using MrModeltest. We implemented an analysed with an ABI Prism 3100 Genetic Analyzer (Applied uncorrelated lognormal clock and node constraints obtained Biosystems). Forward and reverse sequences for each individ- from the fossil and geological record with lognormal distri- ual were edited and manually aligned using BioEdit 5.0.9 butions to estimate divergence dates throughout the tree. (Hall, 1999). Identical sequences for samples from the same Analyses were run for 40 million generations, with samples locality were collapsed into one haplotype. 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 Phylogenetic analyses Carlo (MCMC) analyses, appropriate burn-in, and adequate We analysed our sequence data using Bayesian inference (BI) effective sample sizes (>200 for each estimated parameter). and maximum likelihood (ML) phylogenetic methods. BI Analyses on a partitioned-by-gene dataset resulted in effective analyses were conducted using MrBayes 3.1 (Ronquist & sample sizes below 50 for several parameters. Therefore, the Huelsenbeck, 2003) on the combined mtDNA dataset, imple- final analyses were run on an unpartitioned dataset. After menting separate models for each gene region (ATPase 8, discarding the first 4 million generations (10%) as burn-in, we ATPase 6, ND4, combined tRNAs, 12S, and 16S). MrModel- summarized parameter values of the samples from the test 2.1 (Nylander, 2004) was used to select a best-fit model of posterior on the maximum clade credibility tree using evolution, based on the Akaike information criterion (AIC), TreeAnnotator 1.4.8 (Drummond & Rambaut, 2007) with for each partition. MrBayes settings included random starting the posterior probability limit set to 0.5 and mean node trees, a variable rate prior, a mean branch length exponential heights summarized. prior of 100, and heating temperature of 0.02. Analyses Two fossil calibrations for the tree were obtained for North consisted of four runs (n runs = 4) each conducted with three American pitvipers: (1) the oldest fossil from the genus heated and one cold Markov chain while sampling every 100 Sistrurus from the Late Miocene (Clarendonian; Parmley & generations for 4 million generations. Output parameters were Holman, 2007), and (2) the earliest record of A. contortrix in visualized using the program Tracer v1.4 (Rambaut & the Late Miocene (Late Hemphillian; Holman, 2000). Addi- Drummond, 2007) to ascertain stationarity and whether or tionally, we used the estimated age of divergence between

700 Journal of Biogeography 38, 697–710 ª 2010 Blackwell Publishing Ltd ª Biogeography of Journal 00BakelPbihn Ltd Publishing Blackwell 2010

S. catenatus Ct216 morulus TAM S. miliarius Ct217 morulus TAM Ct150 exiguus GRO Ct223 morulus TAM Ct149 exiguus GRO I Ct31 morulus TAM I Ct151 exiguus GRO Ct9 morulus TAM Ct152 brunneus OAX Ct10 morulus TAM Ct168 brunneus OAX II Ct127 morulus NL Ct153 ravus MEX Ct26 morulus NL morulus 38 Ct225 ravus PUE ravus Ct133 morulus NL Ct204 ravus PUE Ct215 morulus NL 697–710 , Ct139 ravus PUE Ct252 morulus COAH II R44 ravus PUE III Ct20 morulus NL Ct199 ravus TLAX Ct11 morulus NL Ct154 ravus PUE Ct25 morulus NL Ct211 ravus VER Ct164 aquilus QTO I Ct135 armstrongi JAL Ct126 aquilus QTO Ct137,138 armstrongi COL I Ct261 aquilus MEX Ct259 armstrongi MICH Ct269 aquilus MEX Ct254 armstrongi MICH II Ct124 aquilus QTO Ct257 armstrongi MICH armstrongi Ct2 16 aquilus AGS II Ct136 armstrongi JAL Ct239 aquilus GTO Ct193 armstrongi JAL Ct241 aquilus GTO Ct233 armstrongi JAL III 94 Ct209 aquilus MICH 0.05 sub. / site Ct256 armstrongi JAL 88 Ct224 aquilus MICH Ct121,158 Crotalus sp. MICH Ct3 aquilus QTO Ct264 Crotalus sp. MICH Ct160 lepidus x aquilus SLP aquilus Ct267 Crotalus sp. MEX Crotalus sp. Ct165 aquilus SLP Ct268 Crotalus sp. MEX R55 aquilus SLP III Ct144,147 pusillus MICH Ct30 aquilus SLP Ct145 pusillus MICH I Ct197 lepidus x aquilus SLP Ct208 pusillus JAL Ct15 aquilus HID R42 pusillus MICH pusillus Ct266 aquilus HID Ct17 pusillus MICH II Ct123 aquilus HID Ct18 armstrongi MICH Ct14 aquilus HID IV Ct172 triseriatus PUE Ct33,34 aquilus HID Ct155 triseriatus PUE Ct27 aquilus HID Ct157 triseriatus PUE I Ct122 aquilus HID Ct238 triseratus VER Ct283 maculosus NAY I Ct242 triseratus VER Ct118 klauberi ZAC Ct255 triseriatus MICH Ct119 klauberi ZAC Ct262 triseriatus MICH Ct116 klauberi AGS Ct263 triseriatus MICH triseriatus Ct201 lepidus x aquilus AGS Ct230 triseriatus PUE Ct117 klauberi AGS II Ct245 triseratius PUE Ct227 klauberi ZAC Ct1 triseriatus MEX II Ct228 klauberi ZAC R57 triseriatus DF 60 Ct143 klauberi JAL Ct6 triseriatus MEX Ct195 klauberi JAL Ct4 triseriatus MEX 64 Ct12,13 maculosus DUR Ct21 triseriatus DF Ct142 maculosus DUR Ct237 maculosus SIN III Ct32 klauberi DUR Ct220,221 klauberi DUR IV Ct35 lepidus TX Ct140,141 lepidus COAH Ct24 lepidus TX V Ct112 lepidus TX Ct59 lepidus NM lepidus

Ct161 klauberi DUR the of Phylogeography Ct162 klauberi DUR Ct250 klauberi DUR Ct29 klauberi CHIH 88 R21 klauberi CHIH 92 Ct8 klauberi CHIH Ct222 klauberi CHIH Ct226 klauberi SON Ct41 klauberi AZ 92 Ct55 klauberi AZ Ct99 klauberi AZ VI 70 Ct36 klauberi TX Ct39 klauberi NM Ct61 klauberi NM Ct53 klauberi NM Ct46 klauberi NM Ct7 klauberi CHIH Ct163 klauberi CHIH Ct23 klauberi CHIH

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 triseriatus Crotalus sequence data. Sistrurus catenatus and S. miliarus were used as outgroup taxa. Roman numerals indicate major phylogroups nested within major lineages. The single divergent samples of C. aquilus from north-eastern Quere´taro (Ct164) and C. lepidus maculosus from (Ct238) are labelled as phylogroups for simplicity. Numbers at nodes indicate support values (Bayesian posterior probability above, maximum likelihood bootstrap below); black dots represent strongly supported nodes (‡ 95% posterior probability, 70% bootstrap). White dots additionally indicate 12 strongly supported (with one exception) geographically cohesive subgroups clustered within major phylogroups and used in diversification rate analyses. The branch drawn in grey is not to scale. group 701 R. W. Bryson Jr et al.

C. ruber and C. atrox (Castoe et al., 2009) due to the Pliocene trees using yuleSim in laser with the same number of nodes marine incursion of the Sea of Corte´s (Carren˜o & Helenes, and the same speciation rate as that estimated under the pure- 2002; and references therein). The stem of Sistrurus (Guiher & birth model. We additionally generated a lineage-through-time Burbrink, 2008; Wu¨ster et al., 2008) was constrained with a (LTT) plot using the plotLtt function in laser to visualise the zero offset (hard upper bound) of 8 million years ago (Ma), a pattern of accumulation of log-lineages over time. lognormal mean of 0.01, and a lognormal standard deviation Because underestimates of genetic diversity potentially bias of 0.76. This produced a median age centred at 9 Ma and a inferred rates of diversification (Esselstyn et al., 2009), we used 95% prior credible interval (PCI) extending to the beginning two sets of dates estimated in beast for diversification-rate of the Clarendonian at 11.5 Ma (Holman, 2000). The node analyses. The first set consisted of estimated divergence dates representing the most recent common ancestor (MRCA) of between the major phylogroups (Fig. 2), which we considered A. contortrix was given a zero offset of 6 Ma, a lognormal mean to be a conservative approach. The second set included of 0.01, and a lognormal standard deviation of 0.42, producing estimated diversification dates between an additional 12 a median age of 7 Ma and a 95% PCI extending to the start of geographically cohesive monophyletic subgroups clustered the Late Hemphillian at 8 Ma (Holman, 2000). These lognor- within several of the major phylogroups, and this was a more mal distributions with hard lower bounds best reflect the liberal approach. These smaller subgroups represented the prediction, based on the high likelihood of fossil non- finest division of phylogeographical diversity that could be preservation, that any true divergence date will probably be reasonably inferred from our study. older than the oldest known fossil, rather than younger (Ho & Phillips, 2009; Kelly et al., 2009). The node representing the RESULTS 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 Sequence characteristics and phylogenetic estimate a median age centred at the climax of the formation of the Sea of Corte´s and development of the Bouse embayment at 3 Ma, The final dataset used for phylogenetic inference consisted of and a 95% PCI extending to the beginning of the development 2408 aligned nucleotide positions. Models of sequence evolu- of the Sea of Corte´s at 5.5 Ma (Carren˜o & Helenes, 2002, and tion selected for the mtDNA partitions were GTR+I+G references therein). No zero offset was used. (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 Temporal patterns of diversification C. triseriatus group (Figs 2 & 3), five of which corresponded to We analysed temporal shifts in diversification rates using ML- the species C. ravus, C. pusillus, C. triseriatus, C. aquilus and based diversification-rate analysis (Rabosky, 2006a). The fit of C. lepidus (Campbell & Lamar, 1989, 2004). Two lineages different birth–death models implementing two constant rates represented the subspecies C. t. armstrongi and C. l. morulus. (pure-birth, and birth–death) and three variable rates (expo- One lineage represented an undescribed taxon. Strong phylo- nential and logistic density-dependent, and two-rate pure- geographical structure was present within most of these taxa birth) was computed with laser 2.3 (Rabosky, 2006b). Model (Fig. 2). In C. ravus, the geographical distribution of subclades fit was measured using AIC scores. Significance of the change was consistent with recognized subspecies (C. r. ravus, in AIC scores (DAICrc) between the best rate-constant and C. r. brunneus and C. r. exiguus). Relationships among lineages best rate-variable model was determined by creating a null were generally congruent with those of previous molecular distribution for DAICrc. This was done by simulating 1000 studies (Fig. 3; Murphy et al., 2002; Castoe & Parkinson,

ravus ravus ravus

other Crotalus armstrongi pusillus Crotalus sp. pusillus triseriatus pusillus lepidus triseriatus lepidus triseriatus aquilus aquilus aquilus morulus

triseriatus (LG) * triseriatus (LG) * lepidus

(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).

702 Journal of Biogeography 38, 697–710 ª 2010 Blackwell Publishing Ltd Phylogeography of the Crotalus triseriatus group

2006). Differences were largely attributable to our expanded yule2rate models as the best rate-constant and best rate-variable taxonomic and geographic sampling, and the prior use of a models for both the ‘conserved’ and the ‘liberal’ datasets. misidentified sample (see below). 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 Divergence times and tempo of diversification (P = 0.04), suggesting the rate-variable yule2rate model pro- The GTR+I+G model of sequence evolution was selected for vided a better fit. According to the scenario suggested by this the beast analyses. Dating estimates suggested that diversifi- model, the C. triseriatus group had an initial net diversification cation in the C. triseriatus group probably began in the Late rate of 0.53 events per million years. A change in net Miocene, and divergences between most major lineages diversification rate took place 1.02 Ma, when the rate shifted occurred in the Late Miocene and Pliocene (Fig. 4). Phylo- dramatically to 0.09 diversification events per million years. In geographical structure within these lineages appeared to have contrast, the null hypothesis of rate-constancy was not rejected been generated primarily during the Pleistocene. (P = 0.1) for the liberal dataset. The ML estimate of the The LTT plots suggested either a constant rate of diversifi- diversification rate under the best rate-constant pureBirth cation or a decline in the Pleistocene (Fig. 5), depending on the model was 0.45 diversification events per million years. Thus, a scale of phylogeographical diversity recognized by each dataset. strong signal of diversification rate change was not indicated The birth–death likelihood analyses chose the pureBirth and after accounting for fine-scaled phylogeographical diversity.

Gloydius A. piscivorous A. contortrix S. catenatus S. miliarius C. atrox C. ruber Ct150 exiguus GRO Ct149 exiguus GRO I Ct168 brunneus OAX ravus Ct152 brunneus OAX II Ct153 ravus MEX III Ct204 ravus PUE Ct135 armstrongi JAL I Ct137,138 armstrongi COL Ct259 armstrongi MICH II armstrongi 6.3 Ct254 armstrongi MICH Ct233 armstrongi JAL III Ct193 armstrongi JAL Ct264 Crotalus sp. MICH Crotalus sp. Ct268 Crotalus sp. MEX Ct144,147 pusillus MICH I 5.4 3.3 Ct145 pusillus MICH pusillus Ct17 pusillus MICH II Ct208 pusillus JAL Ct155 triseriatus PUE I Ct242 triseratus VER triseriatus Ct245 triseratius PUE II 4.9 Ct262 triseriatus MICH Ct216 morulus TAM I Ct9 morulus TAM 2.1 Ct11 morulus NL morulus II Ct26 morulus NL 4.1 Ct164 aquilus QTO I Ct261 aquilus MEX Ct2,16 aquilus AGS II Ct30 aquilus SLP aquilus Ct3 aquilus QTO III Ct122 aquilus HID 3.4 Ct15 aquilus HID IV Ct283 maculosus NAY I Ct195 klauberi JAL Ct118 klauberi ZAC II Ct12,13 maculosus DUR III Ct32 klauberi DUR IV Ct140,141 lepidus COAH V Ct59 lepidus NM lepidus Ct161 klauberi DUR Ct29 klauberi CHIH Ct46 klauberi NM VI Ct226 klauberi SON Ct55 klauberi AZ 15 10 5 0

Miocene Pliocene Pleistocene

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).

Journal of Biogeography 38, 697–710 703 ª 2010 Blackwell Publishing Ltd R. W. Bryson Jr et al.

Log-Lineages Through Time

(a) (b) r2=0.09

st = 1.02

neages r1=0.53 r1=0.45 i .0 2.5 3.0 3.5 L 2 g Lo .5 1 1.0 1.5 2.0 2.5 3.0 3.5 1.0

0123456 0123456 Time From Basal Divergence (million years ago)

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.

esized filter barriers, such as the Rio Pa´nuco basin (Anducho- DISCUSSION Reyes et al., 2008) and Cerritos-Arista and Saladan Filter Barriers (Morafka, 1977; Bryson et al., 2007) (Fig. 6). Evolutionary drivers of diversity within the Our results revealed strong geographical partitioning of C. triseriatus group genetic diversity within nearly all lineages. Most of this Our analyses indicated that a progressive diversification of the structure appeared to have developed during the Quaternary. C. triseriatus group occurred over the last six million years. Thus, glacial climatic cycles probably contributed to the Both Neogene vicariance and Quaternary climate change had fragmentation of Mexican pine–oak forests and may have comparably strong effects on driving diversification rates. Early driven divergences. This inference was broadly congruent with divergences were temporally and geographically congruent diversification patterns seen in several Middle American with the formation of the Transvolcanic Belt, suggesting that highland pitvipers (Castoe et al., 2009) and other montane the development of this mountain range played a critical role taxa (pines: Moreno-Letelier & Pin˜ero, 2009; Rodrı´guez- in early diversification of this widespread highland group. Five Banderas et al., 2009; insects: Masta, 2000; Smith & Farrell, of the eight major lineages (C. ravus, C. t. triseriatus, 2005; Anducho-Reyes et al., 2008; small mammals: Sullivan C. t. armstrongi, C. pusillus and Crotalus sp.) were distributed et al., 1997; Edwards & Bradley, 2002; Leo´n-Paniagua et al., on or near the Transvolcanic Belt, and estimated divergence 2007; : Zaldivar-Rivero´n et al., 2005; Tennessen & dates between these lineages fell within the orogenic timeframe Zamudio, 2008; birds: Garcı´a-Moreno et al., 2004; McCor- for the area (Ferrusquı´a-Villafranca, 1993; Becerra, 2005). The mack et al., 2008b; Puebla-Olivares et al., 2008). remaining, more northerly three lineages occupied the Central Observed phylogeographical patterns in the C. triseriatus Mexican Plateau and southern Sierra Madre Oriental group, however, might not reflect isolated gene flow. Male (C. aquilus), northern Sierra Madre Oriental (C. l. morulus), rattlesnakes can disperse relatively long distances between and Sierra Madre Occidental (western C. lepidus). Uplift of the populations during the breeding season (e.g. King & Duvall, Central Mexican Plateau coupled with the subsequent aridi- 1990; Clark et al., 2008), and male-biased dispersal cannot be fication and final Pliocene development of the Chihuahuan accounted for in a mtDNA gene tree, unless the dispersed male Desert (Jaeger et al., 2005, and references therein) may have is sampled. Paternally mediated gene flow may occur between separated C. aquilus and C. l. morulus to the east from western seemingly isolated populations. Whereas some phylogroups populations of C. lepidus. The divergence of C. aquilus from appear to be separated by well defined, low elevation breaks C. l. morulus along the Sierra Madre Oriental may have been (Fig. 6, Table 1), others may be marginally separated by mid- facilitated by the development of any one of several hypoth- elevation swathes of oak-, mesquite-, or desert-grassland.

704 Journal of Biogeography 38, 697–710 ª 2010 Blackwell Publishing Ltd Phylogeography of the Crotalus triseriatus group

(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 Pa´nuco 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), Leo´n-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, 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 Quere´taro and adjacent San Luis Potosı´) C. aquilus II/III+IV Unknown C. aquilus III/IV Rio Pa´nuco 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

Boundaries within some northern phylogroups (e.g. western Phylogeographical diversity in the Mexican highlands C. lepidus, C. l. morulus) are not obvious. Future testing with nuclear gene markers is needed to elucidate patterns of gene Despite an overall paucity of phylogeographical studies of flow. Mexican highland taxa, emerging patterns suggest mixed

Journal of Biogeography 38, 697–710 705 ª 2010 Blackwell Publishing Ltd R. W. Bryson Jr et al. responses to past geological and climatic events despite a northern Oaxaca. The Rio Verde additionally divides the Sierra presumed shared history in the same region (Sullivan et al., Madre del Sur in western Oaxaca. To the north, the Rio 2000; Paniagua & Morrone, 2009). The ancient development of Pa´nuco bisects the Sierra Madre Oriental, and the xeric most of the major mountains in Mexico probably pre-dates interior of the Oriental Basin in Puebla and separates diversification of the extant highland species. However, the the highlands of the Sierra Madre Oriental from those of the Neogene formation of the Transvolcanic Belt is undoubtedly a Transvolcanic Belt. Further west, the Rios Lerma and Rio major force driving the evolutionary history of these taxa. In Santiago flank the southern Sierra Madre Occidental and the C. triseriatus group, initial diversification appears to be northern Transvolcanic Belt and separate these two highlands. tightly linked to the uplifting of the Transvolcanic Belt, and Further north, the Rio Mezquital bisects the Sierra Madre several phylogroups are embedded within this mountain range. Occidental. Historical filter barriers across the Central Mexican Deep divergences and high phylogeographical diversity in Plateau may also include large interconnected palaeo-lakes other co-distributed highland taxa (small mammals: Sullivan (Mejı´a-Madrid et al., 2007, and references therein). These et al., 1997; Demastes et al., 2002; Edwards & Bradley, 2002; largely overlooked barriers are probably responsible for Leo´n-Paniagua et al., 2007; birds: McCormack et al., 2008a; disrupting Pleistocene pine–oak forest corridors (Demastes Navarro-Sigu¨enza et al., 2008) are also broadly attributable to et al., 2002) and could, in part, further explain inferred the formation of the Transvolcanic Belt. historical disjunctions between highland taxa distributed In concert, Quaternary glacial–interglacial climatic cycles across the Central Mexican Plateau, such as between phylo- and the complex topography of Mexico had the potential to groups of C. aquilus. produce a myriad of intraspecific phylogeographical patterns in highland taxa. Some pine–oak expansions probably resulted Matrilineal genealogy of the C. triseriatus group in ephemerally contiguous highland biotas in the Mexican sierras (Toledo, 1982; Van Devender, 1990). However, some Utilizing expanded taxonomic and geographical sampling and geographical barriers, such as major river drainages, may have phylogenetic mixed-model analyses of mtDNA, we inferred served as filter barriers, as evidenced by shared genetic breaks several novel historical relationships for the C. triseriatus in co-distributed taxa (Fig. 6, Table 2). In southern Mexico, group. The topology strongly supported the placement of the Sierra Madre del Sur is isolated from the Transvolcanic Belt C. ravus in the C. triseriatus group despite over 100 years of to the north by the Rio Balsas and its associated tributaries inclusion in the genus Sistrurus and a recent transfer into (e.g. Rio Tepalcatepec, Rio Atoyac) that form the Balsas Basin. Crotalus as the sister species to all other Crotalus (Murphy The Rio Ahuijullo isolates these areas to the west. Further, the et al., 2002). Plesiomorphic morphological attributes shared Sierra Madre del Sur is separated from the Transvolcanic Belt with Sistrurus have seemingly confounded the phylogenetic and Sierra Madre Oriental by the Rio Papaloapan basin across 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 Montane rattlesnakes (Crotalus triseriatus group)1, Mexican jays (Aphelocoma ultramarina)*10, Mexican Filter Barriers kingsnakes (Lampropeltis mexicana species group)11 (8) Rio Pa´nuco 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-Sigu¨enza et al., 2008; 5Edwards & Bradley, 2002; 6Puebla-Olivares et al., 2008; 7Garcı´a-Moreno et al., 2004; 8Leo´n-Paniagua et al., 2007; 9Moreno-Letelier & Pin˜ero, 2009; 10McCormack et al., 2008a; 11Bryson et al., 2007.

706 Journal of Biogeography 38, 697–710 ª 2010 Blackwell Publishing Ltd Phylogeography of the Crotalus triseriatus group

We inferred a novel placement for C. t. armstrongi near the years, this study would not have been possible. We thank the base of the tree and distant from C. t. triseriatus. It formed the following people, curators and institutions for providing or sister clade to all other species of the C. triseriatus group except assisting with tissue samples: D.R. Frost (American Museum of for C. ravus. This finding contradicted previous suggestions Natural History), R.D. MacCulloch (Royal Ontario Museum), based on morphological evidence of a close relationship O. Flores-Villela and A. Nieto-Montes (Universidad Nacional between C. t. armstrongi and C. t. triseriatus (Campbell, 1979; Auto´noma de Me´xico), J.A. Campbell, C. Franklin and E.N. Dorcas, 1992). Smith (University of Texas at Arlington), J. Alvarado-Dı´az and Strong support was obtained for a sister relationship A. Quijada-Mascaren˜as (Universidad Michoacana de San between C. aquilus and C. l. morulus, and this clade was sister Nicola´s de Hidalgo, Michoaca´n), A. Kardon (San Antonio to all other C. lepidus. The phylogenetic affinities of C. aquilus ), P. Ponce-Campos, U.O. Garcı´a-Va´zquez, J. Lemos- to other C. triseriatus group taxa have been contentious (see Espinal, L. Canseco-Marquez, G. Swinford, E. Mocin˜o-Deloya, Gutberlet & Harvey, 2004). Crotalus aquilus has oscillated K. Setser and G. Quintero-Dı´az. We thank the numerous between being considered as a subspecies of C. triseriatus (as people who assisted in the field, including S.P. Mackessy, originally described) and a distinct species closely related to M. Torocco, F.R. Mendoza-Paz, B.T. Smith, J. Banda-Leal, C. lepidus. Recent molecular studies (Murphy et al., 2002; G. Ulises de la Rosa-Lozano, R. Romero, D. Hartman, R. Castoe & Parkinson, 2006) proposed a sister relationship Queen, K. Peterson, M. Price, C. Harrison, E. Garcı´a-Padilla between C. aquilus and C. triseriatus. However, this association and E. Beltra´n-Sanchez. B.T. Smith and A. Egan assisted with was based on a misidentified sample, labelled as ‘C. triseriatus use of laser and R. We further thank M.R. Graham, J. Jones, LG’ (ROM 18114; GenBank numbers AF259231, AF259087, C. Gru¨enwald, J.A. Mueller, J.R. Dixon, A. Narvaez and AF259124, AF259161, AF259199), which we determined to be J.A. Campbell for other assistance that greatly improved this C. aquilus. Our finding of a C. aquilus–C. l. morulus sister project. Two anonymous referees and S¸. Proches¸provided relationship suggested that C. l. morulus may be more closely comments that greatly improved the final version of this related to C. aquilus than to other lineages of C. lepidus,in manuscript. Collecting was conducted under permits granted contrast to strong morphological support for a monophyletic by SEMARNAT to R.W.B., R.W.M., D.L.V., D.J. Morafka and C. lepidus (Dorcas, 1992). Other subspecies of C. lepidus in the F. Mendoza-Quijano. All sequencing work was conducted in west (C. l. lepidus, C. l. klauberi and C. l. maculosus) together the laboratory of R.W.M., and funded by the Natural Sciences formed a monophyletic group, but divergences within this and Engineering Research Council of Canada Discovery Grant group appeared to be relatively recent (Fig. 4). Only the A3148. monophyly of C. l. lepidus was supported. Several studies suggest that multiple, geographically isolated REFERENCES species may be represented under the name C. triseriatus (Armstrong & Murphy, 1979; Murphy et al., 2002; Gutberlet & Anducho-Reyes, M.A., Cognato, A.I., Hayes, J.L. & Zuniga, G. Harvey, 2004). Samples of Crotalus obtained along the central (2008) Phylogeography of the bark beetle Dendroctonus portion of the Transvolcanic Belt (Figs 1 & 2) provide clear mexicanus Hopkins (Coleoptera: Curculionidae: Scolytinae). support for this prediction. Although historically considered to Molecular Phylogenetics and Evolution, 49, 930–940. be C. t. triseriatus (Campbell & Lamar, 2004), these genetically Arellano, E., Gonza´lez-Cozatl, F.X. & Rogers, D.S. (2005) distinctsnakesarelikelytorepresentanundescribedcrypticspecies. Molecular systematics of Middle American harvest mice While phylogenetic inferences based on one marker (e.g. Reithrodontomys (), estimated from mitochondrial mtDNA) should be interpreted with caution, maternally inher- cytochrome b gene sequences. Molecular Phylogenetics and ited data can lead to significant biogeographical discoveries (e.g. Evolution, 37, 529–540. Upton & Murphy, 1997; Riddle et al., 2000; Murphy & Aguirre- Are´valo, E., Davis, S.K. & Sites, J.W., Jr (1994) Mitochondrial Leo´n, 2002). An mtDNA-only approach has several limitations DNA sequence divergence and phylogenetic relationships (Funk & Omland, 2003; Ballard & Whitlock, 2004) yet it is much among eight chromosome races of the Sceloporus grammicus more likely to detect geographical limits and cryptic species than complex (Phrynosomatidae) in central Mexico. Systematic studies based on nuclear DNA gene sequences (Moore, 1995; Biology, 43, 387–418. Hudson & Coyne, 2002; Zink & Barrowclough, 2008; Bar- Armstrong, B.L. & Murphy, J.B. (1979) The natural history of rowclough & Zink, 2009). Our genealogical inferences serve as a Mexican rattlesnakes. Special Publication No. 5 of the Museum robust hypothesis of matrilineal relationships within the ofNaturalHistoryUniversityofKansas,Lawrence,Kansas,KS. C. triseriatus species group. Future studies using unlinked Ballard, J.W.O. & Whitlock, M.C. (2004) The incomplete nat- nuclear loci can test whether or not the genealogy also reflects ural history of mitochondria. Molecular Ecology, 13, 729–744. the macroevolutionary species phylogeny. Barrowclough, G.F. & Zink, R.M. (2009) Funds enough, and time: mtDNA, nuDNA and the discovery of divergence. Molecular Ecology, 18, 1–3. ACKNOWLEDGEMENTS Becerra, J.X. (2005) Timing the origin and expansion of the We dedicate this study to the late Fernando Mendoza-Quijano. Mexican tropical dry forests. Proceedings of the National Without his enthusiasm and unfailing support through the Academy of Sciences USA, 102, 10919–10923.

Journal of Biogeography 38, 697–710 707 ª 2010 Blackwell Publishing Ltd R. W. Bryson Jr et al.

Brattstrom, B.H. (1964) Evolution of the pit vipers. Transac- archipelagos? The tempo and mode of diversification in SE tions of the San Diego Society of Natural History, 13, 185–268. Asian shrews. Evolution, 63, 2595–2610. Bryson, R.W., Pastorini, J., Burbrink, F.T. & Forstner, M.R.J. Felsenstein, J. (2004) Inferring phylogenies. Sinauer Associates, (2007) A phylogeny of the Lampropeltis mexicana complex Sunderland, MA. (Serpentes: Colubridae) based on mitochondrial DNA sequences Ferrusquı´a-Villafranca, I. (1993) Geology of Mexico: a syn- suggests evidence for species-level polyphyly within Lampropel- opsis. Biological diversity of Mexico: origins and distribution tis. Molecular Phylogenetics and Evolution, 43, 674–684. (ed. by T.P. Ramamoorthy, R.A. Bye, A. Lot and J. Fa), pp. Campbell, J.A. (1979) A new rattlesnake (Reptilia, Serpentes, 3–107. Oxford University Press, New York. Viperidae) from Jalisco, Mexico. Transactions of the Kansas Ferrusquı´a-Villafranca, I. & Gonza´lez-Guzma´n, L.I. (2005) Academy of Science, 81, 365–370. Northern Mexico’s landscape, part II: the biotic setting Campbell, J.A. & Armstrong, B.L. (1979) Geographic variation across time. Biodiversity, ecosystems, and conservation in in the Mexican pygmy rattlesnake, Sistrurus ravus, with the northern Mexico (ed. by J.-L.E. Cartron, G. Ceballos and R.S. description of a new subspecies. Herpetologica, 35, 304–317. Felger), pp. 39–51. Oxford University Press, Oxford. Campbell, J.A. & Lamar, W.W. (1989) The venomous reptiles of Funk, D.J. & Omland, K.E. (2003) Species-level paraphyly: Latin America. Cornell University Press, Ithaca, NY. frequency, causes, and consequences, with insights from Campbell, J.A. & Lamar, W.W. (2004) Venomous reptiles of the mitochondrial DNA. Annual Review of Ecology and Western Hemisphere. Cornell University Press, Ithaca, NY. Systematics, 34, 397–423. Carren˜o, A.L. & Helenes, J. (2002) Geology and ages of the Garcı´a-Moreno, J., Navarro-Sigu¨enza, A.G., Peterson, A.T. & islands. A new island biogeography of the Sea of Corte´s (ed. by Sa´nchez-Gonza´lez, L.A. (2004) Genetic variation coincides T.J. Case, M.L. Cody and E. Ezcurra), pp. 14–40. Oxford with geographic structure in the common bush-tanager University Press, New York. (Chlorospingus ophthalmicus) complex from Mexico. Castoe, T.A. & Parkinson, C.L. (2006) Bayesian mixed models Molecular Phylogenetics and Evolution, 33, 186–196. and the phylogeny of pitvipers (Serpentes: Viperidae). Gloyd, H.K. (1940) The rattlesnakes, genera Sistrurus and Molecular Phylogenetics and Evolution, 39, 91–110. Crotalus. A study in zoogeography and evolution. Special Castoe, T.A., Daza, J.M., Smith, E.N., Sasa, M.M., Kuch, U., Publication of the Chicago Academy of Sciences, 4, 1–270. Campbell, J.A., Chippindale, P.T. & Parkinson, C.L. (2009) Guiher, T.J. & Burbrink, F.T. (2008) Demographic and phy- Comparative phylogeography of pitvipers suggests a con- logeographic histories of two venomous North American sensus of ancient Middle American highland biogeography. snakes of the genus Agkistrodon. Molecular Phylogenetics and Journal of Biogeography, 36, 88–103. Evolution, 48, 543–553. Clark, R.W., Brown, W.S., Stechert, R. & Zamudio, K.R. Gutberlet, R.L., Jr & Harvey, M.B. (2004) The evolution of (2008) Integrating individual behaviour and landscape New World venomous snakes. The venomous reptiles of the genetics: the population structure of timber rattlesnake Western Hemisphere (ed. by J.A. Campbell and W.W. hibernacula. Molecular Ecology, 17, 719–730. Lamar), pp. 634–682. Cornell University Press, Ithaca, NY. Demastes, J.W., Spradling, T.A., Hafner, M.S., Hafner, D.J. & Hafner, M.S., Light, J.E., Hafner, D.J., Brant, S.V., Spradling, Reed, D.L. (2002) Systematics and phylogeography of T.A. & Demastes, J.W. (2005) Cryptic species in the Mexican pocket gophers in the genera Cratogeomys and Pappogeomys. pocket gopher, Cratogeomys merriami. Journal of - Molecular Phylogenetics and Evolution, 22, 144–154. ogy, 86, 1095–1108. Dorcas, M.E. (1992) Relationships among montane popula- Hall, T.A. (1999) BioEdit: a user-friendly biological sequence tions of Crotalus lepidus and Crotalus triseriatus. Biology of alignment editor and analysis program for Windows 95/98/ the pitvipers (ed. by J.A. Campbell and E.D. Brodie Jr), pp. NT. Nucleic Acids Symposium Series, 41, 95–98. 71–88. Selva, Tyler, TX. Harris, D., Rogers, D.S. & Sullivan, J. (2000) Phylogeography Douglas, M.E., Douglas, M.R., Schuett, G. & Porras, L. (2006) of Peromyscus furvus (Rodentia; Muridae) based on cyto- Evolution of rattlesnakes (Viperidae: Crotalus) in warm chrome b sequence data. Molecular Ecology, 9, 2129–2135. deserts of western North America shaped by Neogene Hillis, D.M. & Bull, J.J. (1993) An empirical test of boot- vicariance and Quaternary climate change. Molecular Ecol- strapping as a method for assessing confidence in phyloge- ogy, 15, 3353–3374. netic analysis. Systematic Biology, 42, 182–192. Drummond, A.J. & Rambaut, A. (2007) BEAST: Bayesian Ho, S.Y.W. & Phillips, M.J. (2009) Accounting for calibration evolutionary analysis by sampling trees. BMC Evolutionary uncertainty in phylogenetic estimation of evolutionary Biology, 7, 214. divergence times. Systematic Biology, 58, 367–380. Dunn, E.R. (1931) The herpetological fauna of the Americas. Holman, J.A. (2000) Fossil snakes of North America: origin, Copeia, 1931, 106–119. evolution, distribution, paleoecology. Indiana University Edwards, C.W. & Bradley, R.D. (2002) Molecular systematics Press, Indianapolis, IN. and historical phylobiogeography of the Neotoma mexicana Hudson, R.R. & Coyne, J.A. (2002) Mathematical consequences species group. Journal of Mammalogy, 83, 20–30. of the genealogical species concept. Evolution, 56, 1557–1565. Esselstyn, J.A., Timm, R.M. & Brown, R.M. (2009) Do geo- Jaeger, J.R., Riddle, B.R. & Bradford, D.F. (2005) Cryptic logical or climatic processes drive speciation in dynamic Neogene vicariance and Quaternary dispersal of the

708 Journal of Biogeography 38, 697–710 ª 2010 Blackwell Publishing Ltd Phylogeography of the Crotalus triseriatus group

red-spotted toad (Bufo punctatus): insights on the evolution Moreno-Letelier, A. & Pin˜ero, D. (2009) Phylogeographic of North American warm desert biotas. Molecular Ecology, structure of Pinus strombiformis Engelm. across the Chi- 14, 3033–3048. huahuan Desert filter-barrier. Journal of Biogeography, 36, Kelly, C.M.R., Barker, N.P., Villet, M.H. & Broadley, D.G. 121–131. (2009) Phylogeny, biogeography and classification of the Murphy, R.W. & Aguirre-Leo´n, G. (2002) The non-avian superfamily Elapoidea: a rapid radiation in the late reptiles: origins and evolution. A new island biogeography of Eocene. Cladistics, 25, 38–63. the Sea of Corte´s (ed. by T.J. Case, M.L. Cody and E. King, M.B. & Duvall, D. (1990) Prairie rattlesnake seasonal Ezcurra), pp. 181–220. Oxford University Press, New York. migrations: episodes of movement, vernal foraging and sex Murphy, R.W., Fu, J., Lathrop, A., Feltham, J.V. & Kovak, V. differences. Animal Behaviour, 39, 924–935. (2002) Phylogeny of the rattlesnakes (Crotalus and Sistrurus) Klauber, L.M. (1952) Taxonomic studies of the rattlesnakes of inferred from sequences of five mitochondrial DNA genes. mainland Mexico. Bulletin of the Zoological Society of San Biology of the vipers (ed. by G.W. Schuett, M. Hoˆggren, M.E. Diego, 26, 1–143. Douglas and H.W. Greene), pp. 69–92. Eagle Mountain Klauber, L.M. (1972) Rattlesnakes: their habits, life histories and Publishing, Eagle Mountain, UT. influence on mankind, 2nd edn. University of California Navarro-Sigu¨enza, A.G., Peterson, A.T., Nyari, A., Garcı´a- Press, Berkeley and Los Angeles, CA. Deras, G. & Garcı´a-Moreno, J. (2008) Phylogeography of the Leo´n-Paniagua, L., Navarro-Sigu¨enza, A.G., Herna´ndez-Ban˜os, Buarremon brush-finch complex (Aves, Emberizidae) in B.E. & Morales, J.C. (2007) Diversification of the arboreal Mesoamerica. Molecular Phylogenetics and Evolution, 47, 21– mice of the genus Habromys (Rodentia: Cricetidae: Neo- 35. tominae) in the Mesoamerican highlands. Molecular Phy- Nylander, J.A.A. (2004) MrModeltest v2. Program distributed logenetics and Evolution, 42, 653–664. by the author. Evolutionary Biology Centre, Uppsala Uni- Martin, P.S. & Harrell, B.E. (1957) The Pleistocene history of versity, Uppsala. temperate biotas in Mexico and eastern United States. Paniagua, L.L. & Morrone, J.J. (2009) Do the Oaxacan High- Ecology, 38, 468–480. lands represent a natural biotic unit? A cladistic biogeo- Masta, S.E. (2000) Phylogeography of the jumping spider graphical test based on vertebrate taxa. Journal of Habronattus pugillis (Araneae: Salticidae): recent vicariance Biogeography, 36, 1939–1944. of sky island populations? Evolution, 54, 1699–1711. Parmley, D. & Holman, J.A. (2007) Earliest fossil record of a McCormack, J.E., Peterson, A.T., Bonaccorso, E. & Smith, T.B. pigmy rattlesnake (Viperidae: Sistrurus Garman). Journal of (2008a) Speciation in the highlands of Mexico: genetic and Herpetology, 41, 141–144. phenotypic divergence in the Mexican jay (Aphelocoma Peterson, A.T., Flores-Villela, O.A., Leo´n-Paniagua, L.S., ultramarina). Molecular Ecology, 17, 2505–2521. Llorente-Bousquets, J.E., Luis-Martinez, M.A., Navarro- McCormack, J.E., Bowen, B.S. & Smith, T.B. (2008b) Inte- Siguenza, A.G., Torres-Chavez, M.G. & Vargas-Fernandez, I. grating paleoecology and genetics of bird populations in two (1993) Conservation priorities in northern Middle America: sky island archipelagos. BMC Biology, 6, 28. moving up in the world. Biodiversity Letters, 1, 33–38. McCranie, J.R. (1988) Description of the hemipenis of Sistrurus Pook, C.E., Wu¨ster, W. & Thorpe, R.S. (2000) Historical ravus (Serpentes: Viperidae). Herpetologica, 44, 123–126. biogeography of the western rattlesnake (Serpentes: Viperi- McDonald, J.A. (1993) Phytogeography and history of the al- dae: Crotalus viridis) inferred from mitochondrial DNA pine–subalpine flora of northeastern Mexico. Biological sequence information. Molecular Phylogenetics and Evolu- diversity in Mexico: origins and distribution (ed. by T.P. tion, 15, 269–282. Ramamoorthy, R. Bye, A. Lot and J. Fa), pp. 681–703. Puebla-Olivares, F., Bonaccorso, E., Espinosa de los Monteros, Oxford University Press, New York. A., Omland, K.E., Llorente-Bousquets, J.E., Peterson, A.T. & Mejı´a-Madrid, H.H., Va´zquez-Domı´nguez, E. & Pe´rez-Ponce de Navarro-Sigu¨enza, A.G. (2008) Speciation in the emerald Leo´n, G. (2007) Phylogeography and freshwater basins in central toucanet (Aulacorhynchus prasinus) complex. The Auk, 125, Mexico: recent history as revealed by the fish parasite Rhabdoch- 39–50. ona lichtenfelsi (Nematoda). Journal of Biogeography, 34, 787–801. Rabosky, D.L. (2006a) Likelihood methods for inferring Mittermeier, R.A., Gil, P.R., Hoffman, M., Pilgrim, J., Brooks, temporal shifts in diversification rates. Evolution, 60, 1152– T., Mittermeier, C.G., Lamoreux, J. & da Fonseca, G.A.B. 1164. (2005) Hotspots revisited: Earth’s biologically richest and most Rabosky, D.L. (2006b) LASER: a maximum likelihood toolkit endangered terrestrial ecoregions. Conservation International, for detecting temporal shifts in diversification rates from Washington, DC. molecular phylogenies. Evolutionary Bioinformatics Online, Moore, W.S. (1995) Inferring phylogenies from mtDNA vari- 2, 257–260. ation: mitochondrial gene trees versus nuclear gene trees. Ramamoorthy, T., Bye, R., Lot, A. & Fa, J. (1993) Biological Evolution, 49, 718–726. diversity of Mexico: origins and distribution. Oxford Univer- Morafka, D.J. (1977) A biogeographical analysis of the Chi- sity Press, Oxford. huahuan desert through its herpetofauna. Biogeographica, 9, Rambaut, A. & Drummond, A.J. (2007) Tracer v1.4. Available 1–313. at: http://beast.bio.ed.ac.uk/Tracer.

Journal of Biogeography 38, 697–710 709 ª 2010 Blackwell Publishing Ltd R. W. Bryson Jr et al.

Riddle, B.R. & Hafner, D.J. (2006) A step-wise approach to Wu¨ster, W., Ferguson, J.E., Quijana-Mascaren˜as, J.A., Pook, integrating phylogeographic and phylogenetic biogeographic C.E., Saloma˜o, M.G. & Thorpe, R.S. (2005) Tracing an perspectives on the history of a core North American warm invasion: landbridges, refugia and the phylogeography of the deserts biota. Journal of Arid Environments, 66, 435–461. Neotropical rattlesnake (Serpentes: Viperidae: Crotalus Riddle, B.R., Hafner, D.J. & Alexander, L.F. (2000) Phyloge- durissus). Molecular Ecology, 14, 1095–1108. ography and systematics of the Peromyscus eremicus species Wu¨ster, W., Peppin, L., Pook, C.E. & Walker, D.E. (2008) A group and the historical biogeography of North American nesting of vipers: phylogeny and historical biogeography of warm regional deserts. Molecular Phylogenetics and Evolu- the Viperidae (: Serpentes). Molecular Phyloge- tion, 17, 145–160. netics and Evolution, 49, 445–459. Rodrı´guez-Banderas, A., Vargas-Mendoza, C.F., Buonamici, A. Zaldivar-Rivero´n, A., Nieto-Montes de Oca, A. & Laclette, J.P. & Vendramin, G.G. (2009) Genetic diversity and phyloge- (2005) Phylogeny and evolution of dorsal pattern in the ographic analysis of Pinus leiophylla: a post-glacial range Mexican endemic genus Barisia (Anguidae: Gerrho- expansion. Journal of Biogeography, 36, 1807–1820. notinae). Journal of Zoological Systematics and Evolutionary Ronquist, F. & Huelsenbeck, J.P. (2003) MRBAYES 3: Bayesian Research, 43, 243–257. phylogenetic inference under mixed models. Bioinformatics, Zink, R.M. & Barrowclough, G.F. (2008) Mitochondrial DNA 19, 1572–1574. under siege in avian phylogeography. Molecular Ecology, 17, Smith, C.I. & Farrell, B.D. (2005) Range expansions in the 2107–2121. flightless longhorn cactus beetles, Moneilema gigas and Moneilema armatum, in response to Pleistocene climate SUPPORTING INFORMATION changes. Molecular Ecology, 14, 1025–1044. Smith, H.M. (1946) Preliminary notes and speculations on the Additional Supporting Information may be found in the Triseriatus group of rattlesnakes in Me´xico. University of online version of this article: Kansas Science Bulletin, 31, 75–101. Appendix S1 Collection and voucher data for genetic Stamatakis, A. (2006) RAxML-VI-HPC: Maximum likelihood- samples used in this study and deposited in the Royal Ontario based phylogenetic analyses with thousands of taxa and Museum (ROM). mixed models. Bioinformatics, 22, 2688–2690. Appendix S2 GenBank accession numbers for genetic Sullivan, J., Markert, J.A. & Kilpatrick, C.W. (1997) Phyloge- samples used in this study. ography and molecular systematics of the Peromyscus aztecus group (Rodentia: Muridae) inferred using parsimony and As a service to our authors and readers, this journal provides likelihood. Systematic Biology, 46, 426–440. supporting information supplied by the authors. Such mate- Sullivan, J., Arellano, E. & Rogers, D.S. (2000) Comparative rials are peer-reviewed and may be re-organized for online phylogeography of Mesoamerican highland : con- delivery, but are not copy-edited or typeset. Technical support certed versus independent response to past climatic fluctu- issues arising from supporting information (other than ations. The American Naturalist, 155, 755–768. missing files) should be addressed to the authors. Tennessen, J.A. & Zamudio, K.R. (2008) Genetic differentia- tion among mountain island populations of the striped plateau lizard, Sceloporus virgatus (Squamata: Phrynoso- matidae). Copeia, 2008, 558–564. Toledo, V.M. (1982) Pleistocene changes of vegetation in trop- BIOSKETCHES ical Mexico. Biological diversification in the tropics (ed. by G.T. Prance), pp. 93–111. Columbia University Press, New York. Robert W. Bryson Jr and Robert W. Murphy began this Upton, D.E. & Murphy, R.W. (1997) Phylogeny of the side- collaborative research while R.W.B. was a Master’s student at blotched lizards (Phrynosomatidae: Uta) based on mtDNA Sul Ross State University, building on their shared interests in sequences: support for a midpeninsular seaway in Baja Cal- rattlesnake systematics and biogeography of Mexico. All ifornia. Molecular Phylogenetics and Evolution, 8, 104–113. authors are broadly interested in better understanding the Van Devender, T.R. (1990) Late Quaternary vegetation and biodiversity of Mexico through evolutionary (R.W.B., R.W.M., climate of the Chihuahuan Desert, United States and Mex- A.L.) and ecological (D.L.V.) studies. ico. Packrat middens. The last 40,000 years of biotic change Author contributions: R.W.B. and R.W.M. conceived the (ed. by J.L. Betancourt, T.R. Van Devender and P.S. Mar- ideas; R.W.B., D.L.V. and A.L. collected the data; R.W.B., tin), pp. 104–133. University of Arizona Press, Tucson, AZ. R.W.M. and A.L. analysed the data; and R.W.B. and R.W.M. Wilgenbusch, J.C., Warren, D.L. & Swofford, D.L. (2004) led the writing. AWTY: a system for graphical exploration of MCMC con- vergence in Bayesian phylogenetic inference. Available at: http://ceb.csit.fsu.edu/awty. Editor: S¸erban Proches¸

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