Molecular Ecology (2006)

15

, 3353–3374 doi: 10.1111/j.1365-294X.2006.03007.x

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

Blackwell Publishing Ltd

Evolution of rattlesnakes (Viperidae;

Crotalus

) in the warm deserts of western North America shaped by Neogene vicariance and Quaternary climate change

MICHAEL E . DOUGLAS,

*

MARLIS R . DOUGLAS,

*

GORDON W. SCHUETT

†

and LOUIS W. PORRAS

‡

*

Department of Fish, Wildlife and Conservation Biology and Graduate Degree Program in Ecology, Colorado State University, Ft. Collins, Colorado 80523-1474 USA,

†

Department of Biology and Center for Behavioural Neuroscience, Georgia State University, Atlanta, Georgia 30303-3088 USA,

‡

7705 N. Wyatt Earp Avenue, Eagle Mountain, Utah 84005 USA

Abstract

During Pleistocene, the Laurentide ice sheet rearranged and diversified biotic distributionsin eastern North America, yet had minimal physical impact in western North Americawhere lineage diversification is instead hypothesized to result from climatic changes. IfPleistocene climatic fluctuations impacted desert species, the latter would reflect patternsof restricted gene flow concomitant with indications of demographic bottlenecks. Accord-ingly, molecular evidence for refugia should be present within these distributions and forsubsequent range expansions as conditions improved. We sought answers to these ques-tions by evaluating mitochondrial DNA (mtDNA) sequences from four species of rattle-snakes [

Crotalus mitchellii

(speckled rattlesnake),

Crotalus cerastes

(sidewinder),

Crotalustigris

(tiger rattlesnake),

Crotalus ruber

(red diamond rattlesnake)] with distributionsrestricted to desert regions of southwestern North America. We inferred relationshipsusing parsimony and maximum likelihood, tested intraspecific clades for populationexpansions, applied an isolation-with-migration model to determine bi-directional migra-tion rates (

m

) among regions, and inferred divergence times for species and clades byapplying a semiparametric penalized likelihood approach to our molecular data. Evidencefor significant range expansion was present in two of eight regions in two species(

Crotalus mitchellii pyrrhus

,

C. tigris

region north). Two species (

C. cerastes

,

C. mitchellii

)showed a distribution concomitant with northward displacement of Baja California frommainland México, followed by vicariant separation into subclades. Effects of Pleistoceneclimate fluctuations were found in the distributions of all four species. Three regionaldiversification patterns were identified: (i) shallow genetic diversity that resulted fromPleistocene climatic events (

C. tigris, C. ruber

); (ii) deep Pleistocene divisions indicatingallopatric segregation of subclades within refugia (

C. mitchellii, C. cerastes)

; and (iii) line-age diversifications that extended to Pliocene or Late Miocene (

C. mitchellii, C. cerastes)

.Clade-diversifying and clade-constraining effects impacted the four species of rattlesnakesunequally. We found relatively high levels of molecular diversification in the two mostbroadly distributed species (

C. mitchellii, C. cerastes

), and lower levels of genetic diversi-fication in the two species (

C. tigris, C. ruber

) whose ranges are relatively more restricted.Furthermore, in several cases, the distributions of subspecies were not congruent with ourmolecular information. We suggest regional conservation perspectives for southwesterndeserts cannot rely upon subspecies as biodiversity surrogates, but must instead employ amolecular and deep historical perspective as a primary mechanism to frame biodiversityreserves within this region.

Keywords

: ATPase, Baja Peninsula, glaciation, mtDNA, phylogeography, Pleistocene, Pliocene

Received 11 December 2005; revision accepted 2 May 2006

Correspondence: Michael E. Douglas, Fax: 970-491-5091; E-mail: michael.douglas@colostate.edu

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Introduction

During glacial cycles, the sheer physical presence of icesheets restructured the biogeography of organisms andseverely impacted global and regional climates (Clark

et al

.1995). Populations of plants and animals were repeatedlyfragmented by abrupt temperature fluctuations as shortinterglacials were initiated or terminated (Bennett 1990).The rapidity of these changes overwhelmed evolutionaryprocess and induced widespread extinctions, except whereclimate remained within tolerance limits for a species (aswithin a refugium; Hewitt 1996, 2000). Despite theseevents, Pleistocene effects on speciation are deemedequivocal (reviewed by Lovette 2005), primarily becausetraces of diversification or collapse are often difficult topinpoint or decipher.

In North America, Pleistocene effects varied amongregions as reflected by the number of biotic studies. A pre-ponderance of research has been conducted in easternNorth America, while fewer have focused on westernregions, or more importantly, compare the two (but seeHoffman & Blouin 2004). This paucity of information maybe partly explained by the fact that the ice sheets had min-imal physical impact on terrain in western North America,yet clearly impacted its climate (in tandem with low atmos-pheric greenhouse gas concentrations, low summer insola-tion, and cold northern sea-surface temperatures; Licciardi

et al

. 2004). During maximum glaciation, summer temper-atures across the western continental interior were at least8–14

°

C below modern levels (Owen

et al

. 2003), and eleva-tional ranges of plants were 600–1200+ m below modern(Thompson

et al

. 1993). With the exception of a fewregional western glaciers [i.e. Wind River (western Wyo-ming), Sierra Nevada (eastern California), and San Ber-nardino (southern California), Licciardi

et al

. 2004)], thosein Late Quaternary displayed reasonably synchronousdynamics across the North American Cordilleras (i.e. thatcomplex of overlapping mountain ranges encompassingthe Rocky Mountains, Sierra Nevada, Sierra Madre Orien-tal, and Sierra Madre Occidental) (Owen

et al

. 2003).Were these climatic effects serious enough to diversify

taxa across western North America? Weir and Schluter(2004) demonstrated that diversification in boreal bird lin-eages was twice as great as that in subboreal or Neotropicalregions, indicating that physical vicariance of the ice sheetwas more important for lineage diversification thancontinent-wide climate shifts that accompanied glacialadvances. Furthermore, major changes in the mammalianbiota of western North America during Late Palaeocene(65–55 Ma), as measured by origination/extinction ratesand proportional taxonomic turnover, were not associatedwith global climate change (Alroy

et al

. 2000). Interest-ingly, no correlation has been found between climaticevents and any episode of mammalian turnover. Most of

the latter, as tracked through the entire Cenozoic (65 Ma–present) occurred during periods of relative faunal stabil-ity (Prothero 2004). Thus, it is difficult to envision thePleistocene as strongly affecting the biota of western NorthAmerica, particularly when compared to eastern biotawhere the Laurentide Ice Sheet held sway (see also Riddle1995). Geophysical processes, nonetheless, have clearlyimpacted western North America and have resonatedthroughout its resident biota (herpetofauna: Morafka 1977;fishes: Minckley

et al

. 1986; Douglas

et al

. 1999; Oakey

et al

.2004; mammals: Riddle

et al

. 2000). Accordingly, a vicari-ant signature should be apparent within the deep historyof this biota (per Avise 2000), whereas shallower (poten-tially climate-induced) effects should be equivocal andopen to question.

The fact that western North America remained unglaci-ated is an advantage when trying to decipher climaticeffects on regional biota. For example, if distributions andabundances of organisms in western North America weresignificantly impacted by Pleistocene climate variation, aneffect would be best ascertained in the warm southwesterndeserts (as per Smith & Farrell 2005). The complex topog-raphy of this area (Oakey

et al

. 2004) provided great poten-tial for development of refugia and associated isolation ofpopulations, thereby allowing climatic effects at theregional level to be most strongly demonstrated. Thus, todetermine whether Pleistocene climate seriously impactedfaunal biogeography in nonglaciated regions, one shouldlook for signatures within the biota of the southwesterndeserts.

Before hypotheses regarding effects of climatic fluctua-tions can be examined in these regions, the manner inwhich deserts evolved must be delineated. Large-scaleorogeny and rifting were major developmental factors.The uplift of the North American Cordilleras essentiallydifferentiated western from eastern and mid-westernNorth America, and was completed in Late Oligocene bythe collision of the East Pacific Rise and the North Ameri-can Plate (summarized in Minckley

et al

. 1986). The rise ofthese mountains disrupted upper level atmospheric circu-lation and truncated moist tropical inflows from the PacificOcean and Gulf of México, thus inducing regional westernaridity (Axelrod 1979). The warm deserts then evolved inresponse to this altered Mid-Miocene climate, with theSonoran Desert developing from pre-existing tropicalthorn-scrub and scattered semidesert in west-centralMéxico. It was well established by Late Miocene (8–5 Ma),thus making it one of the youngest biotic communities ofNorth America (Axelrod 1979, Fig. 18).

An additional and complex factor in Sonoran Desertevolution was development of Baja California (Murphy1983; Grismer 1994, 2002; Murphy & Aguirre-Léon 2002).This peninsula transferred from the North American to thePacific plate

c

. 12.5 Ma, and pulled gradually away from

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mainland México over a 4–5 Myr span. It rifted approxi-mately 500 km northwest along the San Andreas Fault(Gans 1997), allowing the proto Sea of Cortéz to advanceand effectively divide species along its path into Baja and/or western Méxican components (Fig. 1a–e). More south-erly species were bisected first (termed the ‘southernMiocene vicariant complex’) followed by more north-ern species (‘northern Pliocene vicariant complex’) asdescribed by Grismer (1994). By Late Pliocene, the Sea ofCortéz extended deep into California and Arizona (Fig. 1f).Regression of the sea in Mid-Pleistocene again allowedclades to potentially contact one another.

In this study, we searched for impacts of Pleistoceneevents in southwestern North America by deriving phylo-geographical histories of four rattlesnake species with dis-tributions restricted to this region. We assessed more thanone species because each might have responded both indi-vidually and differently to the same climatic or vicariantevent. We predicted (per Avise 2000) that if vicariantevents similarly impacted our four study species, theirphylogeographical patterns would be concordant. If our

study species also were impacted by Pleistocene climatechanges, the effects would be shallower and mitigated bytheir ecologies, with those in similar niches expressing par-allel effects. Additionally, we recognize the differentiationand collapse of clades can be affected by emergent pro-perties specific to each, such as range size, level of frag-mentation, and overall ecology (Stanley 1998; Gould 2002).Comparisons among clades with regard to vicariantand climatic effects must take these characteristics intoconsideration.

Before we examined for codistributed effects, weascertained whether distinct mtDNA lineages were presentwithin each of our study species (as suggested by sub-specific nomenclature), and delineated their geographicalstructure. Once these aspects were clarified, we posed thefollowing questions:

1

Are biogeographical distributions of intraspecific line-ages, the relative timing of their cladogenetic events, andthe observed levels of diversity within regions congruentwith Pleistocene (or older) events?

2

Are local and/or regional levels of genetic diversitysimilar within and congruent across species? A positiveanswer to this question would suggest a unilateral re-sponse to processes in the region.

3

Are molecular data concordant with previously re-cognized subspecies? Can the latter be utilized to developa regional landscape perspective for conserving andmanaging biodiversity within the warm deserts ofsouthwestern North America? This issue is impera-tive primarily because consumptive accesses to scarceresources by agribusiness, coupled with unbridledurban growth, seriously threaten biotic associations andregional structuring in the southwestern deserts. Clarify-ing faunal diversity in these regions (i.e. substantiatingpreviously determined subspecific variation) is a long-overdue first step toward a regional management planthat will conserve these unique biomes and their residentbiotas.

Methods

Study species

We studied four species of rattlesnakes that are endemic orlargely restricted to the southwestern deserts of NorthAmerica:

Crotalus mitchellii, Crotalus cerastes, Crotalus tigris,

and

Crotalus ruber

. Taxonomic and/or natural historysummations for each species are presented in Klauber(1972), McDiarmid

et al

. (1999), Grismer (2002), andCampbell & Lamar (2004). We followed the taxonomicarrangements presented by the latter authors because it isan up-to-date synthesis. Ecological data for these taxa arelargely derived from Klauber (1972) and Stebbins (2003).

Fig. 1 Development of the Baja Peninsula from mainland SonoraMéxico (A–E), where S1 and S2, distributions of the ‘southernMiocene vicariant complex’; N1, distribution of the ‘northernPliocene vicariant complex’ (following Grismer 1994); sc, Sea ofCortéz; black box, northern Sea of Cortéz depicted in the drawingbelow; cross-hatching, areas of overlap among S1, S2 and N1. (F)represents the marine transgression into southeastern Californiaand the lower Colorado River Valley (California and Arizona)during Pliocene (adapted from Dunham & Allison 1960). TheSalton Trough (left-side finger) and the Bouse Embayment(Colorado River Valley) are depicted.

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1

Crotalus mitchellii

(speckled rattlesnake) is a small-to medium-sized (58–111 cm) species, except for theinsular subspecies

Crotalus m. angelensis

, which attains agreater length (137 cm). It occupies rocky areas in deserthabitats in the vicinity of sagebrush, creosote, and succu-lent desert habitats, but it also occurs in thornscrub,chaparral, and pinyon-juniper woodland. Five sub-species are recognized (

C. m. angelensis

,

C. m. mitchellii

,

C. m. muertensis

,

C. m. pyrrhus

, and

C. m. stephensi

), buttwo insular races (

angelensis

and

muertensis

) have beenproposed as full species (Grismer 1999). Insular populationswere not investigated in this study.

2

Crotalus cerastes

(sidewinder) is a small-sized (43–84 cm)species. It is most often found in areas of fine windblownsand where hummocks are topped with creosote,mesquite, and other desert plants, but they also occuron windswept flats, barren dunes, desert hardpan,and rocky hillsides. Three subspecies are recognized(

C. c. cerastes, C. c. cercobombus,

and

C. c. laterorepens

).

3

Crotalus tigris

(tiger rattlesnake) is a medium-sized (46–91 cm) species, largely restricted to rocky canyons, andthe foothills of mountains, up to the oak belt whereit occupies habitats with cactus, mesquite, creosote,ocotillo, saguaro, and palo verde. It does not occur inopen desert flats. No subspecies have been formallyrecognized.

4

Crotalus ruber

(red diamond rattlesnake) is a longand large-bodied (76–165 cm) species. It inhabits desertscrub, thornscrub, coastal sagebrush, chaparral, wood-land, grassland, cultivated areas, rocky alluvial fans, andthe desert floor. Four subspecies are recognized (

C. r.exsul

,

C. r. lorenzoensis

,

C. r. lucasensis

, and

C. r. ruber

), buttheir taxonomic stability is controversial (see Discus-sion). We studied one insular subspecies (

C. r. exsul

).

Generalist vs. specialist species

It is difficult, perhaps even imprudent, to assign species tosimple binary categories such as ‘generalist’ or ‘specialist.’Clearly, by their very definition, all species differ in someaspect of their biology and natural history. Thus, when a

generalist or specialist tag is used to describe an aspect ofa species, the particular character or environmentalassociation must be explicitly stated. Accordingly, of ourfour study rattlesnakes, we suggest two (

C. mitchellii

and

C.cerastes

) are generalists based on broader geographicaldistributions and wider habitat associations, while theothers (

C. tigris

and

C. ruber

) are specialists due torelatively limited geographical distributions and narrowerhabitat associations (Table 1).

With respect to biogeography, both generalists (

C. mitch-ellii

and

C. cerastes

) penetrate higher latitudes, whichinclude the Colorado Plateau and the Great Basin Desert,and occupy a greater diversity of habitats, including sanddunes. Although they share a common northern latitudi-nal limit (Fig. 2),

C. cerastes

has a shallower southern distri-bution in Baja California. Both of the specialists (

C. tigris

and

C. ruber

) are similar in that their ranges are restrictedto warm regions (Sonoran Desert only —

C. tigris

; Sonoranand Mohave deserts —

C. ruber

) within the boundaries ofsimilar northern and southern latitudes (see Fig. 2), buteach differs in the types of habitats occupied, with

C. ruber

being broader.

Collection, DNA extraction and amplification

From 1998 to 2005, we sampled the distributional breadthof each species (Fig. 2) and (except for some insularpopulations) obtained 181 total specimens distributedas follows:

C. mitchellii mitchellii

(

N

= 3),

C. m. pyrrhus

(

N

= 83),

C. m. stephensi

(

N

= 18),

C. cerastes cerastes

(

N

= 6),

C. c. cercobombus

(

N

= 12),

C. c. laterorepens

(

N

= 3),

C. tigris

(

N

= 38),

C. ruber ruber

(

N

= 9),

C. r. lucasensis

(

N

= 1),

C. r.exsul

(

N

= 1). Also, we employed

Crotalus atrox

(

N

= 4),

Crotalus molossus

(

N

= 1),

Crotalus enyo

(

N

= 1), and

Crotaluspolystictus

(

N

= 1) as outgroups, based upon two mtDNA-based phylogenetic hypotheses (Murphy

et al

. 2002,Douglas

et al

., unpublished). For comparative purposes,we employed

C. atrox

as the sister-taxon to

C. ruber

(Murphy

et al

. 2002)

,

and

C. polystictus

/

C. enyo

as sister to

C. cerastes

(Douglas

et al., unpublished). Locality informationis provided in the Appendix.

Table 1 The four species of Crotalus in this study are compared according to extent of their contemporary range (Distribution), level ofecological specialization (Ecology), and number of subspecies (Taxonomy). Also presented are number of molecular clades at sequencedivergence (SD) = 0.5% and 1.0% (or greater)

Species Distribution Ecology Taxonomy Clades (0.5% SD) Clades (1.0% SD)

C. mitchellii broad generalist 3 subspecies* 12 6C. cerastes broad generalist 3 subspecies 5 5C. tigris restricted specialist 0 subspecies 2 1C. ruber restricted specialist 4 subspecies† 1 1

*The two insular subspecies (angelensis and muertensis) from Baja, California were not evaluated in this study.†One of the insular subspecies (lorenzoensis) from Baja, California was not evaluated in this study.

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In live rattlesnakes, c. 0.1 mL whole blood was removedfrom the caudal vein and stored in 1 ml 100% EtOH. Occa-sionally, liver and muscle were obtained from road killsand these were either frozen or preserved as above.Shed skins of captive animals also were used as a DNAsource. Total genomic DNA was isolated using Pure-Gene DNA Isolation Kit (D-70KB; Gentra Systems, Inc.and stored in DNA hydrating solution (same kit).Mitochondrial ATPase 8 and ATPase 6 genes were ampli-fied using primers specified in Douglas et al. (2002).

Double-stranded sequencing reactions were conductedwith fluorescently labelled dideoxy terminators accordingto manufacturer recommendations [Applied BiosystemsInc. (ABI)]. Labeled extension products were separatedand analysed with an automated DNA sequencer (ABIPRISM 3100). Sequences were aligned manually usingsequencher (Gene Codes) and the effectiveness of com-bining these sequences for analyses was tested with thepartition homogeneity test implemented in paup* (Swof-ford 2001).

Fig. 2 Maps depicting distributions ofstudy species with subspecific designations(= shaded areas) labelled. Sample localities(closed circles) and (in some instances)sample numbers correspond to data inAppendix. BCN, Baja California Norte;BCS, Baja California Sur.

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Sequence evolution and neutrality

We determined the model of mtDNA sequence evolutionthat best fit each study species by using modeltest(Posada & Crandall 1998). To ensure selective neutrality,we applied the Hudson–Kreitman–Aguadé (HKA) test(Hudson et al. 1987) based on the fact that both ATPregions are found on the same locus or chromosome. TheHKA test constructs a goodness-of-fit (chi-square) statisticusing observed and expected differences within two sisterspecies, and the differences between the two taxa for theDNA regions being compared. In this test, we evaluatedATP8 vs. ATP6 in C. mitchellii vs. the same regions inC. tigris. We did the same using C. ruber with C. atrox andC. cerastes with C. polystictus and C. enyo.

We tested neutrality using the McDonald–Kreitman (MK)test (McDonald & Kreitman 1991), using dnasp (Rozas et al.2003). This test is similar to the HKA test in that it compareslevels of polymorphism and divergence using two sets ofdata. In the former, these represented two different genes(ATP8 vs. ATP6), but in the MK test they are interspersedwithin each gene. Unlike other tests of neutrality, it doesnot assume panmixia, thus making it appropriate whenpopulation subdivision may exist. Under neutrality, theratio of fixed synonymous-to-nonsynonymous substitu-tions between sister taxa will be equal to the ratio of poly-morphic synonymous-to-nonsynonymous substitutions.Selection is inferred when the variance of this ratio amongloci exceeds expectations.

Phylogenetic analyses and sequence divergence

Maximum parsimony (MP) analysis was used to deriveminimum length gene trees for our study species usingtnt (Tree analysis using New Technology, version 1; P.A.Goloboff, J.S. Farris, K.C. Nixon 2003, www.zmuc.dk/public/phylogeny), with the following parameters selected:random sectorial searches (RSS) = 15/35/3/5; consensus-based sectorial searches (CSS) = same as above; tree-drifting(DFT) = 30/4/0/20/0; and tree-fusing (TF) = 5 (see Goloboff1999). We then used tnt to produce a majority rule consensusof the fused trees. We rooted the C. cerastes tree with C.polystictus/C. enyo, while the remaining trees were rootedwith C. molossus (Murphy et al. 2002, Douglas et al., unpub-lished). We also applied maximum likelihood (ML)analysis to estimate relationships among haplotypes ofstudy species, using program phyml (Guindon & Gascuel2003).

Sequence divergence (p) values, corrected for within-group variance, were generated for each species and eachdefined clade, based on 1000 bootstrapped sequences(mega 3, Kumar et al. 2004). To ascertain clock-like beha-viour in our sequences, we applied Tajima’s (1993) test tocompare representative sequences from study clades vs.

the outgroup. Tajima’s test is based on the expectation thatunder a uniform (i.e. clock-like) rate of substitution, num-bers of sites shared by an outgroup and one of twoingroups should be the same for both ingroups. Webolstered the power of our relative rates test by using areasonable number of base pairs, less distant outgroups,and variable mtDNA markers (per Bromham et al. 2000).Furthermore, we also tested entities within closely relatedspecies-complexes, as opposed to widely divergent clades.We performed five random evaluations per clade, eachinvolving three comparisons.

Molecular diversity at the regional level

To examine broad-scale patterns of regional biodiversity,we pooled samples within those geographical regionsidentified as having a common history based uponphylogenetic analyses. For each region, we calculatedhaplotype (h) and nucleotide diversity (π) using dnasp.We also calculated Tajima’s D-statistic, and (if neutralitywas sustained), applied the latter to infer demographichistory (Tajima 1989). For a stable population, D = zero,whereas it is positive for an excess of high-frequencymutations, as after a population contraction or underbalancing selection. Tajima’s D is negative when there is anexcess of low-frequency mutations, as after a populationexpansion, a recent selective sweep, weak negative selection,or when a sample comes from an admixed population.Causation is difficult to determine when Tajima’s D deviatessignificantly from zero. To clarify, we tested for neutralityand computed Fu’s Fs (Fu 1997) which is particularlygood at detecting population expansion. We derived h, π,Tajima’s D and Fu’s Fs using arlequin (Schneider et al.2000), with 1000 replications.

Range expansions, demographic histories, gene flow, and emergent properties

As a test for range expansions, we used dnasp to con-duct mismatch distribution analyses (MDA: Rogers &Harpending 1992), defined as the number of nucleotidedifferences between all pairs of individuals in a region.Expansion leaves a relatively indelible mark for a longperiod (Rogers 1995) because populations converge butslowly to new equilibria. Hence, a large initial expansionwill obscure for a considerable period of time any effectsof subsequent expansions. Conversely, if a populationshould reduce in size, it will quickly converge to a newequilibrium, thus making this signature difficult to detect.

To ascertain the level of divergence within regions, andwhether they have experienced recent gene flow, weapplied an isolation-with-migration model (program im;Hey & Nielsen 2004) designed to evaluate sequence datagathered from closely related populations (as herein). This

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method employs Markov chain Monte Carlo (MCMC),estimates demographic parameters, and scales them by theneutral mutation rate. Our particular focus in these ana-lyses was to judge bi-directional migration rates (m) betweenregions as a means to gauge gene flow and the propensityfor clade isolation.

Evolutionary history of viperids

The origin of snakes is nebulous, but appears to haveoccurred in the Cretaceous (≈ 144 Ma). Although variousearly lineages (e.g. erycine boids) have been identified, ofinterest here is that acrochordid-like and colubroid-liketaxa are present in the Early Palaeocene (≈ 70–60 Ma; Rage1987). Modern colubroid snakes subsequently originatedand explosively radiated in the Eocene (≈ 58 Ma, Rage et al.2003) and Oligocene (≈ 37 Ma), with modern taxa occurringin the Early to Mid Miocene (≈ 24 Ma) (Rage 1987; Greene1997). These dates are highly conserved in that origins andradiations of snakes are certainly older than the fossilevidence (Rage 1987).

The extant venomous, front-fanged lineages (Atractas-pididae, Elapidae, and Viperidae) do not form a naturalgroup, and viperids consistently fall basal to elapids, andelapids basal to atractaspidids (Vidal & Hedges 2002).Viperids are hypothesized to have originated in Africa(although fossil data are lacking; Szyndlar & Rage 2002)and subsequently invaded Asia and/or India (Lenk et al.2001). The first major split in this lineage (i.e. Causinae +Viperinae) occurred in Oligocene (≈ 40 Ma), followed by arelatively robust centre-of-origin for pitvipers (Crotalinae)in the Oligocene of Asia (Greene 1992; Szyndlar & Rage2002). The sister to the monophyletic Crotalinae is themonotypic Azemiops feae (Knight & Mindell 1993; Heiseet al. 1995; Parkinson 1999).

Viperid history in North America is linked to the BeringLand Bridge (Klauber 1972; Conant 1990; Van Devender& Conant 1990; Holman 2000; Gutberlet & Harvey 2004).Because extant viperine taxa are absent from the NewWorld, an unidentified North American viperid fossilfrom Early Miocene (≈ 22 Ma) is most certainly a pitviper(Crotalinae), and identified pitviper fossils date to theEarly Miocene (≈ 19 Ma). Additionally, fossils unequi-vocally identified as a copperhead (Agkistrodon contortrix) arefrom the Late Miocene (≈ 11 Ma). The existence of theseearly fossils provides reasonable confidence in calibratinga molecular clock for viperid evolution in North America,coupled with the fact that Agkistrodon (sensu stricto) is bothmonophyletic and sister to the rattlesnakes (Crotalus + Sis-trurus; Parkinson et al. 2002). Furthermore, Asian pitvipers[Gloydius (formerly Agkistrodon), Ovophis and/or Protoboth-rops (both formerly Trimeresurus)] are identified as sistersto Agkistrodon + rattlesnakes (Parkinson et al. 2002; Gutberlet& Harvey 2004), thus substantiating an invasion from the

Old World. The split involving Gloydius and Agkistrodon isestimated to have occurred in the Oligocene, ≈ 36–40 Ma(Van Devender & Conant 1990; Gutberlet & Harvey 2004).

However, the origin of Crotalus + Sistrurus is open toquestion. The earliest fossils are Mid-Miocene (≈ 10–8 Ma),and modern species are from the Late Miocene, but theirorigin almost certainly predates this estimate (Holman2000). Knight et al. (1993), for example, suggested thatCrotalus and Sistrurus split as early as 30 Ma, which placesthe origin of rattlesnakes in the Late Oligocene to EarlyMiocene.

Molecular estimation of divergence times

Divergence times for clades, as estimated from DNAsequence data, are becoming common in evolutionary andsystematic studies (Near & Sanderson 2004), and theirincreasing prevalence is being driven by the accelerateddevelopment of optimal estimation procedures (Welch& Bromham 2005; Rutschmann 2006). These proceduresconvert measures of distance among sequences intoestimates of time at which the sequences diverged, yet eachdiffers in its treatment of branch length and rate variationestimates among lineages. Comparisons between methodsare difficult due to varying assumptions, alternate sourcesof error, and inherent methodological biases (Magallón &Sanderson 2005).

Although our sequence data were carefully tested forclock-like behaviour, undetected rate variation can still sig-nificantly over- or underestimate divergence times (Brom-ham et al. 2000). Accordingly, we inferred divergencetimes by applying a semiparametric penalized likelihood(PL) approach (Sanderson 2002), using a truncated New-ton (tn) optimization algorithm as implemented in thesoftware r8s (Sanderson 2003). Time estimates are derivedvia a parametric model having a different substitution rateon every branch, coupled with a nonparametric ‘rough-ness’ penalty that costs the model if rates change tooquickly from branch-to-branch. The relative contributionof each is determined by a smoothing parameter. When itis large, roughness dominates and the model is reason-ably clock-like, yet when it is small, roughness contributeslittle and considerable rate-variation is allowed. Optimalsmoothing is determined by a data-driven cross-validationprocedure that is iteratively run. Each terminal branch is inturn removed, model parameters re-estimated, and theexpected number of substitutions on the removed branchpredicted. The performance of this procedure is evaluatedas a normalized ‘chi-square-like’ score. Once the lowestcross-validation smoothing value was determined, weoptimized the PL approach using this value, the gammashape parameter, and method powell (because of con-strained nodes), using 10 searches and multiple re-startswithin searches. However, rapid rate fluctuations can still

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occur in regions with very short branches, resulting in anover-fitting of data (Rutschmann 2006). We adjusted forthis possibility prior to and during analyses by coalescingzero-length tips and collapsing zero-length interiornodes.

We applied the ML-based Langley-Fitch (LF) method toreconstruct divergence times under the assumption of amolecular clock. Here, one substitution rate is estimatedacross the entire tree, with a set of calibrated divergencestimes derived for all unfixed nodes. LF divergences,derived under a parametric molecular clock method, serveas a useful comparison to the semiparametric resultsderived under a relaxed clock.

r8s allows fossil calibrations on nodes to be either fixedor constrained (minimally or maximally). The node rep-resenting the most recent common ancestor (MRCA) ofAcrochordus (= outgroup) was fixed at 70 Ma, while thenode representing the MRCA of pitvipers (= Azemiops) wasconservatively constrained to a maximum 40 Ma. The MRCAnode for New World Crotalinae (= Gloydius) was conserv-atively constrained to a maximum 34 Ma, while the MRCAnode for North American Agkistrodon was minimallyconstrained at 22 Ma. Finally, the node representing theMRCA of A. contortrix was minimally constrained at 11 Ma.

Results

Polymerase chain reaction (PCR) amplifications andautomated sequencing of ATP8 and ATP6 resulted in 676bp of unambiguously readable sequence with no indels.Combining sequences was supported by a nonsignificantpartition homogeneity test (paup*: P > 0.35). All sequenceswere found to be evolving neutrally [HKA: (0.20 <P < 0.95), MK: (0.37 < P < 0.77)] and in a rate-uniformmanner (nonsignificant Tajima’s test). The best-fittingmodel of sequence evolution for Crotalus ruber and Crotalus

tigris clades was Hasegawa–Kishino–Yano (HKY) whereasfor Crotalus cerastes and Crotalus mitchellii clades it wasTamura–Nei (TrN). The best-fitting model for all cladesand outgroups was general time reversible (GTR).

A single MP tree was derived for C. cerastes, while fourwere derived for the remaining three species and compiledinto majority-rule consensus trees. Using ML and all spe-cies, an optimized tree was attained using GTR, which thenserved as input for a second ML run that yielded a treewith log likelihood = −8847.64426 and a gamma shapeparameter = 0.736. The ML tree was used to derive diver-gence times. Monophyletic groups in this tree coincidedwith those delimited using MP, and only the latter resultsare presented.

Lineages within species and their biogeographic context

1 Analysis of C. mitchellii resulted in three major clades,which geographically corresponded with distribution ofits three subspecies (Figs 3a and 4). The most northernclade (consistent with the subspecies Crotalus mitchelliistephensi) was sister to a clade formed by the remainderof the C. mitchellii group. The latter split into a southernclade restricted to Baja California Sur (correspondingwith C. m. mitchellii), which was sister to a clade locatedin the centre of the species distribution (representingCrotalus mitchellii pyrrhus). Each of the three major cladeswas subdivided into subclades. Both northern andsouthern clades reflected a north–south division (CMSavs. CMSb; CMMa vs. CMMb), whereas the central cladewas partitioned into eastern and western subclades(CMPa vs. CMPb).

2 Analysis of C. cerastes resulted in five clades (CCa–CCd,CCm; Fig. 3b), four of which are diagnosed by strongbootstrap percentages (P > 0.95). Correspondence be-tween geographical distribution of molecular clades

Fig. 3 Maximum parsimony (MP) trees withbootstrap values for Crotalus mitchellii (a),Crotalus cerastes (b), Crotalus tigris (c), andCrotalus ruber (d). Clade designations as inTable 2. CPO, Crotalus polystictus; CMO, Crotalusmolossus.

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and previously designated subspecies was only well definedfor Crotalus cerastes laterorepens (clade CCb; Figs 3b and4), representing the western central range of the species.The northern range of the species, where C. c. cerastesis recognized, revealed two clades representing a deepnorth-and-west (CCc) vs. south-and-east split (CCd);the northwestern clade (CCc) appears restricted toDeath Valley. Similarly, the eastern southcentral area,consistent with the range of Crotalus cerastes cercocombus,revealed a deep north–south divergence, with one cladefound in southwestern Arizona (CCa), and anotherin northwestern Sonora, México (CCm). Furthermore,clades in geographical proximity did not form sistertaxon relationships, which reflected the discordancebetween molecular clades and the described subspecies.

3 Crotalus tigris (CT) revealed three moderately differen-tiated clades (Fig. 3c), roughly corresponding to thenorthern (CTa), central (CTb), and southern (CTm) rangeof the species (Fig. 4). Interestingly, the central clade (CTb;southern border of Arizona) was sister to a clade formedby populations to the north (CTa; Central Arizona) andsouth (CTm; Sonora, México).

4 Crotalus ruber reflected very shallow divergence amongthree clades (Fig. 3d). One, corresponding with the in-sular distribution of Crotalus ruber exsul (CRx) was sisterto a well-defined clade formed by C. ruber occupyingsouthern California and the Baja Peninsula. The latteronly vaguely reflected a north–south differentiation,with specimens collected from areas consistent with therange of C. r. ruber forming a clade (CRa) that was sister

to the specimen from Baja California Sur (CRm), the areaoccupied by Crotalus ruber lucasensis.

Divergence among lineages and cladogenetic events

Differentiation within the two-generalist species waspronounced, with that in C. mitchellii being the mostextensive (Table 2, Fig. 5a). Crotalus m. stephensi and C. m.pyrrhus differed by 5.2–6.7% sequence divergence (SD),whereas C. m. stephensi and C. m. mitchellii separated at6.4–6.8% SD (Table 2, Fig. 5a). Similarly, C. m. mitchelliiand C. m. pyrrhus differed by 5.7–6.4% SD, suggestingrelative ancient splits among these clades.

Divergence was shallower among subclades withinC. m. stephensi and C. m. pyrrhus; CMSa differed fromCMSb at 1.7% SD, and CMPa differed from CMPb at 1.3%SD. In contrast, divergence between subclades of C. m.mitchellii was substantial with CMMa differing fromCMMb at 6.1% SD, suggesting a more ancient cladogeneticevent. In comparison, C. mitchellii separated from C. tigrisby 9.5–11.3% SD.

Crotalus cerastes also revealed substantial divergenceamong its clades, with most separating at 4.0% to 4.3% SD(Table 2, Fig. 5b). The most recently derived clades (CCa,CCb) separated from one another at 1.8% SD.

Differentiation within the specialist species appearsmore recent, ranging from 0.4 to 0.9% SD in C. tigris(Table 2, Fig. 5c), and from 0.3 to 0.7% SD in C. ruber(Table 2, Fig. 5d). The southern (Méxican) populations(CTm) of C. tigris differed from the remainder by 0.7% SD

Fig. 4 Maps designating molecular subclades (indicated by ellipses) superimposed on species-distributions for Crotalus mitchellii (left),Crotalus cerastes (middle), and Crotalus tigris (right). ‘A’, ‘B’ and ‘C’ in the C. cerastes map and ‘A’ and ‘B’ in the C. tigris map refer to molecularclades, as designated in text. Phylogenetic relationships of clades are also depicted for each species.

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Table 2 Pairwise sequence divergence between molecular clades as identified in Figs 3 and 4. Values in lower triangle are percent sequencedivergence (p-distances corrected for within-group variability, as 00/000) while those in upper triangle are standard errors (00/000). Cladeabbreviations are: CMSa, Crotalus mitchellii stephensi clade a; CMSb, C. m. stephensi clade b; CMPa, Crotalus mitchellii pyrrhus clade a;CMPb, C. m. pyrrhus clade b; CMMa, Crotalus mitchellii mitchellii Baja California Sur clade a; CMM, C. m. mitchellii Baja California Sur cladeb; CCa, Crotalus cerastes clade a; CCb, C. cerastes clade b; CCc, C. cerastes clade c; CCd, C. cerastes clade d; CCm, C. cerastes Mexican clade;CTa, Crotalus tigris clade a; CTb, C. tigris clade b; CTm, C. tigris Mexican clade; CR, Crotalus ruber mainland clade; CRm, C. ruber BajaCalifornia Sur; CRx, Crotalus ruber exsul clade

CMSa CMSb CMPa CMPb CMMa CMMb CCa CCb CCc CCd CCm CTa CTb CTm CRa CRm CRx

CMSa 0.005 0.009 0.008 0.010 0.010 0.013 0.013 0.013 0.013 0.013 0.012 0.012 0.012 0.012 0.012 0.012CMSb 0.017 0.009 0.008 0.010 0.009 0.013 0.013 0.014 0.014 0.014 0.012 0.012 0.012 0.012 0.012 0.012CMPa 0.067 0.062 0.003 0.008 0.008 0.013 0.013 0.013 0.014 0.014 0.011 0.011 0.011 0.012 0.012 0.012CMPb 0.058 0.052 0.013 0.009 0.008 0.013 0.013 0.013 0.013 0.014 0.011 0.011 0.011 0.011 0.011 0.011CMMa 0.070 0.064 0.060 0.064 0.008 0.013 0.014 0.014 0.014 0.014 0.012 0.011 0.012 0.012 0.012 0.012CMMb 0.073 0.068 0.050 0.057 0.061 0.013 0.013 0.014 0.013 0.014 0.011 0.011 0.011 0.012 0.012 0.012CCa 0.142 0.141 0.131 0.129 0.145 0.143 0.005 0.006 0.007 0.007 0.013 0.013 0.013 0.013 0.013 0.013CCb 0.144 0.142 0.135 0.133 0.152 0.145 0.018 0.007 0.006 0.007 0.013 0.013 0.013 0.013 0.013 0.012CCc 0.143 0.145 0.127 0.126 0.145 0.144 0.031 0.039 0.007 0.007 0.013 0.013 0.013 0.013 0.013 0.013CCd 0.154 0.154 0.146 0.144 0.156 0.151 0.043 0.040 0.040 0.007 0.013 0.013 0.013 0.014 0.013 0.013CCm 0.155 0.159 0.146 0.145 0.159 0.156 0.042 0.043 0.040 0.041 0.013 0.013 0.013 0.013 0.013 0.013CTa 0.112 0.107 0.101 0.096 0.110 0.101 0.149 0.150 0.144 0.156 0.155 0.002 0.003 0.013 0.013 0.013CTb 0.113 0.108 0.097 0.095 0.108 0.099 0.149 0.149 0.144 0.156 0.154 0.004 0.003 0.013 0.013 0.013CTm 0.111 0.106 0.099 0.096 0.109 0.103 0.149 0.150 0.144 0.156 0.155 0.007 0.009 0.013 0.013 0.013CRa 0.110 0.111 0.115 0.105 0.116 0.114 0.140 0.142 0.144 0.159 0.157 0.135 0.138 0.135 0.002 0.003CRm 0.113 0.113 0.114 0.106 0.117 0.116 0.137 0.139 0.141 0.156 0.156 0.137 0.139 0.136 0.003 0.003CRx 0.115 0.114 0.118 0.107 0.119 0.117 0.135 0.138 0.141 0.154 0.154 0.132 0.135 0.132 0.007 0.007

Fig. 5 Relationships among mtDNAhaplotypes found in Crotalus mitchellii (a),Crotalus cerastes (b), Crotalus tigris (c), andCrotalus ruber (d) based upon p-distancescorrected for within-clade variance. Thesame scale of sequence divergence is usedto plot all networks. These networks aremid-point rooted as opposed to theoutgroup rooting in Fig. 3.

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(CTa) and 0.9% SD (CTb), respectively. The C. rubersampled from Baja California Sur (CRm) showed littledifferentiation from mainland forms (0.3% SD). In fact,C. ruber showed the lowest levels of genetic differentiationamong our four study species, suggesting a more recentdifferentiation among geographical regions.

Regional genetic diversity within and among species

To examine whether genetic diversity at the regional levelwas similar among the four study species, each waspartitioned into geographically defined groups. In mostcases, this involved pooling of molecular sister clades(as identified in Figs 3 and 4). Regional groupings foreach species were: C. mitchellii North (CMS = CMSaand CMSb clades), Central (CMP = CMPa and CMPbclades), and South (CMM = CMMa and CMMb clades);C. cerastes North (CC1 = CCc + CCd clades), Central(CC2 = CCa + CCb clades) and South (CC3 = CCm clade);C. tigris North (CT1 = CTa clade), Central (CT2 = CTbclade), and South (CT3 = CTm clade). Due to its restricteddistribution and shallow within-species divergence (lessthan 0.7% SD) C. ruber was analysed as a single region(CR = CRa + CRx + CRm clades).

For all species, regions revealed high haplotype andlow-to-moderate nucleotide diversity (Table 3), with hvalues ranging from 0.6 (C. ruber) to 0.93 (C. mitchellii —Central region CMP), and π values spanning from 0.0025(C. ruber) to 0.011 (C. mitchellii). Comparably high nucle-otide diversity for CMM and CC2 are due to deep diver-gence of lineages within each region (i.e. in excess of 4%SD). Haplotype networks for all species show starburstpatterns at the regional level (Fig. 5), reflecting the factthat large numbers of haplotypes differ but little withineach.

Historic population demography and gene flow

Parameters that evaluate historic population demographycould only be estimated for six (Tajima’s d-statistic) andseven (Fu′ Fs) out of 10 regional groupings (Table 3).Tajima’s d-statistic was negative for three regions (CR, CMPand CT1), and significant only in the latter two, suggestingvarious levels of population growth within regions. Similarly,Fu’s Fs was negative in two regions (CMP and CT1) andsignificant only in the latter. However, some regions arerepresented by few samples, or contain highly divergentlineages, both of which hamper computation of historicpopulation processes. MDA (Fig. 6), another approachto assess historic changes in population size, detected asignificant signal only in regions CMP and CT1. Highlydivergent lineages within a region will mask a signalof population size change.

To gauge gene flow and propensity for regional isola-tion, an isolation-with-migration model was used to judgebi-directional migration rates (m) between regions. Verylow levels of gene flow were detected only between theNorth (CC1) and Central (CC2) regions in C. cerastes,whereas none was documented between the North (CMS)and Central (CMP) regions for C. mitchellii (Fig. 7).

Table 3 Molecular diversity at the regional level across the fourstudy species of rattlesnakes. Regions are: Crotalus mitchelliiCMS, north; CMP, central; CMM, south; C. cerastes north, CC1;central, CC2; C. tigris north, CT1; central, CT2. Crotalus ruber wasanalysed as a single region. Provided are N, sample size; H,number of haplotypes; h, haplotype diversity (standard devi-ation in parentheses); π, nucleotide diversity (standard deviationin parentheses); T-D, Tajima’s D; Fu-F, Fu’s F

Region N H h π T-D Fu-F

CMS 18 10 0.91 (0.044) 0.019 (0.006) 0.172 1.07 (P < 0.70)CMM 2 2 1.00 (0.500) 0.061 (0.030) n/a 3.71 (P < 0.61)CMP 84 27 0.93 (0.012) 0.011 (0.001) −1.43 −4.21 (P < 0.13)CC1 13 6 0.77 (0.089) 0.009 (0.003) 0.080 2.15 (P < 0.85)CC2 6 5 0.86 (0.137) 0.029 (0.007) 0.441 3.11 (P < 0.91)CT1 28 1 0.71 (0.088) 0.003 (0.001) −1.75 −3.63 (P < 0.02)CT2 6 13 n/a 0.000 (0.000) n/a n/aCR 11 4 0.60 (0.154) 0.003 (0.001) −1.218 0.36 (P < 0.59)

Fig. 6 Results of mismatch distribution analyses (MDA)conducted for Crotalus tigris region 1 (CT1) and Crotalus mitchelliipyrrhus (CMP) depicting pairwise differences among individuals(dashed line) plotted against expected occurrence (solid line)under a model of population expansion.

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Molecular estimation of divergence times

Five iterations of the tn method were needed to establishan optimal smoothing parameter of 6.30957 (log 10 =0.80, × -square = 1213.67). Inserting this and the gammavalue into method powell yielded divergence timesdepicted in Table 4. Also depicted are divergence timesderived under the assumption of a molecular clock, asproduced by the ML-based LF procedure.

The MRCA of pitvipers (Azemiops, maximally con-strained at 40 Ma) resulted in an estimated divergencetime of 28.8 Ma (= Mid Oligocene), while the MRCAnode for New World Crotalinae (= Gloydius, maximallyconstrained to 34 Ma), was estimated at 22.7 Ma (earlyMiocene). The MRCA for the rattlesnakes Sistrurus/Cro-talus was estimated at 12.7 Ma (mid-Miocene), and theseparation of Crotalus polystictus from Crotalus enyo/C.cerastes, and Crotalus atrox from C. ruber was gauged as LateMiocene (7.4 and 7.0 Ma, respectively). Crotalus tigris seem-ingly diverged from C. mitchellii at the Late Miocene/EarlyPliocene boundary (5.6 Ma), and C. m. stephensi and C. m.mitchellii separated from the remainder of C. mitchellii inMid Pliocene (3.7 and 3.2 Ma, respectively). The basal cladeof C. m. pyrrhus did not diverge until Late Pleistocene (1.2Ma).

Discussion

Concordant patterns of regional diversity

Vicariant and climatic processes in the southwesterndeserts of North America appear to have impactedrattlesnakes unevenly, and our data reflect three separatelevels of diversification, each of which is discussed in moredetail below:

1 Broad-scale patterns of lineage diversification were con-cordant with Pliocene/Late Miocene vicariant events(Crotalus mitchellii, Crotalus cerastes).

2 Allopatric segregation of subclades within refugiareflected climatically induced Pleistocene effects (C.mitchellii, C. cerastes).

3 Shallow genetic diversity at the regional level resultedfrom clearly defined impacts of Late Pleistocene climaticevents (Crotalus tigris, Crotalus ruber, and subcladeswithin C. mitchellii, C. cerastes).

We predicted (per Avise 2000) that if vicariant eventssimilarly impacted our study species, then concordant pat-terns would be manifested in their phylogeographies. Thiswas substantiated for the two more broadly distributedspecies (C. mitchellii and C. cerastes). In contrast, effects ofPleistocene climate fluctuations, if apparent, would becodistributed as well, but reflected in genetic architectureat the regional level, and modulated by the ecology of ourstudy species (as per Peterson et al. 1999). This was cor-roborated for the two specialist species with morerestricted distributions (C. tigris and C. ruber), but also at theregional level within C. mitchellii (CMP and CMS) andC. cerastes (CC1).

A comparison of distribution vs. molecular divergence

We found a high level of congruence between the sequencedivergences of our study rattlesnakes and their geogra-phical distributions. Crotalus mitchellii and C. cerastes sharesimilar patterns of distribution and molecular diversity,as do C. ruber and C. tigris. The former displayed relativelydeep geographical divisions among subclades, and bothoccur in the Sonoran and Mohave deserts. Although speciesrestricted to the Sonoran Desert (i.e. C. tigris, C. ruber) are also

Fig. 7 Plots of reciprocal migrationprobabilities (m) calculated betweenregions inhabited by Crotalus cerastes (CC1vs. CC2), and Crotalus mitchellii pyrrhus vs.Crotalus mitchellii stephensi (CMP vs. CMS).P, probability; m, number of migrants.

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congruent with regard to molecular diversity, their patternis demonstrably different from that shown by C. mitchelliiand C. cerastes. However, subclades at the regional levelwithin C. mitchellii and C. cerastes (i.e. C MPa and CCa/b)reveal the same pattern as C. tigris and C. ruber. This wouldsuggest that climate fluctuations affected populations atthe regional level.

Under a fluctuating climatic regime, broadly distributedspecies are more likely to experience population subdivi-sions and greater probabilities of developing molecularsubclades because their densities are often relatively uni-form across habitats and thus exposed to local climaticvagaries. Deep divisions among subclades within majorlineages in C. mitchellii and C. cerastes reflect this trend. Onthe other hand, narrowly distributed species should reflectfewer molecular clades, and this trend is apparent in C.ruber and C. tigris. Distributional breadth is an emergentproperty of a species, much like its ecology, and in thissense, may be a consistent barometer for predicting mole-cular diversity. However, similar evaluations in other taxaare needed to further test this hypothesis.

Lineage diversifications and timing of cladogenetic events

Deep divisions in our molecular data are concordant with(and thus suggest they arose during) periods of vicariantevolution in Baja California. Grismer (1994) recognized thelatter and allocated amphibians and reptiles to ‘southernMiocene’ and ‘northern Pliocene’ vicariant complexes.Conditions for the southern complex are: (i) the lineagemust occur in southern Baja California (ii) it must not havea northerly contact with its sister taxon, and (iii) the sistermust range on the mainland south of Isla Tiburón, México.Conditions for the northern complex are: (i) the lineagemust show a sister-relationship with a nonpeninsularlineage; (ii) both approach and/or contact one another atthe apex of the Sea of Cortéz; and (iii) the peninsularlineage is a Baja-endemic. Grismer (1994) designated C.cerastes and C. mitchellii as ‘Lineages of Uncertain Place-ment’ because their relationship with other rattlesnakespecies was unclear, yet their venom protein and scalecharacteristics suggested a sister relationship. However,recent molecular studies (Murphy et al. 2002; Douglaset al., unpublished) fail to support this hypothesis. Nonethe-less, we suggest that both C. cerastes and C. mitchelliiseemingly fit a Miocene-Pliocene vicariant model, albeitwith modifications.

Our divergence data suggest that a C. enyo/C. cerastesclade separated from C. polystictus (a southern MéxicanPlateau outgroup) during Late Miocene. This may haveoccurred during the initial rifting of the Baja Peninsulafrom mainland México concomitant with northwardadvancement of the Sea of Cortéz. Crotalus enyo/C. cerasteslikely moved northward with the Baja Peninsula andeventually separated from one another in Early Pliocenewhen the peninsula itself became physically isolated fromNorth America, an event triggered by the northernmostand westernmost extension of the Gulf coupled with inun-dation of the Los Angeles Basin by the Pacific Ocean (Grismer1994). These geomorphic events are relatively synchronouswith a Late Miocene/Early Pliocene divergence of C. cerastesfrom C. enyo, as suggested by our molecular data. Thiswould place C. enyo within the ‘Southern Miocene’ complexand C. cerastes within the ‘Northern Pliocene’ complex.Furthermore, Murphy & Aguirre-Léon (2002) indicatedthat the recession of the Pacific Ocean and Sea of Cortéz inEarly Pleistocene may have allowed the Sonoran DesertC. cerastes to disperse into Baja, California. Indeed the separa-tion of central and southern clades of C. cerastes seeminglyoccurred during this period (Table 4), yielding the moderndistributions depicted in Fig. 4. The northernmost subcladeof C. cerastes is also the most divergent lineage within thisspecies, and its evolution reflects a pattern similar to thatfound within C. m. stephensi (discussed below).

Relatively deep divergences are also found among C.mitchellii clades (averaging 5.8–6.4% SD), again indicating

Table 4 Divergence times for snake lineages as determinedusing a semiparametric penalized likelihood (PL) approachimplemented in the software r8s. Method tn was first applied toderive an optimum smoothing factor, then re-run using methodpowell (with random re-starts) to derive age estimates (= tn-age).A fixed rate (Langly Fitch) algorithm was also run to derive timeestimates (= LF-age) under a molecular clock constraint. All agesare in millions of years. Node, most recent common ancestor(MRCA) for a given taxon; -F, fixed age; -Ma, maximumconstraint; -Mi, minimum constraint

Node tn-age LF-age

Acrochordus (70-F) 70 70Viperidae 33.63 32.34Daboia russelii 23.96 24.37Crotalinae (40-Ma) 28.76 27.56Gloydius (34-Ma) 22.73 22.52Agkistrodon (22-Mi) 22 22A. contortrix (11-Mi) 11 11Crotalus/Sistrurus 12.68 11.97S. miliarius/S. catenatus 10.23 9.58C. polystictus/C. enyo 7.36 6.96C. enyo/C. cerastes-North 5.36 5.11C. cerastes-South/C. cerastes-Central 1.53 1.48C. atrox/C. ruber 7.01 6.46C. atrox 2.20 2.01C. ruber/C. r. exsul 0.21 0.19C. tigris 5.85 5.32C. tigris-North/C. tigris-South 0.19 0.17C.m. stephensi 3.69 3.26C.m. stephensi-Basal 0.93 0.82C.m. mitchellii 3.15 2.77C.m. mitchellii-Baja North vs. South 2.53 2.22C. m. pyrrhus-Basal 1.25 1.08

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an older vicariant evolution. Murphy (1983) suggestedC. mitchellii and C. tigris diverged from a common ancestordue to the initial formation of the Baja Peninsula. In thissense, both C. m. stephensi and C. m. mitchellii may havebeen part of an earlier (prepeninsular) Méxican distri-bution that included C. tigris, and this hypothesis issupported by the Late Miocene (5.85 Ma) divergence time forthe latter, as estimated from our molecular data. As thepeninsula formed, we posit that C. m. mitchellii split fromthe mainland and became resident within Baja Californiaas part of the ‘Southern Miocene’ complex (Grismer 1994).Crotalus m. stephensi, much like C. cerastes, may have beenpart of the ‘Northern Pliocene’ complex. Subsequentdevelopment of the Bouse Embayment (as above) inun-dated the lower Colorado River Valley to southern Nevada(Lucchitta 1979) and submerged 39 000 km2 of terrain. ByMid-Pliocene, it had tracked west through the ImperialValley (California) to within 80 km of the Pliocene coast-line (Dunham & Allison 1960), reaching to the base of theTransverse Range in southern California (Grismer 1994).We suggest this event would not only have separated C. m.stephensi (which reflects a Mid-Pliocene divergence time),but could also have reduced its range while displacing itfurther northward. Haplotypes at the most northern extentof the C. m. stephensi distribution (i.e. Mono and north-ern Inyo counties, California; Appendix) are sister to theremainder of that clade. Again, a similar situation is foundin C. cerastes, with the most basal clade reflecting a com-parable pattern of evolution.

Our molecular data also agree with other hypothesizedaspects of peninsular biogeography. For example, C. m.mitchellii is deeply divided between northern and southernBaja California Sur, a circumstance that supports the for-mation of a mid-peninsular break (i.e. Vizcaino Seaway)(Murphy 1983). Indeed, our molecular data suggest a Mid-Pliocene (2.5 Ma) separation for northern and southernC. m. mitchellii. Finally, the sister relationship of C. r. exsul(an insular form) with mainland C. ruber supports the‘Peninsular Archipelago’ hypothesis that posits Baja,California was represented as a scattering of islands priorto its coalescence (Murphy & Aguirre-Léon 2002). Ourtentative date for separation of C. r. exsul from mainlandpopulations falls at 0.21 Ma.

Climate fluctuations, refugia, and allopatric segregation

Three allopatric desert refugia are postulated for C.mitchellii and C. cerastes: one in the northern part of theirrange, near Death Valley (clades CMSa and CMSb, CCcand CCd), one east of the Sea of Cortéz apex (clades CMPa,CCa), and one west of this apex (clades CMPb, CCb). Bothspecies must have effectively used these refugia whenclimatic conditions deteriorated, then re-expanded asfavourable conditions returned (per Stewart & Lister 2001;

Ayoub & Reichert 2004). Our insights into this process areaugmented by an evaluation of data on climate changerecorded post-last glacial maximum (LGM). Four largeglacial advances were recorded in the San GregorioMountains (Transverse Range of southern California) at20 000–18 000, 16 000–15 000, 13 000–12 000 and 9000–5000 years ago (Owen et al. 2003), composing 74% ofthat 15 000 year period. Thus, local and regional climaticconditions in southern California must have fluctuatedmore than previously thought, possibly in a recursivemanner throughout Pleistocene (per Smith & Farrell 2005).Given this variance in climate, both C. mitchellii and C.cerastes must have experienced an extended temporalpersistence within or near these refugia as evidenced bydeep sequence divergences among clades.

Refugia locations for C. tigris and C. ruber are more diffi-cult to infer, primarily due to the shallower molecular his-tories evinced by these species. Likely, they persisted insimilar areas as the above species, with C. tigris restrictedto the east and C. ruber to the west of the Sea of Cortéz apex.Both are primarily restricted to Sonoran Desert habitat,and this provides evidence of climatic limits for these twospecies. Thus, an alternative hypothesis would be that bothwere indeed more broadly distributed prior to Pleistoceneclimate fluctuations, but vanished from northern refugiaduring adverse conditions and have not since re-colonizedthe area. Jaeger et al. (2005) invoked similar arguments toexplain distributions of divergent lineages in red spottedtoad (Bufo punctatus) across the southwestern deserts.

Shallow genetic diversity reflects demographic history and gene flow

At the regional level, all four rattlesnake species westudied herein revealed signs of recent population growth,albeit to varying degrees. As climate became milder in thepost-Pleistocene, favourable conditions facilitated spreadof suitable habitat and allowed distributions of desert-adapted species to expand, particularly into higherlatitudes. This pattern is most pronounced (and supportedby significant Tajima’s D and Fu’s F) in C. m. pyrrus (CMP)and C. tigris (CT1), whereas C. ruber (CR) showed anonsignificant trend (Table 4). The notion of populationexpansion for the former two species is also supported byMDA (Fig. 6).

Patterns are more complicated and thus less obvious inother regions. The northern area of distribution (MohaveDesert) for C. m. stephensii and C. cerastes, as well as the cen-tral region (Sonoran Desert) for the latter, harbour quitedivergent lineages that suggest isolation in allopatric re-fugia during periods of Pleistocene climate fluctuation (asdiscussed above). This ‘admixture’ masks a signal of recentpopulation growth in C. m. stephensii (CMS) and C. cerastes(CC1 and CC2), as indicated by a positive value of Tajima’s

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D (Table 3). However, shallow genetic architecture withinsubclades (CMSa, CCa-CCd) of both species hints at recentpopulation expansion (Fig. 5). It was not possible to exam-ine patterns within each separate subclade due to samplesize restrictions.

Regional patterns of molecular diversity reflect a ‘star-burst’ phylogeny, with low variability among populationsand gradual differentiation extending across broader areas(Douglas et al. 2003). This pattern is most pronounced in C.tigris, but also apparent in C. ruber and within subclades ofthe two broadly distributed species (as above). As a meansof identifying historic processes that shape moleculardiversity, Grant & Bowen (1998) classified marine fishesinto four groups based upon different combinations ofhaplotype (h) and nucleotide diversity (π). Their categorieswere defined according to demographic events that alterthe likelihood of mtDNA lineage survival and the time toancestral coalescence. Crotalus tigris clearly falls within cat-egory 2 of Grant & Bowen (1998), a species with high h butquite low π. This situation is represented in a phylogeneticsense by several prevalent haplotypes (as above) embed-ded within a cluster of twigs, one or a few mutationsremoved. Demographic events that would most likely giverise to this condition are a period of low effective popula-tion size followed by rapid expansion. Our explanation isthat C. tigris (and possibly C. ruber) endured a severe LatePleistocene bottleneck followed by a relatively recentrange expansion that, in essence, reset their mtDNA clocks.

Our data also suggest that C. m. pyrrhus has recentlyexpanded eastward, in particular along the ColoradoRiver, where individuals from lower Grand Canyon(northwestern Arizona) represent some of its most derivedhaplotypes. Similarly, based on MDA and other statisticaltests, C. tigris showed evidence of recent geographicalexpansion as well.

Pleistocene environmental changes in the southwestern deserts

What evidence do we have for rapid climatic changesin southwestern deserts during Pleistocene, and thusthe propensity to incur population bottlenecks amongorganisms? At termination of the last glacial maximum(LGM), the desert biome was virtually nonexistent, and inits stead was open woodland of Pinyon Pine and Juniper(Thompson & Anderson 2000). Desert vegetation wasrestricted to minor areas below 300 m in Death Valley andat the confluence of the Sea of Cortéz (Betancourt 2004),both hypothesized as refugial areas. At 14 000 years bp,an apparent mega-drought at the Pleistocene–Holoceneinterface (Betancourt 2004) dramatically decreased pluviallake and groundwater levels. Biotic distributions beganto shift broadly, with desert replacing woodlands,and woodlands replacing forest. By Mid-Holocene, the

southwestern deserts had reached their northern limits(Thompson & Anderson 2000). Although our under-standing of these events is based primarily on vegeta-tional histories derived from Late Pleistocene packratmiddens (Betancourt 2004), we suggest that similar andequally rapid climatic transitions likewise occurred muchearlier in the Pleistocene (Adams et al. 1999; Lockwood 2001),which negatively impacted distributions of C. tigris andC. ruber.

Interestingly, reciprocal migration probabilities (Fig. 7)suggest little to no gene flow between north and centralregions in C. mitchelii and C. cerastes. In light of the deepdivergence among subclades within both species, this isnot surprising. Extended isolation in refugia during Pleis-tocene not only allowed regional populations to diverge,but likely also promoted local adaptation to specifics ofMohave vs. Sonoran desert microclimates. Consequently,regional populations are not only divergent at the mole-cular level, but also with regard to their ecologies. Both areconsidered important attributes of lineages on separateevolutionary trajectories (Crandall et al. 2000).

Taxonomy as a conservation issue

Arguments about conservation are almost alwaysarguments about species, for these are the currency ofbiodiversity. Yet, there is no phylogenetic level thatequates to a species. Researchers have often invoked thegenealogical distinctiveness of populations as a basis forconservation priorities, simply because most variabilitywithin species is subdivided spatially among geographicalsets of populations (i.e. among regions, as herein) (Avise2000). While these often form distinctive branches on agene tree, they are provisional as taxonomic units untilcorroborated with a second, independent marker system(principle of genealogical concordance; Avise & Ball 1990).An additional aspect of this principle arises (as herein) whencodistributed species display similarities in phylogeographicalarchitecture, again indicative of a shared historicalbiogeography. Yet in the past, ambiguous and poorly definedcriteria have often been used to diagnose taxonomicgroupings (Douglas et al. 2002), and subsequent conservation-related efforts directed towards the latter may be, in fact,misapplied. This underscores the domination of taxonomyin conservation biology and, when coupled with theelevated cost of species conservation, aptly demonstratesthe pitfalls of a taxon-centric view.

Klauber (1972) provided a substantial foundation forunderstanding species-level systematics in rattlesnakes,and his taxonomies for C. cerastes and C. tigris haveremained unchanged. Our parsimony and ML analyses,however, divided C. cerastes into five clades, only one ofwhich is congruent with the subspecies that Klauberdefined. Such disagreement is not particularly unusual in

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that numerous studies have documented little (or no)congruence between molecular clades and traditionalsubspecific designations (Burbrink et al. 2000; Demboski& Cook 2001; Janzen et al. 2002). This suggests that infra-specific taxa are indeed a taxonomic problem (see below).

Klauber (1972) recognized C. mitchelli as a polytypictaxon composed of five subspecies. Although the deepphylogeographical divisions we discovered (on average6.5% SD) agree with previously designated subspecies,we argue these divisions represent more substantialtaxonomic breaks, and in fact, have used the principle ofgenealogical concordance (Avise & Ball 1990) to diagnoseC. m. stephensi as a biological species (Douglas et al. 2006).We also uncovered a major phylogeographic divisionbetween populations of C. mitchellii from Baja California,with SD of 6.1%. This exceeds the 5.3% SD (± 0.3) thatseparates the sister-species C. ruber and C. atrox (Douglaset al., unpublished) and (as below) suggests the taxonomyof these species should be revisited.

Two subspecies of C. ruber were also diagnosed byKlauber (1972). Since then, McDiarmid et al. (1999) have con-sidered these as separate species (i.e. C. exsul and C. ruber),whereas to other researchers (Grismer et al. 1994; Murphyet al. 1995) they remain conspecific. Campbell & Lamar(2004) recognized C. exsul as one of four C. ruber subspe-cies. Our molecular data shed some light on this contro-versy. Although we did not evaluate all insular subspecies,we uncovered quite shallow subdivisions within C. ruber.Furthermore, our data do not validate C. r. exsul as a fullspecies, in spite of its insular nature. We also questionwhether genetic divisions within C. ruber are substantialenough to warrant subspecific designations.

A similar situation was revealed in C. tigris, a taxon withlevels of SD comparable to those of C. ruber. In sharp con-trast, deeper and more substantial breaks were found inboth C. m. stephensi and C. m pyrrhus. These discrepanciescast doubt not only on the diagnostic threshold for recog-nizing subspecies, but on the entire concept of trinomials(per Douglas et al. 2002). An important concern, as dis-cussed in Zink (2004), is whether subspecies are a poortaxonomic category (as in C. cerastes and C. mitchellii), or arethere instead just poor subspecies (as in C. ruber).

Phylogenetic diversity and regional conservation

Species problems (as above) could be avoided bypreserving areas (Agapow 2005), rather than waiting fora perfect understanding of species-boundaries that maynever, in fact, arrive. The intent of setting aside regions(deduced phylogeographically across taxa) would beto preserve regional hotspots of evolutionary diversity,rather than applying geological features as a surrogate forbiological diversity (a format previously implemented inthe U.S. National Park system). Conservation genetics

displayed early in its development such a broader andtheoretically more comprehensive approach to biodiversitydistribution (Avise 1992, 1996; Riddle et al. 2000). Yet, morerecent studies have often been relatively nonsynthetic andhave centred primarily on single species.

Our research was driven by holistic questions andapproaches, and we focused on regions rather than taxa.Accordingly, we attempted to integrate the phylogeo-graphies of several relatively sedentary species within thewarm deserts of southwestern North America, and to seekcausation for their patterns as a baseline for the develop-ment of an integrated regional conservation and manage-ment plan. Yet, if resident biotas lack requisite biodiversityto serve as an index, they cannot serve as surrogates for alandscape-based conservation perspective.

Herein, we fixed on specialized clades whose memberstend to show low vagility and reduced geographicalranges. These, in turn, are often found in regions where‘orbitally induced range dynamics’ (ORD: Dynesius &Jansson 2000) have a weak history (like the southwesterndeserts) ( Jansson & Dynesius 2002). The latter are also par-ticularly susceptible to habitat degradation (Dobson et al.1997), and consequently must be considered of highconservation priority. Here, molecular analyses can be ofassistance for they clearly identify the deep histories ofregional biotas, and underscore the biogeography of theirrelevant biodiversity. Landscape planners can employ theresults of these analyses as a means to incorporate crypticbiodiversity within the design of regional desert preserves.However, such a perspective has been consistently ignoredas a North American conservation strategy (Donlan &Martin 2004). Instead, national and regional parks andpreserves are designed solely by geographical criteria (asabove). The latter, while aesthetically pleasing, are oftenless effective in encapsulating biodiversity.

An additional layer of complexity for this issue stemsfrom the fact that genetically defined provinces in south-eastern United States juxtapose well with traditionalbiogeography (Avise 1992, 1996, 2000). This suggests inturn that the latter may serve as a proxy for the former. Wecontend this guideline is invalid for making conservationdecisions within the warm deserts of southwestern NorthAmerica, because intraspecific molecular boundaries withinthis region are much more variable and, in most cases, failto associate with traditional biogeography. Accordinglyregional conservation plans for southwestern deserts mustinstead employ a molecular (and deep historical) perspec-tive as a primary mechanism in framing the biodiversityreserves of this unique region.

Acknowledgements

Numerous individuals provided field assistance, additional sam-ples, and/or critical advice. They are: J. Badman, D. Beck, M. Cardwell,

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G. Carpenter, D. DeNardo, M. Feldner, W. Hayes, A. Holycross, C.Jurebe, D. Langella, H. Lawler, B. Mackin, C. Meachum, C. Mill-branth, B. Montgomery, D. Morafka, L. Nienaber, J. O’Leile,J. Perez, C. Ponder, C. Porras, D. Porras, R. Reed, R. Reiserer,R. Repp, E. Schuett, J. Servoss, R. Strom, M. Spille, B. Starrett, E.Taylor, B. Thomason, P. Viehoever, W. White, and M. Widemann.We also acknowledge assistance of personnel managing livecollections located at: Arizona State University (Tempe), CaliforniaPolytechnical University (Pomona), Centro Ecologica de Sonora(México), Texas A & M University (Kingsville), and ZoologicalSociety of San Diego. Collecting permits were provided by: Ari-zona, Nevada, Utah, The Navajo Nation, The Hualapai Tribe, andGrand Canyon NP. Animal Care permits were granted by ArizonaState University and Colorado State University. GenBank Acces-sion nos are: DQ49376–DQ493824.

References

Adams J, Maslin M, Thomas E (1999) Sudden climatic transitionsduring the Quaternary. Progress in Physical Geography, 23, 1–36.

Agapow P-M (2005) Species: demarcation and diversity. In: Phylo-geny and Conservation (eds Purvis A, Gittleman JL, Brooks T),pp. 57–75. Cambridge University Press, UK.

Alroy J, Koch PL, Zachos JC (2000) Global climate change andNorth American mammalian evolution. Paleobiology, 26, 259–288.

Avise JC (1992) Molecular population structure and the biogeo-graphic history of a regional fauna: a case history with lessonsfor conservation biology. Oikos, 63, 62–76.

Avise JC (1996) Towards a regional conservation genetics perspec-tive: phylogeography of faunas in the southeastern UnitedStates. In: Conservation Genetics: Case Histories from Nature (edsAvise JC, Hamrick JL), pp. 431–470. Chapman & Hall, NewYork.

Avise JC (2000) Phylogeography, the History and Formation of Species.Harvard University Press, Cambridge MA.

Avise JC, Ball RM (1990) Principles of genealogical concordance inspecies concepts and biological taxonomy. Oxford Surveys inEvolutionary Biology, 7, 45–67.

Axelrod DI (1979) Age and origin of the Sonoran Desert vegeta-tion. Occasional Papers of the California Academy of Sciences, 132, 1–74.

Ayoub NA, Reichert SE (2004) Molecular evidence for Pleistoceneglacial cycles driving diversification of a North American desertspider, Agelenopsis aperta. Molecular Ecology, 13, 3453–3465.

Bennett KD (1990) Milankovitch cycles and their effects on speciesin ecological and evolutionary time. Paleobiology, 16, 11–21.

Betancourt JL (2004) Arid lands paleobiogeography: the rodentmidden record in the Americas. In: Frontiers in Biogeography (edsLomolino MV, Heaney LR), pp. 27–65. Sinauer Associates,Sunderland MA.

Bromham L, Penny D, Rambaut A, Hendy MD (2000) The powerof relative rates tests depend on the data. Journal of MolecularEvolution, 50, 296–301.

Burbrink FT, Lawson R, Slowinski JB (2000) Mitochondrial DNAphylogeography of the polytypic North American rat snake(Elaphe obsoleta): a critique of the subspecies concept. Evolution,54, 2107–2118.

Campbell JA, Lamar WL (2004) The Venomous Reptiles of the West-ern Hemisphere, 2 volumes. Comstock Publishing Associates,Ithaca, NY.

Clark PU, MacAyeal DR, Andrews JT, Barltein PJ (1995) Ice sheetsplay important role in climate change. Eos, 76, 265–270.

Conant R (1990) The fossil history of the genus Agkistrodonin North America. In: Snakes of the Agkistrodon Complex: AMonographic Review (eds Gloyd HK, Conant R), pp. 539–545.Contributions to Herpetology Number 6, Society for the Studyof Amphibians and Reptiles, Oxford, Ohio.

Crandall KA, Bininda-Edmonds ORP, Mace GM, Wayne RK(2000) Considering evolutionary processes in conservation bio-logy. Trends in Ecology & Evolution, 15, 290–295.

Demboski JR, Cook JA (2001) Phylogeography of the dusky shrew,Sorex monticolus, (Insectivora, Soricidae): insights into deep andshallow history in northwestern North America. Molecular Eco-logy, 10, 1227–1240.

Dobson AP, Rodriguez JP, Roberts WM, Wilcove DS (1997) Geo-graphic distribution of endangered species in the United States.Science, 275, 550–553.

Donlan CJ, Martin PS (2004) Role of ecological history in invasivespecies management and conservation. Conservation Biology, 18,267–269.

Douglas ME, Minckley WL, DeMarais BD (1999) Did vicarianceshape phenotypes of western North American fishes? Evidencefrom Gila River cyprinids. Evolution, 53, 238–246.

Douglas ME, Douglas MR, Schuett GW, Porras LW, Holycross AT(2002) Phylogeography of the western rattlesnake (Crotalus viridis)complex, with emphasis on the Colorado Plateau. In: Biology of theVipers (eds Schuett GW, Höggren M, Douglas ME, Greene HW),pp. 11–50. Eagle Mountain Publishing LC., Eagle Mountain, UT.

Douglas MR, Brunner PC, Douglas ME (2003) Drought in an evo-lutionary context: molecular variability in flannelmouth sucker(Catostomus latipinnis) from the Colorado River Basin of westernNorth America. Freshwater Biology, 48, 1254–1273.

Douglas ME, Douglas MR, Schuett GW, Porras LW, Thomason BL(2006) Genealogical concordance between mitochondrial andnuclear DNAs supports species recognition of the PanamintRattlesnake (Crotalus mitchellii stephensi Klauber). Copeia, 2006,submitted.

Dunham JW, Allison EC (1960) The geologic history of BajaCalifornia and its marine faunas. Systematic Zoology, 9, 47–91.

Dynesius M, Jansson R (2000) Evolutionary consequences ofchanges in species’ geographical distributions driven byMilankovitch climate oscillations. Proceedings of the NationalAcademy of Science, USA, 97, 9115–9120.

Fu YX (1997) Statistical test of neutrality of mutations againstpopulation growth, hitchhiking and background selection.Genetics, 147, 915–925.

Gans PB (1997) Large-magnitude Oligo-Miocene extension insouthern Sonora: implications for the tectonic evolution ofnorthwest México. Tectonics, 16, 388–408.

Goloboff PA (1999) Analyzing large data sets in reasonable times:solutions for composite optima. Cladistics, 15, 415–428.

Gould SJ (2002) The Structure of Evolutionary Theory. Harvard Uni-versity Press, Cambridge MA.

Grant WS, Bowen BW (1998) Shallow population histories in deepevolutionary lineages of marine fishes: insights from sardinesand anchovies and lessons for conservation. Journal of Heredity,89, 415–426.

Greene HW (1992) The ecological and behavioral context forpitviper evolution. In: Biology of the Pitvipers (eds Campbell JA,Brodie ED Jr), pp. 107–118. Selva, Tyler TX.

Greene HW (1997) Snakes: The Evolution of Mystery in Nature.University of California Press, Berkeley CA.

3370 M . E . D O U G L A S E T A L .

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

Grismer LL (1994) The origin and evolution of the peninsular her-petofauna of Baja California, México. Herpetological Natural His-tory, 2, 51–106.

Grismer LL (1999) An evolutionary classification of reptiles onislands in the Gulf of California, México. Herpetologia, 55, 446–469.

Grismer LL (2002) Amphibians and Reptiles of Baja California Includ-ing its Pacific Islands and the Islands in the Sea of Cortéz. Universityof California Press, Berkeley CA.

Grismer LL, MacGuire JA, Hollingsworth BD (1994) A report onthe herpetofauna of the Viscaíno Peninsula, Baja California,México, with a discussion of its biogeographic and taxonomicimplications. Bulletin of the Southern California Academy of Science,93, 45–80.

Guindon S, Gascuel O (2003) A simple, fast and accurate algorithmto estimate large phylogenies by maximum likelihood. System-atic Biology, 52, 696–704.

Gutberlet RL Jr, Harvey MB (2004) The evolution of New Worldvenomous snakes. In: Venomous Reptiles of the Western Hemi-sphere, 2 volumes (eds Campbell JA, Lamar WW), pp. 634–682.Cornell University Press, Ithaca NY.

Heise PJ, Maxson LR, Dowling HG, Hedges SB (1995) Higher-levelsnake phylogeny inferred from mitochondrial DNA sequencesof 12S rRNA and 16S rRNA genes. Molecular Biology and Evolu-tion, 12, 259–265.

Hewitt GM (1996) Some genetic consequences of of ice ages, andtheir role in divergence and speciation. Biological Journal of theLinnean Society, 58, 247–276.

Hewitt GM (2000) The genetic legacy of the Quaternary ice ages.Nature, 405, 907–913.

Hey J, Nielsen R (2004) Multilocus methods for estimatingpopulation sizes, migration rates and divergence time, withapplications to the divergence of Drosophila pseudoobscuraand D. persimilis. Genetics, 167, 747–760.

Hoffman EA, Blouin MS (2004) Evolutionary history of the North-ern Leopard Frog: reconstruction of phylogeny, phylogeo-graphy, and historical changes in population demography frommitochondrial DNA. Evolution, 58, 145–159.

Holman JA (2000) Fossil Snakes of North America: Origin, Evolution,Distribution, Paleocology. Indiana University Press, Bloomington IN.

Hudson RR, Kreitman M, Aguade M (1987) A test of neutralmolecular evolution based on nucleotide data. Genetics, 116,153–159.

Jaeger JJ, Riddle BR, Bradford DF (2005) Cryptic Neogene vicari-ance and Quaternary dispersal of the red-spotted toad (Bufopunctatus): insights on the evolution of North American warmdesert biotas. Molecular Ecology, 14, 3033–3048.

Jansson R, Dynesius M (2002) The fate of clades in a world ofrecurrent climatic change: Milankovitch oscillations and evolu-tion. Annual Review of Ecology and Systematics, 33, 741–777.

Janzen FJ, Krenz JG, Haselkorn TS, Brodie ED Jr (2002) Molecularphylogeography of common garter snakes (Thamnophis sirtalis)in western North America: implications for regional historicalforces. Molecular Ecology, 11, 1739–1751.

Klauber LM (1972) Rattlesnakes: Their Habits, Life Histories, andInfluence on Mankind, 2nd edn. 2 volumes. University of Califo-ria Press, Berkeley CA.

Knight A, Mindell DP (1993) Substitution bias, weighting of DNA-sequence evolution, and the phylogenetic position of Fea’s Viper.Systematic Biology, 42, 18–31.

Knight A, Styer D, Pelikan S, Campbell JA, Densmore III LD,Mindell DP (1993) Choosing among hypotheses of rattlesnake

phylogeny: a best-fit rate test for DNA sequence data. SystematicBiology, 42, 356–367.

Kumar S, Tamura K, Nei M (2004) mega 3: integrated software formolecular evolutionary genetics analysis and sequence align-ment. Bioinformatics, 5, 150–163.

Lenk P, Kalyabina S, Wink M, Joger U (2001) Evolutionary rela-tionships among the true vipers (Reptilia: Viperidae) inferredfrom mitochondrial DNA sequences. Molecular Phylogeneticsand Evolution, 19, 94–104.

Licciardi JM, Clark PU, Brook EJ, Elmore D, Sharma P (2004)Variable response of western U.S. glaciers during the last deglac-iation. Geology, 32, 81–84.

Lockwood JG (2001) Abrupt and sudden climatic transitions andfluctuations: a review. International Journal of Climatology, 21,1153–1179.

Lovette IJ (2005) Glacial cycles and the tempo of avian speciation.Trends in Ecology & Evolution, 8, 57–59.

Lucchitta I (1979) Late Cenozoic uplift of the southwesternColorado Plateau and adjacent lower Colorado River region.Technophysics, 61, 63–95.

Magallón SA, Sanderson MJ (2005) Angiosperm divergence times:the effects of genes, codon positions, and time constraints. Evo-lution, 59, 1653–1670.

McDiarmid RW, Campbell JA, Touré TA (1999) Snake Species ofthe World: a Taxonomic and Geographic Reference, Vol. 1. TheHerpetologists’ League. Washington, DC.

McDonald JH, Kreitman M (1991) Adaptive protein evolution atthe adh locus in Drosophila. Nature, 352, 652–654.

Minckley WL, Hendrickson DA, Bond CE (1986) Geography ofwestern North American freshwater fishes: description andrelationships to intracontinental tectonism. In: The Zoogeographyof North American Freshwater Fishes (eds Hocutt CH, Wiley EO),pp. 519–613. John Wiley & Sons Publishing, New York, NY.

Morafka DJ (1977) A Biogeographical Analysis of the ChihuahuanDesert Through its Herpetofauna. Dr. W. Junk B.V., Publishers,The Hague, Netherlands.

Murphy RW (1983) Paleobiogeography and genetic differenti-ation of the Baja California herpetofauna. Occasional Papers of theCalifornia Academy of Sciences, 137, 1–48.

Murphy RW, Aguirre-Léon G (2002) The nonavian reptiles. In: ANew Island Biogeography of the Sea of Cortés (eds Case TJ, Cody ML,Ezcurra E), pp. 181–220. Oxford University Press, New York.

Murphy RW, Kovac V, Haddrath O, Allen GS, Fishbein A,Mandrak NE (1995) mtDNA gene sequence, allozyme, andmorphological uniformity among red diamond rattlesnakes,Crotalus ruber and Crotalus exsul. Canadian Journal of Zoology, 73,270–281.

Murphy RW, Fu J, Lathrop A, Feltham JV, Kovak V (2002) Phylo-geny of the rattlesnakes (Crotalus and Sistrurus) inferred fromsequences of five mitochondrial DNA genes. In: Biology of theVipers (eds Schuett GW, Hôggren M, Douglas ME, Greene HW),pp. 69–92. Eagle Mountain Publishing LC., Eagle Mountain, UT.

Near TJ, Sanderson MJ (2004) Assessing the quality of moleculardivergence time estimates by fossil calibrations and fossil-basedmodel selection. Philosophical Transactions of the Royal Society ofLondon. Series B, Biological Sciences, 359, 1477–1483.

Oakey DD, Douglas ME, Douglas MR (2004) Small fish in a largelandscape: diversification of Rhinichthys osculus (Cyprinidae) inwestern North America. Copeia, 2004, 207–221.

Owen LA, Finkel RC, Minnick RA, Perez AE (2003) Extremesouthwestern margin of late Quaternary glaciation in NorthAmerica: timing and controls. Geology, 31, 729–732.

R A T T L E S N A K E E V O L U T I O N I N N O R T H A M E R I C A N D E S E R T S 3371

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

Parkinson CL (1999) Molecular systematics and biogeographicalhistory of pitvipers as determined by mitochondrial ribosomalDNA sequences. Copeia, 1999, 576–586.

Parkinson CL, Campebell JA, Chippendale PT (2002) Multigenephylogenetic analysis of pitvipers, with comments on their bio-geography. In: Biology of the Vipers (eds Schuett GW, HöggrenM, Douglas ME, Greene HW), pp. 93–110. Eagle Mountain Pub-lishing LC., Eagle Mountain UT.

Peterson AT, Soberón J, Sánchez-Cordero V (1999) Conservatismof ecological niches in evolutionary time. Science, 285, 1265–1267.

Posada D, Crandall KA (1998) modeltest: testing the model ofDNA substitution. Bioinformatics, 14, 817–818.

Prothero DR (2004) Did impacts, volcanic eruptions, or climatechange affect mammalian evolution? Palaeogeography, Palaeo-climatology, Palaeoecology, 214, 283–294.

Rage J-C (1987) Fossil history. In: Snakes: Ecology and EvolutionaryBiology (eds Siegel RA, Collins JT, Novak SS), pp. 51–76.MacMillan, New York NY.

Rage J-C, Bajpai S, Thewissen JGM, Tiwari BN (2003) Early Eocenesnakes from Kutch, Western India, with a review of the Palaeo-phiidae. Geodiversitas, 25, 695–716.

Riddle BR (1995) Molecular biogeography in the pocket mice(Perognathus and Chaetodipus) and grasshopper mice (Onychomys)–the Cenozoic development of a North American aridlandsrodent guild. Journal of Mammalogy, 76, 283–301.

Riddle BR, Hafner DJ, Alexander LF, Jaeger JR (2000) Crypticvicariance in the historical assembly of a Baja California desertbiota. Proceedings of the National Academy of Sciences, USA, 97,14438–14443.

Rogers AR (1995) Genetic evidence for a Pleistocene populationexplosion. Evolution, 49, 608–615.

Rogers AR, Harpending H (1992) Population growth makeswaves in the distribution of pairwise genetic differences. Mole-cular Biology and Evolution, 9, 552–569.

Rozas J, Sánchez-DeI, Barrio JC, Messenguer X, Rozas R (2003)dnasp, DNA polymorphism analyses by the coalescent andother methods. Bioinformatics, 19, 2496–2497.

Rutschmann F (2006) Molecular dating of phylogenetic trees: abrief review of current methods that estimate divergence times.Diverstity and Distributions, 12, 35–48.

Sanderson MJ (2002) Estimating absolute rates of molecular evo-lution and divergence times: a penalized likelihood approach.Molecular Biology and Evolution, 19, 101–109.

Sanderson MJ (2003) r8s: inferring absolute rates of molecularevolution and divergence times in the absence of a molecularclock. Bioinformatics, 19, 301–302.

Schneider S, Roessli D, Excoffier L (2000) arlequin, ver. 2.001.Department of Anthropology & Ecology, University of Geneva,Switzerland.

Smith CI, Farrell BD (2005) Range expansions in the flightlesslonghorn cactus beetles, Moneilema gigas and Moneilema armatum,in response to Pleistocene climate changes. Molecular Ecology,14, 1025–1044.

Stanley SM (1998) Macroevolution: Patterns and Process. JohnsHopkins University Press, Baltimore MD.

Stebbins RC (2003) A Field Guide to Western Reptiles and Amphibians,3rd edn. Houghton Mifflin Company, Boston, MA.

Stewart JR, Lister AM (2001) Cryptic northern refugia and theorigins of the modern biota. Trends in Ecology & Evolution, 16,608–613.

Swofford DL (2001) paup*, Phylogenetic Analysis Using Parsi-mony (and other methods), Version 4.04b. Sinauer Publishers,Sunderland, MA.

Szyndlar Z, Rage J-C (2002) Fossil records of the true vipers. In:Biology of the Vipers (eds Schuett GW, Höggren M, Douglas ME,Greene HW), pp. 419–444. Eagle Mountain Publishing LC,Eagle Mountain UT.

Tajima F (1989) The effects of change in population size on DNApolymorphism. Genetics, 123, 597–601.

Tajima F (1993) Simple methods for testing the molecular evolu-tionary clock hypothesis. Genetics, 135, 599–607.

Thompson RS, Anderson KH (2000) Biomes of western NorthAmerica at 18 000, 6000 and 0 14C yr bp reconstructed frompollen and packrat midden data. Journal of Biogeography, 27,555–584.

Thompson RS, Whitlock C, Bartlein PJ, Harrison SP, SpauldingWG (1993) Climatic changes in the western United States since18,000 year bp. In: Global Climates Since the Last Glacial Maximum(eds Wright HE Jr, Kutzback JE, Webb III T, Ruddman WE,Street-Perrott FA, Bartlein PJ), pp. 468–513. University ofMinnesota Press, Minneapolis MN.

Van Devender TR, Conant R (1990) Pleistocene forests andcopperheads in the eastern United States, and the historical bio-geography of New World Agkistrodon. In: Snakes of the AgkistrodonComplex: a Monographic Review (eds Gloyd HK, Conant R),pp. 601–614. Contributions to Herpetology Number 6, Societyfor the Study of Amphibians and Reptiles, Oxford OH.

Vidal N, Hedges SB (2002) Higher-level relationships of caen-ophidian snakes inferred from four nuclear and mitochondrialgenes. Comptes Rendus Biologies, 325, 987–995.

Weir J, Schluter D (2004) Ice sheets promote speciation in borealbirds. Proceedings of the Royal Society of London. Series B, BiologicalSciences, 271, 1881–1887.

Welch JJ, Bromham L (2005) Molecular dating when rates vary.Trends in Ecology & Evolution, 20, 320–327.

Zink RM (2004) The role of subspecies in obscuring avian biolo-gical diversity and misleading conservation policy. Proceedingsof the Royal Society of London. Series B, Biological Sciences, 271, 561–564.

The authors share a common interest in deciphering the evolutionof the western North American deserts through evaluation of theirresident biotas, which are endemic, isolated and with uniqueadaptations subsumed within a landscape where ‘. . . every creaturehas a sting and every plant a thorn.’ MED and MRD jointly run theConservation Genetics and Molecular Ecology Laboratory atColorado State University, while the research interests of GWS focuson behavioral ecology and mating systems of reptiles, particularlypitvipers, using phylogenetic comparative approaches. LWPis proprietor of Eagle Mountain Publishing, LC, specializing inbiological titles, and his research interest centers on the naturalhistory of the New World herpetofauna.

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Appendix

Specimens employed in this study. Individuals were diagnosed to species or subspecies (SP) at time of collection, individually enumerated(IND), and gender identified (SX) where possible. Locality information is provided as state (ST), county (CO) and specific locale(LOCATION). Based on mtDNA sequence analysis, each specimen was allocated to a molecular clade (MC) as specified in Fig. 3. Speciesabbreviations are: CMP, Crotalus mitchellii pyrrhus; CMS, Crotalus mitchellii stephensi; CMM, Crotalus mitchellii mitchellii; CC, Crotalus cerastes;CT, Crotalus tigris; CR, Crotalus ruber; CX, Crotalus ruber exsul. For gender M, male; F, female

SP IND MC SX ST CO LOCATION

CMP 1 CMPa M AZ Coconino Grand Cn. Nat. Park, Havasu CK (RM 158S)CMP 2 CMPa M AZ Coconino Grand Cn. Nat. Park, Whitmore Wash (RM 188N)CMP 3 CMPa M AZ Coconino Grand Cn. Nat. Park, Spring Cn. (RM 204N)CMP 4 CMPa M AZ Coconino Grand Cn. Nat. Park 220 mi. Cn. (RM. 220N)CMP 5 CMPa M AZ Coconino Grand Cn. Nat. Park 220 mi. Cn. (RM 220N)CMP 6 CMPa M AZ Coconino Grand Cn. Nat. Park 220 mi. Cn. (RM 220N)CMP 7 CMPa M AZ Coconino Grand Cn. Nat. Park 220 mi. Cn. (RM 220N)CMP 8 CMPa AZ Maricopa Phoenix, South Mt.CMP 9 CMPa M AZ Maricopa South Mt. (South Side)CMP 10 CMPa F AZ Maricopa North Mt. PreserveCMP 11 CMPa AZ Mohave Diamond CreekCMP 12 CMPa AZ Maricopa McDowell ParkCMP 13 CMPa AZ Yavapai Rt. 96, 3 mi. E. Santa Maria RiverCMP 14 CMPa AZ Mohave Nr. OatmanCMP 15 CMPa AZ Mohave Nr. OatmanCMP 16 CMPb F CA Imperial Ogilby Hills, W. AZ/CA border on I-8CMP 17 CMPa M AZ Maricopa Estrella Mts.CMP 18 CMPa AZ Yavapai Rt. 96, 18 mi. E. BagdadCMP 19 CMPa F AZ Maricopa Estrella Mt. ParkCMP 20 CMPa F AZ Maricopa Estrella Mt. ParkCMP 21 CMPb CA San Bernardino Rt. 247CMP 22 CMPb CA San Bernardino Rt. 247CMP 23 CMPb CA San Diego Rt. 188CMP 24 CMPa AZ Maricopa Camelback Mt.CMP 25 CMPa F AZ Maricopa Lake PleasantCMP 26 CMPa M AZ Maricopa Lake PleasantCMP 27 CMPa M AZ Maricopa White Tank Mts.CMP 28 CMPa AZ Maricopa SE end of Estrella Mts.CMP 29 CMPb M CA San Bernardino Hwy. 18 & Apple Valley Road.CMP 30 CMPa AZ Yavapai Rt. 96, 1.8 mi. W HillsideCMP 31 CMPa F AZ Maricopa South Mt.CMP 32 CMPa M AZ Maricopa South Mt.CMP 33 CMPa M AZ La Paz Harquahala Mts.CMP 34 CMPb F CA Imperial Laizo Muchacho Mts.CMP 35 CMPa M AZ Maricopa Gila Bend Mts.CMP 36 CMPa F AZ La Paz Harquahala Mts.CMP 37 CMPa M AZ Mohave Hualapai Mts.CMP 38 CMPa AZ Mohave Grand Cn. Nat. Park at Spring Cn. (RM 204N)CMP 39 CMPa M AZ Mohave Hualapai Mts. Chicken Springs Road.CMP 40 CMPa F AZ Maricopa Vulture Mts.CMP 41 CMPa M AZ Maricopa Vulture Mts.CMP 42 CMPa M AZ Maricopa Estrella Mts.CMP 43 CMPa M AZ Maricopa Estrella Mts.CMP 44 CMPb CA San Bernardino Hwy. 247, 15 mi. S. BarstowCMP 45 CMPb CA Riverside Bautista Cn., 10 mi. ESE HemetCMP 46 CMPb CA San Bernardino Mill Ck., 10 mi. ENE RedlandsCMP 47 CMPb CA San Bernardino Apple ValleyCMP 48 CMPb CA San Bernardino City limits of Joshua TreeCMP 49 CMPa AZ Mohave Grand Cn. Nat. Park (RM 202N)CMP 50 CMPa AZ Mohave Grand Cn. Nat. Park RM 204.5NCMP 51 CMPa AZ Mohave Grand Cn. Nat. Park RM 204.5NCMP 52 CMPa AZ Coconino Grand Cn. Nat. Park RM 209SCMP 53 CMPa AZ Coconino Grand Cn. Nat. Park RM 209SCMP 54 CMPa F AZ La Paz Harquahala Mts.CMP 55 CMPa F AZ La Paz Harquahala Mts.

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CMP 56 CMPa AZ Mohave Grand Cn. Nat. Park (Rm. 204N)CMP 57 CMPa AZ Mohave Grand Cn. Nat. Park (Rm. 211N)CMP 58 CMPa AZ Mohave Grand Cn. Nat. Park (Rm. 211N)CMP 59 CMPa F AZ Maricopa White Tank Mts.CMP 60 CMPa M AZ Yavapai Bumble Bee Road. at I-17CMP 61 CMPa M AZ Mohave Grand Cn. Nat. Park (RM 204.5)CMP 62 CMPb CA San Bernardino Pisgah CraterCMP 63 CMPb CA Orange Silverado Cn.CMP 64 CMPb F CA San Diego San DiegoCMP 65 CMPb M CA San Diego San DiegoCMP 66 CMPa AZ Mohave OatmanCMP 67 CMPa AZ Maricopa Vulture Mine Road., 2 mi. SW WickenbergCMP 68 CMPa F MX Sonora Sierra de Pinacate, nr. SonoitaCMP 69 CMPa M AZ Mohave Grand Cn. Nat. Park (RM 193.3R)CMP 70 CMPa M AZ Mohave Grand Cn. Nat. Park (RM 204R)CMP 71 CMPa M AZ Mohave Grand Cn. Nat. Park (RM 204R)CMP 72 CMPa M AZ Mohave Grand Cn. Nat. Park (RM 204R)CMP 73 CMPa M AZ Mohave Grand Cn. Nat. Park (RM 209R)CMP 74 CMPa M AZ Mohave Grand Cn. Nat. Park (RM 211R)CMP 75 CMPa F AZ Mohave Peach Springs Cn., 6 km N. Rt.66CMP 76 CMPa F AZ Maricopa Phoenix North Mt. ParkCMP 77 CMPa AZ Coconino Grand Cn. Nat. Park (RM 198R)CMP 78 CMPa F AZ LaPaz Plomosa Mts., 7 mi. E. QuartsiteCMP 79 CMPa M AZ Yavapai HillsideCMP 80 CMPa F AZ Yavapai HillsideCMP 81 CMPa AZ Mohave Grand Cn. Nat. Park (RM 198R)CMP 82 CMPa AZ Mohave Grand Cn. Nat. Park (RM 202.3R)CMP 83 CMPb M CA San Diego Anza Borrego State ParkCMP 84 CMPa NV Clark New Gold Butte Rd. 8.3 mi. SW. Hwy. 170CMM 1 CMPb M MX BCN Valle de GuadalupeCMM 2 CMMa M MX BCS Canon Agua Caliente, Sierra de la LagunaCMM 3 CMMb MX BCS 26.1 mi. S. MulegeCMS 1 CMSa NV Esmeralda 4.7 mi. S. Goldfield SummitCMS 2 CMSa NV Esmeralda 4.7 mi. S. Goldfield SummitCMS 3 CMSa NV Esmeralda 4.7 mi. S. Goldfield SummitCMS 4 CMSa CA Inyo Hwy. 168, White Mts. E. IndependenceCMS 5 CMSa M CA Inyo 15 mi. N. BishopCMS 6 CMSa M CA Inyo 15 mi. N. BishopCMS 7 CMSa NV Clark Spring Mts. just W. Las VegasCMS 8 CMSb J CA Mono Gorge Road.CMS 9 CMSb M CA Mono Gorge Road.CMS 10 CMSb M CA Mono Gorge Road.CMS 11 CMSa CA Inyo Panamint Mts. at Wildrose Cn.CMS 12 CMSa CA Kern El Paso Mts. at Last Chance Cn.CMS 13 CMSa F CA San Bernardino 5 km WSW Death Valley Nat. ParkCMS 14 CMSa F CA Inyo Death Valley Road. nr. Nicholas Eureka MineCMS 15 CMSb F CA San Bernardino N. of BarstowCMS 16 CMSa NV Nye 5 mi. S. BeattyCMS 17 CMSa M NV Clark Las VegasCMS 18 CMSb CA Inyo Gorge Road. 12.4 mi. NW BishopCC 1 CCa M AZ Maricopa South PhoenixCC 2 CCa AZ Maricopa Hwy. 283 nr. MobileCC 3 CCa AZ Maricopa Sun Valley ParkwayCC 4 CCa AZ Maricopa Sun Valley ParkwayCC 5 CCa AZ Maricopa I-10 at Sun Valley Pkwy.CC 6 CCd CA San Bernardino East Ord Mt., S. BarstowCC 7 CCa F AZ Pinal Park Link DriveCC 8 CCa F AZ Pinal Park Link DriveCC 9 CCa M AZ Maricopa Rainbow Valley Road.CC 10 CCa F AZ Maricopa Rainbow Valley Road.CC 11 CCa AZ La Paz Nr. Salome

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CC 12 CCb M CA San Diego Anza Borrego State ParkCC 13 CCb F CA Riverside Whitewater Cn. Road., W. Palm SpringsCC 14 CCd CA San Bernardino Shadow Mt. Road.CC 15 CCd CA San Bernardino Shadow ValleyCC 16 CCd M CA San Bernardino 3 miles E. Twenty-nine PalmsCC 17 CCc M CA Inyo Hwy. 190, btw. Owens Lake and Death ValleyCC 18 CCb J CA Riverside Hwy. 177 1 mi. N. Desert CenterCC 19 CCc CA Mono Fish Slough Road., nr. BishopCC 20 CCm F MX Sonora Near Bahia Kino (coastal Sonora)CC 21 CCm F MX Sonora Punta Chueca, N. of Bahia KinoCC 22 CCd NV Clark New Gold Butte Rd. 18.6 mi. S. Hwy. 170CT 1 CTb AZ Cochise Peloncillo Mts.CT 2 CTa M AZ Maricopa Mummy Mt.CT 3 CTa M AZ Maricopa Pioneer MuseumCT 4 CTa M AZ Maricopa Hwy. 88 nr Canyon LakeCT 5 CTa F AZ Pinal Owl Head ButtesCT 6 CTa AZ Maricopa Squaw PeakCT 7 CTa M AZ Maricopa Squaw PeakCT 8 CTa AZ Maricopa South Mt. ParkCT 9 CTa AZ Maricopa South Mt.CT 10 CTb AZ Santa Cruz Mt. Hopkins, western Santa Rita Mts.CT 11 CTb AZ Santa Cruz Mt. Hopkins, western Santa Rita Mts.CT 12 CTa AZ Maricopa Near Pinnacle PeakCT 13 CTa F AZ Maricopa Hwy. 88 9.5 mi. E jct Apache TrailCT 14 CTa M AZ Pima Hwy. 386 3.5 mi. S. Hwy. 86CT 15 CTa M AZ Pima Tres Bellotes Road. 2.7 mi. S. Arivaca Road.CT 16 CTa M AZ Maricopa South Mt.CT 17 CTa M AZ Pima Pima Cn., Catalina Mts.CT 18 CTa M AZ Pinal N32 36.256 W111 07.462CT 19 CTa M AZ Maricopa Squaw PeakCT 20 CTa F AZ Pinal N32 36.260 W111 07.939CT 21a CTa M AZ Pinal Suizo MountainsCT 21 CTa F AZ Pinal Suizo Mts.CT 22 CTa AZ Maricopa Maricopa Mts.CT 23 CTa M AZ Maricopa Apache Trail btw. Overlook and Canyon LakeCT 24 CTa F AZ Maricopa Apache Trail near Tortilla FlatsCT 25 CTa M AZ Maricopa McDowell Mts.CT 26 CTa M AZ Maricopa McDowell Mts.CT 27 CTb F AZ Santa Cruz Pajarito Mts.CT 28 CTb M AZ Santa Cruz Pajarito Mts.CT 29 CTa F AZ Pima Little Ajo Mts.CT 30 CTa AZ Maricopa Butterfield TrailCT 31 CTa M AZ Maricopa Superstition Mts.CT 32 CTa AZ Pinal FlorenceCT 33 CTb AZ Santa Cruz Mt. Hopkins RoadCT 34 CTb AZ Santa Cruz Mt. Hopkins RoadCT 35 CTm M MX Sonora Alamos areaCT 36 CTm M MX Sonora HermosilloCT 37 CTm F MX Sonora HermosilloCR 1 CRa F MX BCN Ojos NegrosCR 2 CRm M MX BCS Canon Agua Caliente. Sierra de la LagunaCR 3 CRa CA Riverside Redlands city limitsCR 4 CRa CA Riverside Whitewater Cn. just N. Palm SpringsCR 5 CRa M CA San Bernardino Bryn Mawr (E. Loma Linda)CR 6 CRa M CA Riverside Box Springs Mts.CR 7 CRa F CA Riverside Box Springs Mts.CR 8 CRa CA San Diego Fallbrook (∼50 mi. NNW San Diego)CR 9 CRa M CA Riverside Off Hwy. 60 nr. UC-RiversideCR 10 CRa M CA San Diego Anza Borrego State ParkCX 1 CRx M MX BCN Isla Cedros BCN

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Appendix Continued