Journal of Biogeography (J. Biogeogr.) (2011) ORIGINAL Ephemeral Pleistocene woodlands ARTICLE connect the dots for highland rattlesnakes of the Crotalus intermedius group Robert W. Bryson Jr1*, Robert W. Murphy2,3, Matthew R. Graham1, Amy Lathrop2 and David Lazcano4 1School of Life Sciences, University of Nevada, ABSTRACT Las Vegas, 4505 Maryland Parkway, Las Aim To test how Pleistocene climatic changes affected diversification of the Vegas, NV 89154-4004, USA, 2Centre for Biodiversity and Conservation Biology, Royal Crotalus intermedius species complex. Ontario Museum, Toronto, ON M5S 2C6, Location Highlands of Mexico and the south-western United States (Arizona). Canada, 3State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Methods We synthesize the matrilineal genealogy based on 2406 base pairs of Zoology, The Chinese Academy of Sciences, mitochondrial DNA sequences, fossil-calibrated molecular dating, reconstruction Kunming 650223, China, 4Laboratorio de of ancestral geographic ranges, and climate-based modelling of species Herpetologı´a, Universidad Auto´noma de distributions to evaluate the history of female dispersion. Nuevo Leo´n, San Nicolas de los Garza, Nuevo Results The presently fragmented distribution of the C. intermedius group is the Leo´n CP 66440, Mexico result of both Neogene vicariance and Pleistocene pine–oak habitat fragmentation. Most lineages appear to have a Quaternary origin. The Sierra Madre del Sur and northern Sierra Madre Oriental are likely to have been colonized during this time. Species distribution models for the Last Glacial Maximum predict expansions of suitable habitat for taxa in the southern Sierra Madre Occidental and northern Sierra Madre Oriental. Main conclusions Lineage diversification in the C. intermedius group is a consequence of Pleistocene climate cycling. Distribution models for two sister taxa in the northern and southern Sierra Madre Occidental and northern Sierra Madre Oriental during the Last Glacial Maximum provide evidence for the expansion of pine–oak habitat across the Central Mexican Plateau. Downward displacement and subsequent expansions of highland vegetation across Mexico during cooler glacial cycles may have allowed dispersal between highlands, which resulted in contact between previously isolated taxa and the colonization of new *Correspondence: Robert W. Bryson Jr, School habitats. of Life Sciences, University of Nevada, Las Keywords Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA. Ancestral area reconstruction, Crotalus intermedius, divergence dating, Mexico, E-mail: [email protected] niche modelling, phylogeography, Quaternary, rattlesnakes. del Sur (SMS) in central and southern Mexico. The highland INTRODUCTION biotas on each range are isolated by intervening dry lowlands. The Mexican highlands, which encompass a complex assem- Most notably, the highlands of the northern Sierra Madres are blage of montane biotas characterized by high endemism and separated by hundreds of kilometres of xeric habitat formed by diversity (Ramamoorthy et al., 1993; Campbell, 1999), are one the Chihuahuan Desert and Central Mexican Plateau. of Earth’s biodiversity hotspots (Mittermeier et al., 2005). They Although few data detail climatic shifts in Mexico during the consist of four major mountain ranges predominantly covered Pleistocene (see Metcalfe et al., 2000), available evidence by mixed pine–oak (Pinus–Quercus) woodlands, namely the suggests that conditions across northern and central Mexico north–south-trending Sierra Madre Occidental (SMOc) and were wetter and cooler during the Last Glacial Maximum Sierra Madre Oriental (SMOr) of northern Mexico, and the (LGM) than they are now. McDonald (1993) reports a east–west-trending Transvolcanic Belt (TVB) and Sierra Madre downward displacement of montane vegetation of at least ª 2011 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/jbi 1 doi:10.1111/j.1365-2699.2011.02565.x R. W. Bryson Jr et al. 1000 m during glacial times. This displacement may have approach is controversial (e.g. Edwards & Bensch, 2009), yet united highland biotas via pine–oak corridors across the these data can detect recent geographic patterns (Moore, 1995; Central Mexican Plateau (Baker, 1956; Martin, 1958; Duell- Hudson & Coyne, 2002; Zink & Barrowclough, 2008; Bar- man, 1965; Morafka, 1977; Schmidly, 1977; McCranie & rowclough & Zink, 2009) and lead to significant biogeographic Wilson, 1987; Fa & Morales, 1993; McDonald, 1993; Marshall discoveries (e.g. Upton & Murphy, 1997; Riddle et al., 2000). & Liebherr, 2000). During post-glacial episodes throughout the Maternal history remains an important tool for exploring the Pleistocene, montane woodlands seem to have retracted to the genetic consequences of ecological history (e.g. Wiens et al., highlands, thus isolating the associated biota (Metcalfe et al., 2007; Burney & Brumfield, 2009; Pyron & Burbrink, 2009), 2000; Anducho-Reyes et al., 2008). partly because genetic recombination and gene sorting do not The highland biotas of the northern Sierra Madres appear to occur. have had a recent connection (Marshall & Liebherr, 2000). Here, we explore the effects of Pleistocene climatic change Several dispersal corridors across central Mexico are postulated on the diversification of the highland rattlesnakes of the for the Pleistocene (Duellman, 1965; McCranie & Wilson, C. intermedius group. Montane Pleistocene corridors may have 1987; Fa & Morales, 1993; McDonald, 1993) or Pliocene facilitated contact of these rattlesnakes through dispersal. We (Morafka, 1977). The best evidence for these corridors comes model the distribution of C. pricei because this taxon currently from multiple species with closely related, disjunct, highland shows the greatest extent of geographic isolation of any species populations. These species are either absent or replaced by in the C. intermedius group. If populations were connected sister taxa in the Transvolcanic Belt. Martin (1958) notes during the last glaciation, then distribution models projected numerous taxa that fit this pattern, including pines, snails, onto Late Pleistocene climates should demonstrate habitat non-avian reptiles and mammals. However, confirmation of connectivity. these patterns using molecular data is largely lacking (see Appendix S1 in Supporting Information). Only two studies, MATERIALS AND METHODS focused on alligator lizards (Zaldivar-Rivero´n et al., 2005) and Mexican jays (McCormack et al., 2008, 2010), estimate dates of Taxon sampling and laboratory methods divergence between isolated sister taxa in the northern Sierra Madres, and both pre-date Pleistocene cycles. Thus, despite Between 1999 and 2009 we collected 60 samples that repre- being over 50 years old, the hypothesis of a Pleistocene woodland sented all taxa in the C. intermedius group from throughout corridor across central Mexico remains largely untested. their distributions (Fig. 1; Appendix S2). Sampling was con- In this study, we examine the historical diversification of the centrated along the northern Sierra Madres to maximize Crotalus intermedius species complex, a group of rattlesnakes geographic coverage of C. pricei pricei and C. pricei miquihu- endemic to the Mexican highlands. This group is closely anus. Based on recent phylogenetic analyses (Murphy et al., associated with mixed pine–oak forests above 1900 m eleva- 2002; Castoe & Parkinson, 2006; Wu¨ster et al., 2008), we used tion, and it is distributed throughout all of the major Sistrurus catenatus and S. miliarus as outgroup taxa. All mountains of Mexico (Campbell & Lamar, 2004). Crotalus handling of animals followed animal use protocols approved intermedius contains three subspecies (Campbell & Lamar, by the University of Nevada at Las Vegas Animal Care 2004) disjunctly distributed primarily in pine–oak forest above Committee (R701-1105-203). 2000 m along the southern SMOr (C. intermedius intermedius) We sequenced three regions of the mitochondrial genome, and SMS (C. intermedius gloydi and C. intermedius omiltem- namely a portion of the 12S and 16S ribosomal RNA genes, anus). Similarly, Crotalus pricei pricei is isolated above 1900 m NADH dehydrogenase subunit 4 (ND4) and its flanking in the SMOc, and Crotalus pricei miquihuanus occurs in the tRNAs, and the complete ATPase subunits 8 and 6 (ATPase 8, SMOr (Campbell & Lamar, 2004). Finally, the sister species ATPase 6), following methods specified in Bryson et al. Crotalus transversus and C. tancitarensis are distributed in (2011a). These gene regions have been shown to be informa- pine–oak and pine–fir (Pinus–Abies) forests at elevations above tive at inter- and intraspecific levels within rattlesnakes 2900 m on the TVB (Alvarado-Dı´az & Campbell, 2004; (Murphy et al., 2002; Wu¨ster et al., 2005; Bryson et al., Campbell & Lamar, 2004). These patterns offer an opportunity 2011a). Forward and reverse sequences for each individual to test hypotheses on the role of Pleistocene pine–oak were edited and manually aligned using BioEdit 5.0.9 (Hall, fragmentation in shaping patterns of genetic variation across 1999). Identical sequences for samples from the same locality the highlands of Mexico. were collapsed into one haplotype. Average uncorrected Recent methodological advances provide useful tools in the pairwise divergences between these groups (p-distances) were reconstruction of the evolutionary history of species. Mito- determined using mega 4 (Kumar et al., 2008). chondrial DNA (mtDNA) sequences can be used for fossil- calibrated