molecular systematics of new world gopher, bull, and pinesnakes (pituophis: colubridae), a...

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Molecular Phylogenetics and EvolutionVol. 14, No. 1, January, pp. 35–50, 2000Article ID mpev.1999.0698, available online at http://www.idealibrary.com on

Molecular Systematics of New World Gopher, Bull, and Pinesnakes(Pituophis: Colubridae), a Transcontinental Species Complex

Javier A. Rodrıguez-Robles*,1 and Jose M. De Jesus-Escobar†

*Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, California 94720-3160;and †Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California,

Berkeley, California 94720-3204

Received October 14, 1998; revised June 17, 1999

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Pituophis melanoleucus (gopher, bull, and pine-nakes) is among the most widely distributed poly-ypic species complexes in North America, with mostuthors recognizing from a single transcontinentalpecies (the melanoleucus complex, composed of 15ubspecies) to four (monotypic and polytypic) species.e used mitochondrial gene sequences from the twoiddle American species, P. deppei and P. lineaticollis,

nd from 13 subspecies from most of the range of theelanoleucus complex to test various phylogeneticypotheses for Pituophis. Maximum parsimony andaximum likelihood methods identified the same ma-

or clades within Pituophis and indicated that twoegments of the melanoleucus complex, the lodingi–elanoleucus–mugitus eastern pinesnake clade and

he affinis–annectens–bimaris–catenifer–deserticola–ayi–ruthveni–vertebralis clade from central and west-rn United States and northern Mexico, representivergent, allopatric lineages with no known intergra-ation zone. We recognize each of these two groupingss a different species. Our data also indicate that someuthveni are more closely related to sayi than to otheruthveni. Nonetheless, ruthveni is an allopatric taxoniagnosable from its closest relatives by a combinationf morphometric characters, and because it is likelyhat at least some of these traits are independent andenetically inherited, we interpret this as evidencehat ruthveni has attained the status of independentvolutionary lineage, despite the fact that it retainstrong genetic affinities with sayi. The endemic Bajaalifornian gopher snakes (bimaris and vertebralis)re considered by some taxonomists as a differentpecies, P. vertebralis, but we discovered that theseerpents belong to two different clades and hence weo not agree with the recognition of P. vertebralis asresently defined. In summary, we believe that threeistinct species are included in the melanoleucus com-lex, Pituophis melanoleucus (sensu stricto), P. cateni-er, and P. ruthveni, and that their recognition better

1 To whom correspondence should be addressed. Fax: (510) 643-
238. E-mail: [email protected].
35

epresents the evolutionary diversity within this spe-ies complex. r 2000 Academic Press

Key Words: biogeography; Colubridae; mitochondrialNA; phylogenetics; Pituophis; snakes; species bound-ries; subspecies.

INTRODUCTION

The question as to the number of species includedin this genus [Pituophis] is a difficult one to decide.(Cope, 1900, p. 866)Boundaries between species and subspecies have

eceived considerable attention from systematists (Ballnd Avise, 1992; Cracraft, 1983; McKitrick and Zink,988; Patton and Smith, 1994; Smith et al., 1997;uster and Thorpe, 1992) and have often generated

nstructive and sometimes intense discussions (Avisend Wollenberg, 1997; Collins, 1992; Dowling, 1993;dwards, 1954; Frost and Hillis 1990; Frost et al., 1992;ighton, 1998; Inger, 1961; Wake and Schneider, 1998;iens, 1982; Wilson and Brown, 1953). During the

‘molecular systematics revolution’’ of the last threeecades (Avise, 1994; Hillis et al., 1996; Soltis et al.,998), genetic data have sometimes contradicted previ-us ideas about species integrity or taxonomic distinc-ions that were based largely on morphological descrip-ions and thus led authors to reject named subspecies,ecognize new ones, or elevate some to the species levele.g., Alcobendas et al., 1996; Cicero, 1996; Garcıa-

oreno and Fjeldså, 1999; Good and Wake, 1992;ohnson, 1995; Miththapala et al., 1996; Shaffer andcKnight, 1996; Wuster and Thorpe, 1994; Zamudio

nd Greene, 1997; Zamudio et al., 1997; Zink et al.,997). Determining which populations warrant taxo-omic recognition contributes to our understanding ofhe evolution of biodiversity and has significant, practi-al implications, e.g., for sociology (Keita, 1993) andonservation policies (Avise, 1989; Geist, 1992; Greene,994; O’Brien and Mayr, 1991; Sites and Crandall,997; Walker et al., 1998; Zhu et al., 1998).
Evolutionary relationships of transcontinental taxa
1055-7903/00 $35.00Copyright r 2000 by Academic PressAll rights of reproduction in any form reserved.

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36 RODRIGUEZ-ROBLES AND DE JESUS-ESCOBAR

ave rarely been studied in detail (e.g., Bernatchez andilson, 1998). Taxa with such broad geographic ranges

rovide an excellent opportunity to investigate theractical issues in naming population assemblages.nakes of the colubrid genus Pituophis Holbrook, 1842re among the most widely distributed polytypic spe-ies complexes in North America. Commonly known innglish as gopher, bull, and pinesnakes and in Spanishs ‘‘alicantes,’’ ‘‘cincuates,’’ ‘‘coralillos,’’ and ‘‘mazacua-es,’’ these serpents range from the Pacific to thetlantic coast of the United States (US) and fromouthwestern Canada to central Guatemala and the tipf the Baja California peninsula in Mexico (Figs. 1 and). Species of Pituophis are nonvenomous constrictors;hey occur in a wide variety of habitats, includingeserts, sandhills, prairies, shrublands, and forestsDegenhardt et al., 1996; Palmer and Braswell, 1995;weet and Parker, 1990); they attain body sizes in theild of up to 2.7 m; they exhibit notable variation in

nout morphology (Knight, 1986) and reproductiveiology (Fitch, 1985; Palmer and Braswell, 1995; Rei-hling, 1990; Zappalorti et al., 1983); and they may beatesian mimics of rattlesnakes (Kardong, 1980;anchez-Herrera et al., 1981; but see Sweet, 1985).

FIG. 1. Approximate distribution of the subspecies of the Pituophionant and Collins, 1991; Reichling, 1995; Stebbins, 1985; Sweet a

ocalities of the specimens included in this study. The black barypothesized Pleistocene midpeninsular seaway (after Upton and Mcale.

espite the fact that Pituophis has figured prominently i

n the anatomical, ecological, ethological, and herpeto-ulturist literature, the genus has had a contentiousaxonomic history, which we briefly review as back-round for our systematic study of these snakes.The monophyly of Pituophis is strongly supported by

t least two unique morphological characters, an en-arged epiglottal keel and a laryngeal septum (Cope,891; White, 1884; Young et al., 1995), which togetherroduce the unusual defensive hissing made by Pituo-his (Martin and Huey, 1971; Young et al., 1995), asell as by phylogenetic analyses of mtDNA sequencesf the genus and its closest relatives (i.e., Arizona,ogertophis, Cemophora, Elaphe, Lampropeltis, Rhino-

heilus, and Senticolis; Rodrıguez-Robles and De Jesus-scobar, 1999). In these analyses the monophyly ofituophis was supported by very high ($96%) boot-trap values in maximum parsimony trees.Beyond the issue of the monophyly of the genus,

here has been little agreement among different au-hors about the number of species and subspeciesecognized within Pituophis. The most common argu-ents presented can be summarized in five main

heses.(1) The most taxonomically conservative view regard-

elanoleucus complex in Canada, the United States, and Mexico (afterParker, 1990; Tennant, 1984). Numbers indicate the approximate

oss central Baja California indicates the inferred location of thehy, 1997). The four islands indicated with arrows are not drawn to

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37SYSTEMATICS AND BIOGEOGRAPHY OF Pituophis

us species complex (hereafter, the melanoleucus com-lex) in southwestern Canada, the US, and northernexico recognizes a single, polytypic species, P. melano-

eucus, composed of 15 subspecies, including four taxandemic to islands off the west coast of California andaja California (Conant, 1956; Dowling, 1958; Smithnd Kennedy, 1951; Fig. 1). This taxonomic arrange-ent is followed in three widely used field guides to

mphibians and reptiles from the US (Behler, 1995;onant and Collins, 1991; Stebbins, 1985), as well as in

he Catalogue of American Amphibians and ReptilesSweet and Parker, 1990).

(2) Some authors (e.g., Fugler, 1955; Holman, 1996;lauber, 1947; Wright and Wright, 1957) have at least

uggested that the melanoleucus complex is a compos-te taxonomic unit consisting of two distinct species,ituophis melanoleucus (sensu stricto, including theubspecies lodingi, melanoleucus, and mugitus) in theastern US and P. catenifer (including the subspeciesffinis, annectens, bimaris, catenifer, coronalis, deserti-ola, fuliginatus, insulanus, pumilus, sayi, and vertebra-is) in the central and western US, northern Mexico,nd offshore islands. The Louisiana pinesnake, ruthveni,as been assigned to either P. melanoleucus (sensutricto; Stull, 1940; Wright and Wright, 1957) or P.atenifer (Fugler, 1955).(3) The Cape gopher snake, vertebralis, has sometimes

een claimed to be a distinct species (Pituophis vertebralis;.g., Cope, 1900; Grismer, 1994; Stull, 1940; Sweet, 1984).(4) Based on a phenetic analysis of 14 morphometric

haracters and on its geographic distribution, Reich-

FIG. 2. Approximate distribution of Pituophis deppei and P. lineaumbers indicate the approximate localities of the specimens include

ing (1995; see also Collins, 1991) argued for recogniz- m

ng ruthveni as a distinct species, Pituophis ruthveni,herefore effectively implying the recognition of P.elanoleucus (sensu stricto) and P. catenifer as well.(5) The taxonomic status of the middle American

opher snakes (Fig. 2), Pituophis deppei and P. lineati-ollis, each with two named subspecies, has been fairlytable since Duellman’s (1960) review. Nevertheless,ixon et al. (1962) questioned the validity of the two

ubspecies of P. deppei (P. d. deppei and P. d. jani),hereas Conant (1965) suggested that intergradationr introgression occurred between P. deppei and P. m.ffinis in northern Mexico, perhaps implying that P.eppei was not a valid species, a suggestion with whichorafka (1977) agreed.The taxonomic status of the various forms of Pituo-

his thus has provided systematists with an unre-olved controversy, particularly in the melanoleucusomplex, within which different authors have recog-ized from a single transcontinental polytypic specieso four (monotypic and polytypic) species. However,ith the exception of Cope’s (1900) and Stull’s (1940)

tudies, all these taxonomic arrangements were pro-osed after examining only a subset of the describedorms of this species complex, or were suggested basedn the geographic distribution and presumed diagnos-bility of the named subspecies. Our study tests theaxonomic hypotheses presented above with phyloge-etic trees inferred from mitochondrial DNA (mtDNA)equences from 16 of the 19 described forms of Pituo-his, the greatest number of described forms includedn any previous taxonomic study of the genus. Our

llis in Mexico and Guatemala (after Duellman, 1960; Grismer, 1994).this study.

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mong the currently recognized species and subspeciesf Pituophis, assuming that our gene genealogy accu-ately reflects these relationships (see Brower et al.,996; Moore, 1995, 1997). We also compare our findingso previous phylogenetic hypotheses for Pituophis (Fig.) and discuss some of the biogeographical implicationsf our results.

MATERIALS AND METHODS

axon Sampling, DNA Isolation, and Sequencing

We obtained tissue samples from one to four individu-ls of Arizona elegans (glossy snake), Bogertophis sub-cularis (Trans Pecos ratsnake), Lampropeltis getula

FIG. 3. Evolutionary relationships proposed for Pituophis: (a)fter Stull (1940); (b) after Klauber (1947).

common kingsnake), Pituophis d. deppei (Mexican (

ullsnake), P. deppei jani (northern Mexican bull-nake), P. lineaticollis gibsoni (Guatemalan gophernake), and within the melanoleucus complex, fromffinis (Sonoran desert gopher snake), annectens (Saniego gopher snake), bimaris (northern Baja Californiaopher snake), catenifer (Pacific gopher snake), deserti-ola (Great Basin gopher snake), fuliginatus (Sanartın Island gopher snake), lodingi (black pine-

nake), pumilus (Santa Cruz Island gopher snake),uthveni, sayi (bullsnake), and vertebralis (Table 1; Fig.). We could not secure samples of coronalis (Southoronado Island gopher snake) or insulanus (Cedros

sland gopher snake) but we believe that the omissionf these two taxa does not alter the major findings ofur study.We extracted total genomic DNA from ventral scale

lips preserved in 95% ethanol or from tissue samplesblood, liver, muscle) stored frozen at 274°C using theodium dodecyl sulphate–proteinase K/phenol/RNAaseethod (Sambrook et al., 1989). Using total cellularNA as template, we amplified (with the polymerase

hain reaction, PCR [Saiki et al., 1986, 1988]) and usedor phylogenetic analyses an 893-bp fragment of mtDNAhat encompassed a 697-bp portion of the 38 end of theicotinamide adenine dinucleotide dehydrogenase sub-nit 4 (Ndh4, or ‘‘ND4’’ gene) and a 196-bp section ofhree transfer ribonucleic acid (tRNA) genes (tRNAHis,RNASer, tRNALeu), using primers labeled ND4 and LeuArevalo et al., 1994). ND4 is a reliable tracer ofvolutionary history (Russo, 1997; Russo et al., 1996;ardoya and Meyer, 1996) and a relatively fast-volving gene useful for resolving relationships amonglosely related taxa (Cracraft and Helm-Bychowski,991). PCR was carried out in a programmable thermalycler in 100-µl reactions consisting of 2 µl of templateNA (50 ng/µl), 2.5 µl of primers (40 µM), 10 µl of 103CR reaction buffer (Stratagene), 2 µl of MgCl2 (25M), 2 µl of deoxynucleoside triphosphates (10 mM), 4

l of Thermus aquaticus DNA polymerase (5 U/µl), and7.5 µl of H2O. DNA was denatured initially at 94°C formin; then 33 cycles of amplification were carried outnder the following conditions: 94°C denaturation for0 s, 55°C annealing for 30 s, and 72°C extension for 1in, followed by a final 5-min extension at 72°C. Tenicroliters of the resulting PCR product were electro-

horesed on a 1% agarose gel and stained with ethidiumromide to verify product band size. For each indi-idual, we cloned its PCR product into a phosphatasedcoRV pBluescript II SK 6 phagemid vector (Strata-ene) using Escherichia coli as the vector and se-uenced both DNA strands in an automated sequencersing the dideoxy chain-termination method (Sanger etl., 1977). The sequence of Elaphe vulpina (fox snake)ncluded in this study was provided by R. Lawson
California Academy of Sciences, San Francisco).

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hylogenetic Analyses

Sequences from the light and heavy DNA strandsere input into the Sequence Navigator (version 1.0.1)rogram and aligned to each other and to the referenceequence of the colubrid snake Dinodon semicarinatusKumazawa et al., 1998). This initial alignment wasefined with the MacDNASIS Pro software (version.0). Pairwise comparisons of observed proportionalequence divergence ( p-distance), corrected sequenceivergence (with the Tamura–Nei model; Tamura andei, 1993), and number of transitions and transver-

ions by codon position were obtained using the com-uter program PAUP* 4.0b2a (Swofford, 1999).To estimate the phylogenetic information content of

he mtDNA character matrix, we used the g test (Hillisnd Huelsenbeck, 1992; Huelsenbeck, 1991; but seeallersjo et al., 1992) to assess the skewness of the tree

ength distribution of 100,000 trees randomly generatedith PAUP*. Probability of phylogenetic structure wasssessed using the values provided by Hillis and Huelsen-eck (1992).We used two methods of phylogenetic reconstruction:aximum parsimony (MP; Camin and Sokal, 1965;wofford et al., 1996) and maximum likelihood (ML;elsenstein, 1981; Huelsenbeck and Crandall, 1997), as

mplemented by PAUP*. For MP analyses, we used twoharacter weighting schemes: equal-weighting, in whichll nucleotide substitutions were weighted equally re-ardless of type or codon position, and differentialodon position weighting, in which we down-weightedhird position transitions (see below). Sites with inser-ion or deletion events were removed from the analyses.ach base position was treated as an unordered charac-

er with four alternative states. Ancestral charactertates were determined via outgroup comparison (Far-is, 1982; Maddison et al., 1984; Watrous and Wheeler,981). We used Arizona elegans, Bogertophis subocu-aris, Elaphe vulpina, and Lampropeltis getula asutgroups because previous molecular systematic stud-es (Rodrıguez-Robles and De Jesus-Escobar, 1999)dentified these taxa as close relatives of Pituophis.

Because the number of terminal taxa was too large toermit evaluating all trees or employing the branch-and-ound algorithm (Hendy and Penny, 1982), we usedeuristic search strategies for each tree-building meth-dology. We used 100 repeated randomized input ordersf taxa for all MP analyses to minimize the effects ofntry sequence on the topology of the resulting clado-ram(s). MP analyses were conducted without theteepest descent option and with accelerated characterransformation (ACCTRAN) optimization, tree bisec-ion–reconnection (TBR) branch swapping, save allinimal trees (MULPARS), and zero-length branches

ollapsed to yield polytomies settings in place. We usedonparametric bootstrapping (100 pseudoreplicates, 10
ddition-sequence replicates for MP, 50% majority rule) s
o assess the stability of internal branches in clado-rams (Berry and Gascuel, 1996; Felsenstein, 1985;elsenstein and Kishino, 1993; Sanderson, 1995). Non-arametric bootstrap values generally are a conserva-ive measure of the probability that a recovered groupepresents a true clade (Hillis and Bull, 1993; Li, 1997;harkikh and Li, 1992). Alternate topologies wereested for significance at the 95% level using the TempletonLarson, 1994; Templeton, 1983) and Kishino–HasegawaKishino and Hasegawa, 1989) tests for MP and ML trees,espectively, as implemented in PAUP*.

For ML analyses we randomly selected as the start-ng tree one of the trees found during the MP searches.sing empirical nucleotide frequencies and five rate

ategories, we fixed the probabilities of the six possibleucleotide transformations (A & C, A & G, A & T,& G, C& T, G& T), the proportion of invariable

ites u, and the a ‘‘shape’’ parameter of the gammaistribution of rate heterogeneity across nucleotideositions (Yang, 1996) to the empirical values calcu-ated from the starting tree in a search for a better MLree (a tree with a higher log-likelihood value), underhe general time-reversible model of nucleotide substi-ution (Gu et al., 1995; Swofford et al., 1996; Yang,994). In other words, we used the most parameter-richodel available to search for ML trees. When a tree of

igher likelihood was found, we reoptimized and fixedhe parameters for a subsequent ML search. We re-eated this procedure until the same tree was found inuccessive iterations (Swofford et al., 1996).Because ND4 is a protein-coding gene, we plotted p-

istance (y) versus corrected (with the Tamura–Nei model)stimates of proportional sequence divergence (x) for first,econd, and third codon positions. This was done sepa-ately for transitions and transversions to test for theossibility that some types of nucleotide substitutions haveecome saturated. Points that fall along the y 5 x line havehe same observed and estimated numbers of changes andhus have not been subjected to multiple hits. Points thatall below the y 5 x line indicate that multiple hits haveccurred; saturation is reached when observed sequenceivergence does not continue to increase, despite the facthat corrected estimates do. Conventional statistical testsf the relationship between estimated and observed se-uence divergence are not appropriate because of noninde-endence of the data points, due to the inclusion of eachoint in more than one pairwise comparison. Accordingly,he plots were used as heuristic devices to help identifylasses of changes occurring at different rates, whichhould be weighted differently in phylogenetic analyses.

RESULTS

equence Variation

There were 348 variable and 225 potentially phyloge-etically informative characters (sites with at least two
hared differences among all taxa) in the 893-bp mtDNA

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TABLE 1

Taxon, Sample Number (If Necessary), GenBank Ac-ession No., Voucher Number (If Available), and Local-ty of the Taxa Used in This Study

TaxonSamplenumber

GenBank Accession no.,voucher number, and locality

utgroupsArizona elegans — AF138749; MVZ 137685; U.S.: Cali-

fornia, Riverside Co., Hwy. 195,21.1 mi W junction with I-10 atChiriaco Summit

ogertophis sub-ocularis

— AF138752; CME 116; U.S.: Texas,Culberson Co., 18.1 road mi NVan Horne on Hwy. 54

laphe vulpina — AF138758; CAS 184362; U.S.: Ohio,Ottawa Co., East Harbor StatePark

ampropeltisgetula

— AF138759; HWG 1485; U.S.: Cali-fornia, San Benito Co., Hwy. 25,2.6 mi SE junction of Hwy. 146and Pinnacles National Monu-ment

ituophiseppei deppei 1 AF138765; Mexico: Durangoeppei deppei 2 AF138766; Mexico: Michoacaneppei jani — AF141096; specimen from the pet

tradeineaticollis gibsoni 3 AF138767; CJF 1500; Guatemala:

Departamento Zacapa, Sierra delas Minas

ineaticollis gibsoni 4 AF141097; CJF 1501; Guatemala:Departamento Zacapa, Sierra delas Minas

elanoleucus com-plex

ffinis 5 AF141098; MVZ 137697; U.S.: Ari-zona, Cochise Co., 12 mi NEDouglas via Route 80

ffinis 6 AF141099; HBS 1511; U.S.: NewMexico, Luna Co., 21 mi NColumbus on Route 11

ffinis 7 AF141100; MVZ 162369; U.S.: Ari-zona, Maricopa Co., southeastside of Salt River Mountains

nnectens 8 AF138763; MVZ 150206; U.S.: Cali-fornia, San Diego Co., UniversityCity

nnectens 9 AF141101; MVZ 149983; U.S.: Cali-fornia, San Diego Co., S.E. slopeof Mount Palomar, near southend of Jeff Valley, between Hwy.S-7 and Cedar Creek

imaris 10 AF141102; Mexico: Baja CaliforniaSur, Km marker 10 at SanIgnacio

imaris 11 AF141103; Mexico: Baja CaliforniaSur, Ciudad Constitucion

imaris 12 AF141104; Mexico: Baja California,Km. marker 154, S. San Quintın

imaris 13 AF141105; specimen from the pettrade

TABLE 1—Continued

TaxonSamplenumber


atenifer 14 AF141106; CAS 201258; U.S.: Cali-fornia, Mendocino Co., Mendo-cino National Forest, Forest RoadM1, 5.1 mi NE (by road) of Men-docino Pass Road at Eel RiverWork Center

atenifer 15 AF141107; JAR 75; U.S.: Cali-fornia, Monterey Co., HastingsNatural History Reservation,Carmel Valley, 38601 E. CarmelValley Road

atenifer 16 AF141108; JAR 77; U.S.: Cali-fornia, Alameda Co., 9618 LupinWay

atenifer 17 AF141109; U.S.: California, NapaCo., Napa, 1.8 mi W 2930 Red-wood Road

eserticola 18 AF138764; MVZ 137577; U.S.:Nevada, Mineral Co., Hwy. 31,6.6 mi SW Hawthorne

eserticola 19 AF141110; MVZ 150216; U.S.: Colo-rado, Garfield Co., on ColoradoRiver, 40 mi E Grand Junction

eserticola 20 AF141111; RSR 115; U.S.: Cali-fornia, Kern Co., 5 mi N and 2.5mi E junction Neuralia and Cali-fornia City Boulevard

eserticola 21 AF141112; U.S.: Utah, Utah Co.18–27 m W Hwy. 89, aboutmidway between Provo andSpringville

uliginatus — AF141113; Mexico: Baja California,San Martın Island

odingi 22 AF141114; HWG 2651; specimenfrom the pet trade

odingi 23 AF141115; HWG 2652; specimenfrom the pet trade

elanoleucus 24 AF138770; MVZ 150219; U.S.:North Carolina, Brunswick Co.,3.5 mi N Southport

elanoleucus 25 AF141116; MVZ 225520; U.S.: NewJersey, Cumberland Co., TheNature Conservancy, Warner Site

elanoleucus 26 AF141117; MVZ 225521; U.S.: NewJersey, Burlington Co., BassRiver State Forest

ugitus 27 AF138769; USNM 211452; U.S.:Florida, Wakulla Co., St. Mark’sWildlife Refuge, about 1.5 mi SWOtter Lake

ugitus 28 AF141118; MVZFC 13063; U.S.:Florida, Hillsborough Co.,Tampa, 1 mi SE University ofSouth Florida campus

umilus 29 AF141119; U.S.: California, SantaBarbara Co., Santa Cruz Island,near Stanton Ranch

umilus 30 AF141120; U.S.: California, SantaBarbara Co., Santa Cruz Island

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ata matrix. Of the informative characters, 29 were atrst codon positions, 13 at second positions, 144 athird positions, and 39 at noncoding positions. Withinituophis there were 21, 9, 121, and 33 informativeharacters at first, second, third, and noncoding posi-ions, respectively. Significant phylogenetic signal wasresent in the data set (g1 5 20.6222, P ,,, 0.01;ean 6 SD tree length 5 1817.3 6 31.3, range 1625–

905); thus, inferring cladograms was justified.Scatter plots of observed versus estimated sequence

ivergences indicated that these relationships are lin-ar for first and second position transitions and trans-ersions and third position transversions (Fig. 4). Thirdosition transitions deviated greatly from a linearattern, suggesting that these mutations are satu-ated. To estimate the transition-to-transversion biasor third position transitions, we fitted a least-squaresegression line, forced through the origin, to the part ofhe curve of third position transitions versus thirdosition transversions that is roughly linear. The slopef the regression line, 0.648, is an estimate of the

TABLE 1—Continued

TaxonSamplenumber


uthveni 31 AF138771; U.S.: Louisiana, Bien-ville Parish, 2 km E Kepler CreekLake Bridge

uthveni 32 AF141121; U.S.: Louisiana, Bien-ville Parish, 7 km E Kepler CreekLake Bridge

uthveni 33 AF138772; U.S.: Louisiana, Bien-ville Parish, 2 km S junction ofLA 154 and 507

ayi 34 AF141122; MVZ 150218; U.S.:Texas, Jeff Davis Co., on U.S.Route 90, 43.8 mi SE junction ofI-10 and U.S. Route 90

ayi 35 AF141123; U.S.: Colorado, Jef-ferson Co., SE junction of W. CoalMine Road and Wadsworth Bou-levard

ayi 36 AF141124; MVZ 226247; U.S.: Mis-souri, Saint Louis Co.

ayi 37 AF141125; U.S.: Oklahoma, Cleve-land Co.

ertebralis 38 AF141126; Mexico: Baja CaliforniaSur, Rancho Buena Vista

ertebralis 39 AF141127; JAR 78; Mexico: BajaCalifornia Sur, Cape region

ertebralis 40 AF141128; specimen from the pettrade

Note. Museum and collector abbreviations are: CAS, Californiacademy of Sciences, San Francisco; MVZ, Museum of Vertebrateoology, University of California, Berkeley; MVZFC, MVZ frozenollection; USNM, National Museum of Natural History, Smithso-ian Institution, Washington, DC; CJF, Carl J. Franklin; CME,urtis M. Eckerman; HBS, H. Bradley Shaffer; HWG, Harry W.reene; JAR, Javier A. Rodrıguez; RSR, Randall S. Reiserer.

ransition-to-transversion ratio (Lara et al., 1996; Moore t

nd DeFilippis, 1997). Therefore, we down-weightedhird codon transitional changes by a factor of 6 using a:1:0.17 codon position weighting (first, second, andhird codon position, respectively) to correct for theiased substitution rates at this position.

hylogenetic Relationships

The two phylogenetic methods that we used recov-red the same major nodes (Figs. 5 and 6), regardless ofhe weighting scheme used, which suggests that theroupings represent true clades. Most of these nodesere supported by relatively high bootstrap values

$80%) in the two MP consensus trees but the relation-hips among the clades were almost completely unre-olved. Although the single ML tree obtained (Fig. 6)esolved nearly all the relationships among the cladesf Pituophis, some of the internal branches of this treere short, indicating that they are supported by fewharacters, which may explain why these nodes col-apse during the random resampling of characters thatakes place during bootstrapping. The short internodeslso suggest that the divergence of the major lineages ofituophis occurred rapidly and near simultaneously.P and ML trees constrained to include only monophy-

etic subspecies of the melanoleucus complex wereignificantly poorer estimates of phylogenetic relation-hips than the unconstrained trees (Table 2).MP and ML methods identified monophyletic clades

f the two species of middle American gopher snakes (P.eppei and P. lineaticollis). Nonetheless, the lack ofpecimens of affinis and sayi from Mexico and ourimited sampling of the two subspecies of P. deppeirevented us from evaluating the disputed monophylyf the latter relative to the melanoleucus complexConant, 1965; Dixon et al., 1962; Morafka, 1977).learly, further studies that include representatives ofexican affinis and sayi, of P. d. deppei and P. d. jani

rom additional localities, and of P. l. lineaticollis fromouthern Mexico are needed to better understand thevolutionary history of middle American Pituophis.The MP and ML trees also showed that the eastern

inesnakes, lodingi, melanoleucus, and mugitus, formmonophyletic clade, and these trees are significantly

horter or have a higher likelihood ratio, respectively,han those constrained to include nonmonophyleticastern pinesnakes (Table 3). The MP bootstrap treenferred with equally weighted characters and the MLree identified the eastern pinesnake lineage as theister taxon to all other Pituophis, but this topology wasot significantly different from that on which theastern pinesnakes were constrained to not be theister taxon to all other Pituophis (Table 4).All the topologies recovered by the MP and MLethods identified separate, monophyletic clades of theelanoleucus complex (Figs. 5 and 6). Within these,

here is clear geographic structuring. As mentioned,
he eastern pinesnakes formed a distinct clade, and so

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id the gopher snakes from southern Baja Californiavertebralis and southern bimaris); affinis from south-rn Arizona; catenifer and deserticola from northernalifornia and western Nevada; annectens, bimaris,

uliginatus, and pumilus from southern California,orthern Baja California, and offshore islands; and sayind ruthveni from the central US. To test whether thewo recovered clades of endemic Baja Californian go-her snakes (bimaris and vertebralis) represent a reli-ble estimate of the relationships of these peninsularnakes, we compared the MP and ML trees (Figs. 5 and) with trees obtained using the same search param-ters but constrained to form a single monophyleticlade of bimaris and vertebralis. The unconstrainedrees were significantly better estimates of relationshan the constrained phylogenies (Table 5).

DISCUSSION

Our analyses indicated that some populations of theelanoleucus complex are more closely related to geo-

FIG. 4. Scatter plots of pairwise sequence differences (uncorrecositions versus Tamura–Nei estimates of pairwise divergence for the

raphically closer populations of different subspecies r

han to more distant, consubspecific populations. Taxare recognized as subspecies, not full species, preciselyecause they intergrade with neighbors, and this geneow will blur the boundaries of subspecies, keepinghem from attaining reciprocal monophyly at thetDNA level (Patton and Smith, 1994). Consequently,

he observed pattern of geographic distribution of genea-ogical lineages within Pituophis from the US is noturprising, as there is evidence of intergradation be-ween affinis and sayi, affinis and deserticola, affinisnd annectens, annectens and catenifer, annectens andeserticola, bimaris and vertebralis, catenifer and deser-icola, and among lodingi, melanoleucus, and mugitusGrismer, 1997; Klauber, 1946, 1947; Sweet and Parker,990). Furthermore, the subspecies of the melanoleu-us complex, especially those from the western US,ere originally recognized on the basis of rather minorifferences in squamation, number of tail spots, andumber, shape, and coloration (e.g., brown versuslack) of body blotches (Klauber, 1947), which suggestshat some of these taxa may not deserve taxonomic

) in transitions and transversions at first, second, and third codonme class of substitutions.

ted

ecognition.

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The mtDNA sequences resolved several lineagesithin the melanoleucus complex, reflecting the evolu-

ionary history of independent units within this group.e used a conservative approach and based the follow-

ng discussion of phylogenetic and biogeographic pat-erns within Pituophis on those clades common to theP and ML trees.

he Taxonomic Status of the Eastern Pinesnakes

Our genetic data, irrespective of the phylogeneticethod used, indicate that the eastern pinesnakes,

odingi, melanoleucus, and mugitus, form a distinctlade within Pituophis. Collectively, these three taxare allopatric and diagnosable from other subspecies ofhe melanoleucus complex using a combination of mor-hometric characters (Reichling, 1995). We interprethese findings as evidence that the eastern pinesnakesepresent a distinct evolutionary lineage, and thereforee agree with previous suggestions (e.g., Cope, 1900;ugler, 1955; Klauber, 1947; Wright and Wright, 1957)

hat they should be considered as a distinct species, forhich the name Pituophis melanoleucus (sensu stricto)

s available. By recognizing P. melanoleucus (sensutricto), we effectively imply the existence of at leastne more species within the melanoleucus complex, forhich the name Pituophis catenifer is available, andhich includes the subspecies annectens, affinis, cateni-

FIG. 6. Maximum likelihood tree (LnL 5 25539.18903) for 42tDNA haplotypes of Pituophis. Branches are drawn proportional to

ranch lengths (expected amount of character change) estimated byhe maximum likelihood algorithm. The proposed specific taxonomys indicated for each taxon.

FIG. 5. Maximum parsimony bootstrap consensus trees for 42tDNA haplotypes of Pituophis. Numbers on the tree indicate

ercentage of nonparametric bootstrap support for nodes retained byore than 50% of bootstrap replicates; (a) with all characterseighted equally; (b) with third position transitions down-weightedy a factor of 6:1 and with the proposed specific taxonomy indicated

er, coronalis, deserticola, fuliginatus, insulanus, pumi-

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us, sayi, and perhaps bimaris, vertebralis, and ruthvenibut see below).

Knight (1986) suggested that the shape of the premax-lla–nasal articulation in the skull of Pituophis fromhe US was a useful character to distinguish between‘eastern’’ (melanoleucus, mugitus, and sayi) and ‘‘west-rn’’ (affinis and deserticola) taxa. Regardless of the facthat our results unambiguously indicate that sayi doesot belong within the eastern pinesnake clade, ourxamination of several skulls from most of the range ofhe melanoleucus complex indicates that the shape ofhe premaxilla–nasal articulation is so variable that itannot be reliably used to differentiate between snakesrom this species complex.

The ranges of lodingi and ruthveni, the geographi-ally closest taxa of Pituophis melanoleucus (sensutricto) and the rest of the melanoleucus complex,espectively, are separated by the broad alluvial plainf the Mississippi River. The alluvium, the swamps,nd the periodic flooding to which the big river valley isubjected seemingly combine to form an effective bar-ier to contact between P. melanoleucus (sensu stricto)nd the rest of the melanoleucus complex. The Missis-ippi River, especially its lower drainage in the south-astern US, also delimits the eastern or western limits

TAB

Comparison of Maximum Parsimony (MP; with theaximum Likelihood (ML; with the Kishino–Hasegawa

nd Constrained to Include Monophyletic Subspecies o

hylogeneticmethod

Weightingscheme Topology

Treelength R

MP EW Unconstrained 851 0.Constrained 979 0.

MP TS/TV 1:6 Unconstrained 1300 0.Constrained 1490 0.

ML — Unconstrained 855Constrained 984

Note. EW, equally weighted characters; RCI, rescaled consistency in

TAB


nd Constrained to Include a Nonmonophyletic Grond mugitus)

hylogeneticmethod


Treelength

MP EW Unconstrained 851Constrained 864

MP TS/TV 1:6 Unconstrained 1300Constrained 1328


Note. EW, equally weighted characters; RCI, rescaled consistency inde

f the distribution of subspecies of other amphibian andeptile taxa (e.g., Scaphiopus holbrookii [eastern spade-oot toad; Wasserman, 1968], Deirochelys reticulariachicken turtle; Zug and Schwartz, 1971], Ophisaurusttenuatus [slender glass lizard; Holman, 1971], Micru-us fulvius [harlequin coralsnake; Roze and Tilger,983; see also Blair, 1958), among which there are nonown zones of intergradation. It would be of interest toonduct phylogenetic studies of these taxa to determinehether populations on each side of the Mississippiiver also represent different evolutionary lineages.

he Phylogenetic Position of the Louisiana Pinesnake

The taxonomic status of ruthveni, the Louisianainesnake, has long been controversial. Some authorse.g., Stull, 1940; Wright and Wright, 1957) have claimedhat ruthveni is more closely related to the pinesnakesrom the eastern US, whereas others (e.g., Fugler, 1955)tated that its closest relatives are the gopher snakesrom the central and western US and Mexico. Espous-ng Wiley’s (1978:18) version of the evolutionary speciesoncept (‘‘a species is a single lineage of ancestralescendant populations of organisms which maintainsts identity from other such lineages and which has its

E 2

empleton Test [Larson, 1994; Templeton, 1983]) andst [Kishino and Hasegawa, 1989]) Trees Unconstrainedhe melanoleucus Complex

n TS 2ln L SD t P

101 220.5 — — — ,0.0001

103 268 — — — ,0.0001

— — 5539.19 40.3 9.58 ,0.00015925.08

x; TS/TV, transition-to-transversion ratio.

E 3

empleton Test [Larson, 1994; Templeton, 1983]) andst [Kishino and Hasegawa, 1989]) Trees Unconstrained

of Eastern Pinesnakes (i.e., lodingi, melanoleucus,

CI n TS 2ln L SD t P

67 35 198 — — — 0.035716 24 55 — — — 0.00399

— — 5539.19 7.99 2.31 0.025557.68

L

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wn evolutionary tendencies and historical fate’’),eichling (1995) elevated ruthveni to the species level.Both morphologically and geographically, the Louisi-

na pinesnake lies between the eastern pinesnakes andhe gopher snakes, with lodingi its nearest neighbor tohe east and sayi to the west (Reichling, 1995). Appar-ntly, there is no evidence that ruthveni intergradesith sayi, and there are only three specimens from

outheastern Louisiana and western Mississippi touggest that intergradation may take place betweenuthveni and lodingi (Conant, 1956; Crain and Cliburn,971). (The specimen discussed in Conant’s article wasntil recently believed to be missing but it is at theerpetological collection of Cornell University [CU2953].) Nonetheless, our analyses (as well as those ofllozyme data; J. Himes, pers. comm.) unambiguouslyupport placement of ruthveni within the clade ofopher snakes from the central and western US andorthern Mexico; they indicate as well that ruthvenind three of the four sayi included in this study form aell-supported clade (Figs. 5 and 6). Considering that

he ranges of ruthveni and sayi probably were parapat-ic in the recent past (Conant, 1956; Reichling, 1995),heir close relationship could be the result of recent

TAB


nd Constrained to Not Depict a Sister Group Reelanoleucus, and mugitus) and All Other Pituophis

hylogeneticmethod


Treelength




Note. EW, equally weighted characters; RCI, rescaled consistency in

TAB


nd Constrained to Include a Monophyletic Groupnd vertebralis

hylogeneticmethod


Treelength




Note. EW, equally weighted characters; RCI, rescaled consistency inde

ene flow or of stochastic lineage sorting of sharedncestral polymorphisms.Is ruthveni a distinct species? The answer to this

uestion is the most difficult phylogenetic problemithin Pituophis, and the arguments presented in

upport of either position once again underscore thathe criteria that we use to identify species and delin-ate species boundaries in nature are determined byur particular philosophy about species (e.g., de Quei-oz and Donoghue, 1988; Frost and Hillis, 1990; Pattonnd Smith, 1994; Wake and Schneider, 1998; Zink andcKitrick, 1995). Provided that our gene tree repre-

ents the evolutionary relationships of Pituophis, ourata indicate that the recognition of Pituophis ruthveniould render P. catenifer a paraphyletic species (Figs. 5nd 6). Consequently, systematists who prefer to definepecies by monophyly would probably disagree with thelevation of ruthveni to species status. On the otherand, authors who adhere to the evolutionary speciesoncept (ESC) perhaps would feel that such a taxo-omic decision is warranted, as ruthveni is an allopat-ic taxon diagnosable from its nearest neighbors by aombination of morphometric characters (Reichling,995). Proponents of the ESC could also claim that the

4

empleton Test [Larson, 1994; Templeton, 1983]) andst [Kishino and Hasegawa, 1989]) Trees Unconstrainedionship between Eastern Pinesnakes (i.e., lodingi,

CI n TS 2ln L SD t P

367 22 110 — — — 0.55365416 11 24 — — — 0.37414

— — 5539.19 4.52 0.49 0.625541.41


5

empleton Test [Larson, 1994; Templeton, 1983]) andst [Kishino and Hasegawa, 1989]) Trees UnconstrainedEndemic Baja Californian Gopher Snakes, bimaris

I n TS 2ln L SD t P

7 36 140 — — — 0.000706 34 112 — — — 0.00050

— — 5539.19 17.52 3.71 0.00025604.13

LE

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0.0.0.0.

——

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——


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attern of genetic relationship between ruthveni andayi revealed by our analyses simply reflects the re-ency of their divergence and lack of time for lineageorting to result in reciprocal monophyly. However,ritics might still contend that Reichling’s taxonomictudy of ruthveni did not include all the subspecies ofhe melanoleucus complex and accordingly cannot besed to argue in favor of or against recognizing theouisiana pinesnake as a distinct species.Two arguments are relevant when considering the

forementioned views regarding the taxonomic statusf ruthveni. First, the theoretical basis for the empha-is on naming monophyletic species is not clear, as anypecies has evolved from a preexisting species, and thisncestral species necessarily ceased to be monophyletict the moment of speciation (Baum, 1992; de Queiroznd Donoghue, 1988, 1990; Graybeal, 1995). Hence, its impossible to avoid recognizing paraphyletic species.The practice of assigning new species status to allaughters of a cladogenetic event is simply a taxonomiconvention and does not necessarily indicate biologicaleality.)Second, although Reichling’s (1995) taxonomic study

f ruthveni did not include the western-most subspeciesf the melanoleucus complex (i.e., annectens, bimaris,ertebralis, and the four island taxa), it included theeographically and phenotypically closer relatives ofuthveni (lodingi, melanoleucus, mugitus, and sayi), asell as affinis, deserticola, and catenifer. Hence, weaintain that Reichling’s findings should be taken into

ccount when judging the taxonomic status of theouisiana pinesnake and that not doing so is tanta-ount to ignoring available evidence. Therefore, be-

ause it is likely that at least some of the morphometricharacters shown by Reichling to collectively and unam-iguously distinguish ruthveni from its closer relativesre independent and genetically inherited, we believehat the Louisiana pinesnake has attained the status ofndependent evolutionary lineage. We then agree witheichling’s (1995) suggestion (see also Collins, 1991;onant, 1956) of recognizing ruthveni as a distinctpecies, Pituophis ruthveni. One reason why our mtDNAequence data do not yet reflect the independent evolu-ionary trajectory of P. ruthveni may be that popula-ions of this snake constitute an example of whatraybeal (1995) called ‘‘ferespecies,’’ interbreedingroups of organisms that do not yet possess exclusivityf descent. Nevertheless, it is possible that DNA se-uences of other genetic markers (e.g., from the nuclearenome or even other mitochondrial genes) wouldndicate a higher degree of genetic cohesiveness amongopulations of ruthveni.Despite the above arguments, the taxonomic status

f the Louisiana pinesnake is likely to remain controver-ial, not so much due to lack of relevant data, but ratherecause of different interpretations of available evi-
ence, and although we recognize these snakes at full m
pecies rank, we understand why other workers mayisagree with our position. If Louisiana pinesnakes arendergoing ‘‘the speciation process,’’ their debatabletatus illustrates the continuing difficulty in makingaxonomic assignments in such cases.

he Taxonomic Status and Biogeography of theEndemic Baja Californian Gopher Snakes

The results of our phylogenetic analyses regardinghe endemic gopher snakes from Baja California, bima-is and vertebralis, are in disagreement with the cur-ent taxonomy of these serpents. Traditionally, theopher snakes from central and southern Baja Califor-ia were assigned to bimaris, with snakes from theutskirts of the town of La Paz south to the tip of theeninsula (the Cape region) assigned to vertebralisFig. 1). Recently, Grismer (1994, 1997) synonymizedimaris with vertebralis and elevated the latter topecies level. We discovered that the southernmostimaris included in our study was more closely relatedo vertebralis (sensu stricto) than to more northernopulations of bimaris, which nested within a separatelade composed of two island subspecies (fuliginatusnd pumilus), and of annectens and catenifer (sample5) from southern California (Figs. 5 and 6). Addition-lly, the two distinct clades that include the endemicaja Californian gopher snakes are not sister taxa toach other, and when we constrained all bimaris andertebralis to form a monophyletic group, the resultingP and ML trees were significantly poorer estimates of

volutionary relationships within Pituophis (Table 2).hese findings led us to reject the recognition ofituophis vertebralis as defined by Grismer.The notable genetic break between northern and

outhern Baja Californian populations of Pituophisirrors the phylogeographic pattern displayed by Uta

tansburiana (side-blotched lizard) on the peninsulaUpton and Murphy, 1997). Upton and Murphy sug-ested that the disruption of mtDNAhaplotype distribu-ion between side-blotched lizards from northern andouthern Baja California was consistent with the exis-ence of a temporary midpeninsular seaway (Durhamnd Allison, 1960; Fig. 1). Presumably, this barrieraused the cessation of gene flow between populationsn the northern and southern portions of the peninsula,llowing them to evolve independently. Based on vari-us assumptions, Upton and Murphy hypothesizedhat the seaway existed one million years ago (Mya).he smallest uncorrected percentage sequence diver-ence between specimens of bimaris and vertebralisrom the northern and southern clades is 6.2%. Esti-ates of mtDNA sequence divergence for reptile spe-

ies for which branching events have been confidentlyated using fossil records or geological events rangerom 0.47 to 1.32% per million years (Zamudio andreene, 1997). Relying on these lower and upper esti-
ates, the two clades of endemic Baja Californian

gfioe

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opher snakes diverged 13.2–4.7 Mya. This estimate isor the divergence of the mtDNA lineages, not necessar-ly of the populations, and consequently it indicatesnly a maximum age for the split of the two clades ofndemic Baja Californian gopher snakes.Nevertheless, the above estimate suggests that the

enetic breakup between northern and southern Bajaalifornian gopher snakes was caused by a physiogeo-raphic event older than the formation of the midpenin-ular seaway. Elaborating on an idea apparently firstdvanced by Klauber (1947) and later restated byeviton and Tanner (1960), and relying on updatedaleogeographic evidence of the tectonic origin of Bajaalifornia, Murphy (1975, 1983a,b) proposed a ‘‘trans-ulfian vicariance model’’ for the origin of the herpeto-auna of the peninsula (see also Grismer, 1994). In

urphy’s scenario, several taxa migrated along withaja California when the area that became the Capeegion started rifting away from the western coast ofainland Mexico beginning approximately 11 Mya

late Miocene). If Murphy’s vicariance model applies toituophis, the endemic Baja Californian gopher snakeshould be the sister group of at least one of the middlemerican species of Pituophis, P. deppei and P. lineati-

ollis, a prediction not supported by our data. Hence,he historical events responsible for the genetic discon-inuity between northern and southern Baja Californiaopher snake populations remain unknown.

omparisons with Previous Hypothesesof Relationships within Pituophis

Although phylogenetic hypotheses do not depict an-estor–descendant relationships among the taxa stud-ed, our findings agree in part with two previousypotheses of relationships within Pituophis (Fig. 3),oth of which were based on morphological data andnferred using noncladistic methods. In Stull’s (1940)iew, sayi evolved from an affinis stock and in turn gaveise to ruthveni, which was the ancestor of the easterninesnakes (lodingi, melanoleucus, and mugitus; Fig.a). Our data support a close relationship betweenome affinis and sayi and between sayi and ruthveni.owever, contrary to Stull’s hypothesis, the easterninesnakes form a monophyletic group that is notlosely related to ruthveni. The mtDNA sequence datalso agree with Stull’s suggestion of a close relationshipetween affinis and deserticola, between catenifer andome deserticola, and between P. deppei and P. lineati-ollis but do not support a clade composed of affinis andertebralis, nor of annectens and deserticola.Klauber (1947) presented a hypothesis of relation-

hips within Pituophis (Fig. 3b) similar to Stull’s (1940)ut thought that the most primitive forms of the genusere P. deppei and P. lineaticollis. As Stull did, Klauber

onsidered affinis the most primitive taxon of theelanoleucus complex, from which he believed sayi,

uthveni, and the eastern pinesnakes evolved. In Klaub- D

r’s view, a second major offshoot of affinis was deserti-ola, from which first catenifer and then annectens wereerived. Our results support a close relationship be-ween some affinis and some sayi and between someatenifer and some deserticola but not between annec-ens and deserticola. Although Klauber considered thatimaris and vertebralis evolved from an affinis-likencestor that crossed the area now occupied by the Gulff California, he admitted that the derivation of thesewo forms was the most difficult phylogenetic problemithin Pituophis. As discussed above, the evolutionaryistory of the endemic Baja Californian gopher snakes

s indeed complex, and the two clades of bimaris andertebralis identified in our analyses are closely relatedo affinis, annectens, catenifer, and deserticola. Klauberlso believed that the insular forms pumilus (fromanta Cruz Island) and fuliginatus (from San Martınsland) evolved from nearby mainland forms, and ourP and ML trees indicate that these two taxa are in

act closely related to mainland populations of theelanoleucus complex geographically close to the is-

ands. Although specimens of coronalis (from Southoronado Island) and insulanus (from Cedros Island)ere not available for this study, we hypothesize that

hey are also closely related to populations on thedjacent mainland.

CONCLUSION

The view of a single, widely distributed, polytypicituophis melanoleucus is inconsistent with the in-

erred evolutionary history of these snakes. Our phylo-enetic analyses indicated that two segments of theelanoleucus complex, the lodingi–melanoleucus–ugitus eastern pinesnake clade and the affinis–

nnectens–bimaris–catenifer–deserticola–sayi–ruthveni–ertebralis clade from central and western Unitedtates and northern Mexico, represent divergent, allopa-ric lineages with no known intergradation zone. Wenterpret the available evidence as suggesting that theouisiana pinesnake has attained the status of indepen-ent evolutionary lineage. We therefore recognize threeistinct species in the melanoleucus complex, P. melano-eucus (sensu stricto), P. catenifer, and P. ruthveni;hether additional species should be recognized within. catenifer remains questionable. The quotation at theeginning of this paper was fitting in 1900, and thendings and arguments herein presented demonstratehat, despite several additional taxonomic studies, itemains so a century later.

ACKNOWLEDGMENTS

We thank H. W. Greene, D. L. Hardy, Sr., J. Gee, L. L. Grismer,. D. Hollingsworth, the late B. E. Dial, J. A. Campbell, M. Sasa,. M. Hardy, J. G. Himes, H. K. Reinert, G. M. Bolen, T. Benson, S. M.
eban, A. de Queiroz, W. I. Lutterschmidt, R. E. Jones, M. D. Matocq,

TCsaHRJMdaoNMaGaaH

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. Moise, D. A. Creer, R. S. Reiserer, K. R. Zamudio, J. V. Vindum, D.hiszar, H. M. Smith, and C. J. Bell for kindly providing all thepecimens or tissue samples used in this study; R. M. Harland forllowing us to use his laboratory facilities; K. R. Zamudio and M.aggart for providing the primers; C. D. Ortiz, G. M. Bolen, and Y.odrıguez for assistance; H. W. Greene, J. L. Patton, K. R. Zamudio,. P. Huelsenbeck, M. Garcıa-Parıs, M. J. Mahoney, A. Graybeal, A.eyer, D. Parks, and S. K. Davis for innumerable, enlightening

iscussions on phylogenetic analyses; and H. W. Greene, J. L. Patton,nd D. B. Wake for valuable suggestions to improve previous versionsf the manuscript. This study was supported by a grant from theorthern California Chapter of the ARCS Foundation, by an Annie. Alexander Fellowship from the Museum of Vertebrate Zoology,

nd by a Mentored Research Award from the Office of the Dean of theraduate Division, University of California at Berkeley, to J.A.R.; byNational Science Foundation predoctoral fellowship to J.M.D.J.;

nd by a National Institutes of Health grant (GM 49346) to R. M.arland.

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