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Mitochondrial-DNA Analysis of the Systematic Relationships within the Peromyscusmaniculatus Species GroupAuthor(s): Kelly M. Hogan, Scott K. Davis and Ira F. GreenbaumSource: Journal of Mammalogy, Vol. 78, No. 3 (Aug., 1997), pp. 733-743Published by: American Society of MammalogistsStable URL: http://www.jstor.org/stable/1382932.

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MITOCHONDRIAL-DNA ANALYSIS OF THE SYSTEMATICRELATIONSHIPS WITHIN THE PEROMYSCUS MANICULATUS

SPECIES GROUPKELLYM. HOGAN,coTTK. DAVIS,AND RAE GREENBAUM

Departmentof Biology, Texas A&M University,College Station,IX 77843 (KMH,IFG)Departmentof AnimalSciences, TexasA&M University,College Station,TX77843 (SKD).Present address of KMH:Departmentof Biology, South Texas CommunityCollege,3201 WestPecan Boulevard,McAllen,TX 78501Both parsimony and distance-based phylogenetic analyses of sequence data from threemitochondrial DNA (mtDNA) genes (ND3, ND4L, and ND4) revealed that, as currentlyrecognized, the Peromyscus maniculatus species group is polyphyletic. This apparent poly-phyly is resolved when P. slevini is removed from the species-group, thus limiting thegroup to P. maniculatus, P. keeni, P. polionotus, P. sejugis, and P. melanotis. Additionally,the phylogenetic analysis revealed that P. m. coolidgei is closer to P. sejugis than to theother subspecies of P. maniculatus examined. The systematic integrity of P. maniculatusis further complicated by the sister-species relationship between P. keeni and P. sejugis-P.m. coolidgei. These relationships suggest either a high degree of lineage sorting within P.maniculatus or that some of the currently recognized subspecies may be distinct species.Key words: Peromyscus maniculatus, mitochondrial DNA, systematics

The attributes of mitochondrial DNA(mtDNA), particularly its rapid rate of evo-lution and simple clonal mode of inheri-tance (Brown, 1983, 1985; Brown et al.,1979; Honeycutt and Wheeler, 1990; Mo-ritz et al., 1987), make it a popular choicefor studies involving the systematics of ro-dents, including Peromyscus (Avise et al.,1983). These attributes are particularly rel-evant to Peromyscus given the diversity(Hall, 1981) and recent divergence (Hib-bard, 1968) of taxa in the genus. To com-municate the diversity within, and generalpattern of relationships among taxa ofPeromyscus, Osgood (1909) employed thespecies-group concept. Of the eight species-groups recognized (Osgood, 1909), thePeromyscus maniculatus group has thewidest distribution and serves as a naturalmodel for numerous theories in the fields ofsystematics, ecology, behavior, and evolu-tion (Kirkland and Layne, 1989). However,Our confidence in such theories demands,if not presupposes, a sound framework of

relationship and pattern of descent (Carle-ton, 1989:54). With the recent systematicrealignments in the P. maniculatus speciesgroup under the specific epithet keeni (Ho-gan et al., 1993), the systematic scope ofthe species-group warrants further investi-gation.The P. maniculatus species group initial-ly consisted of P. maniculatus, P. sitkensis,P. polionotus, and P. melanotis, rangingfrom the highlands of central Mexico northto southern Alaska and the Canadian taiga(Osgood, 1909). Subsequent to Osgood's(1909) revision, the number of species inthe P maniculatus species group increasedto seven, with the recognition of P. sleviniand P. sejugis from the islands in the Gulfof California, Mexico, and P. oreas fromthe Pacific Northwest.

Peromyscus slevini was described byMaillaird (1924) as endemic to Santa Cat-alina Island, Baja California del Sur, Mex-ico. Based on body size and molar mor-phology, Maillaird (1924) aligned P. slevini

Journal of Mammalogy, 78(3):733-743, 1997 733

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734 JOURNALOF MAMMALOGY Vol. 78, No. 3

with the P. californicus group of the sub-genus Haplomylomys. Burt (1934) disputedthis arrangement, citing that the cranialmorphology of P. slevini was similar to thatof P. maniculatus, and that P. slevini be-longed in the subgenus Peromyscus. Theinclusion of P. slevini within the P. mani-culatus species group, although not specif-ically addressed by Burt (1934), appears tohave resulted from the mention of its sim-ilarity with P. maniculatus. Hooper (1968)considered the placement of P. slevini with-in the P. maniculatus species group as in-certae sedis, noting that the supraorbitalshelf is well expressed in P. slevini and,thus, is similar to species in the P. mexi-canus species group.The affiliation of P. sejugis within the P.maniculatus species group is less problem-atic. Originally described by Burt (1932), P.sejugis is endemic to the islands of San Di-ego and Santa Cruz, in the Sea of Cortez,Mexico. The inclusion of P. sejugis withinthe P. maniculatus species group is sup-ported by an analysis of both phallic mor-phology (Hooper and Musser, 1964) and al-lozymes (Avise et al., 1974, 1979). In theiranalysis of allozymic variation, Avise et al.(1979) supported the cohesiveness (and in-ferred monophyly) of P. maniculatus, P.melanotis, P. polionotus, and P. sejugis rel-ative to 20 other species of peromyscine ro-dents. Cytological data, particularly C- andG-banding, have further supported themonophyletic relationships of P. manicu-latus, P. melanotis, and P. polionotus(Greenbaum and Baker, 1978; Greenbaumet al., 1978; Robbins and Baker, 1981; Rog-ers et al., 1984; Stangl and Baker, 1984;Yates et al., 1979). Cytological data are notavailable for P. sejugis or P. slevini.Past cytological (Gunn, 1988; Gunn andGreenbaum, 1986; Hedin, 1989; Pengilly etal., 1983; Thomas, 1973), ecological (Dice,1949; Liu, 1954; Sheppe, 1961), morpho-logic (Allard and Greenbaum, 1988; Allardet al., 1987; Gunn and Greenbaum, 1986;Sheppe, 1961; Sullivan et al., 1990), allo-zymic (Calhoun and Greenbaum, 1991; He-

din, 1989; Hogan et al., 1993), and mtDNA(Hogan et al., 1993) investigations of the P.maniculatus species group have revealed adichotomy consistent with the recognitionof two species in the Pacific Northwest. Asa result of these studies, Hogan et al. (1993)recognized those mice with a karyotypecharacterized by a large proportion of biar-med chromosomes as P. keeni, whereasthose with a reduced number of autosomalarms corresponded to P. maniculatus. Thischange places the species P. oreas and P.sitkensis in synonymy under P. keeni, there-by reducing the number of species in the P.maniculatus species group to six.In this paper, we examine systematic re-lationships within the P. maniculatus spe-cies group using sequence data from a1,439 base-pair (bp) region of mtDNA. Weseek to resolve the uncertainty regardingthe systematic position of P. slevini, as wellas elucidate the phylogenetic relationship ofP. keeni within the species-group. In addi-tion, we provide insight into the degree ofintraspecific variation in the nucleotide se-quences examined by an analysis of restric-tion fragment length polymorphisms(RFLP) generated from a restriction-en-zyme analysis of the amplified fragment.

METHODSAND MATERIALSDNA was isolatedfrom all species, and sev-eral subspecies, in the P. maniculatusspeciesgroup,as well as the outgroup axaP. leucopus,

P. gossypinus, and Reithrodontomys megalotis.Correspondingcollecting localities, and speci-men numbersare listedin AppendixI. DNA wasextracted rom heartmuscle, liver,orkidneytis-sue using the methodsof Maniatiset al. (1982).A 1,439 base-pair bp) regionof mtDNA ex-tending from the 3' end of the glycine tRNA(glYtRNA) hrough672 bp of the 5' end of ND4was amplifiedusing the polymerasechainreac-tion (PCR)withAmplitaq PerkinElmer/Cetus).Primersused in theseamplifications re listed inAppendixII. Amplificationof templatemtDNAfollowedtheprocedures f Ar6valoet al. (1994).The amplifiedmtDNA fragmentwas then ligat-ed into an Eco RV digestedBluescriptSK plas-mid modifiedby addition of dT overhangsby



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August1997 HOGAN ET AL.--MITOCHONDRIALOF PEROMYSCUSDNA 735

the procedure of Marchuk et al. (1991), trans-formed into Escherichia coli DH5 (Gibco BRL),and grown for 16 h at 370C. Procedures for re-covering the amplified DNA, and denaturationprior to sequencing, followed Kraft et al. (1988)and Ar6valo et al. (1994).Dideoxy-DNA chain-termination sequencing(Sanger et al., 1977) with [35S]ATP abelling fol-lowed protocols included with the Sequenase 2.0sequencing kit (United States Biochemical Cor-poration), which employs the bacteriophage T7DNA polymerase method of Tabor and Richard-son (1987). With the exception of the universalM13 primers (-40: gtt ttc cca gtc acg and Re-verse: ttc aca cag gaa a, Sequenase Version 2.0,United States Biochemical Company) used forthe initial sequencing of the 5' and 3' ends ofthe fragment, the position and orientation of thesequencing primers used in this study are listedin Appendix II. These primers allowed for com-plete sequencing of both strands of the 1,439 bpfragment. Sequencing gels were run on an IBImodel STS60 sequencer, dried under vacuum for30 min and exposed to Kodak Diagnostic FilmSB 100 for ca. 24 h at -800C. Sequences wereread manually from the exposed film and en-tered directly into the computer programMacVector 4.17 (IBI-Kodak). Complete se-quences were aligned manually using the Mussequence (Bibb et al., 1981) as a guide.

Phylogenetic reconstructions were performedunder the principle of maximum parsimony withthe aid of the Phylogenetic Analysis Using Par-simony (PAUP) computer program of Swofford(1993, version 3.1.1). Because transitions andchanges at third-base positions in codons tend toaccumulate rapidly relative to transversions andchanges at first- and second-base positions(Brown, 1983; Brown et al., 1982; Honeycuttand Wheeler, 1990), several approaches to char-acter weighting were employed. The first ap-proach weighted all substitutions equally, re-gardless of type of substitution or nucleotide po-sition. A second analysis in which transversionswere weighted over transitions according to ob-served frequency difference between the twotypes of substitutions was conducted using thecomputer program McClade 3.01 (Maddison andMaddison, 1992). Additionally, substitutions atthird positions were weighted to avoid the affectof positional bias. Gaps observed in the se-quence were treated as missing data.The number of taxa examined (13) rendered

an exact search for the shortest topology com-putationally unfeasible. Instead, the method ofHendy and Penny (1982) was employed usingthe branch-and-bound option in PAUP (Swof-ford, 1993) to generate the most parsimoniousphylogenetic tree(s). A strict consensus tree wasproduced if two or more equally parsimonioustrees were generated in either analysis. Robust-ness of the inferred phylogeny was evaluated bybootstrap resampling (Felsenstein, 1985) usingthe bootstrap option in PAUP (Swofford, 1993)with 1,000 replications. In addition, the distri-bution (g, statistic-Hillis, 1991; Hillis andHuelsenbeck, 1992; Huelsenbeck, 1991) of themost parsimonious tree relative to the distribu-tion of randomly produced topologies also wascalculated.

Relationships among taxa also were investi-gated using an analysis of distance data derivedfrom nucleotide substitutions among taxa em-ploying the two-parameter model (Kimura,1980). Distance data were analyzed using theneighbor-joining algorithm (Saitou and Nei,1987) to derive the shortest phylogenetic tree.These analyses were conducted using the Mo-lecular Evolutionary Genetics Analysis (MEGA)computer program (Kumar et al., 1993).To assess potential intraspecific variation inthe fragment used for phylogenetic analyses, andthereby establish reliability of the fragment inrepresenting taxa, an analysis of restriction-sitepolymorphism was conducted. The number ofindividuals sampled in this analysis were: P.keeni (localities 1, 2, 3, 4, 5, 6, 7, and 8, n =20); P. maniculatus (localities 9, 11, and 12, n= 8); P. melanotis (locality 13, n = 9); P. po-lionotus (locality 14, n = 12); P. sejugis (local-ities 15 and 16, n = 8); P. slevini (locality 17,n = 9); and P. leucopus (localities 18, 19, 20,21, and 22, n = 10). Collecting localities foreach of the taxa are listed in Appendix I.A comparative analysis (using the MacVector4.17 program) of sequence data for each speciesin the P. maniculatus species group revealedfour restriction enzymes, Dde I, Dpn II, Rsa I,and Taq I, with restriction sites that were poly-morphic among species. After PCR amplifica-tion, the 1,439 bp fragment was digested witheach restriction enzyme following the manufac-turer's recommendations (New England Biol-abs). The digests were separated by electropho-resis on 2.0% agarose gels for 10-15 h, stainedwith ethidium bromide, and photographed under



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736 JOURNAL OF MAMMALOGY Vol. 78, No. 3

UV light (Davis, 1986;Hillis andDavis, 1986).A standard f lambdaphageDNA, cut withDraI, was included on each gel as a size marker.The resulting restriction-fragmentatternsweremappedto restrictionsites using the sequencedata andcoded as binarycharactersor analysis.RESULTS

The sequences of mtDNA examined inthis study for R. megalotis, P. leucopus, P.gossypinus, P. m. austerus, P. m. coolidgei,P. m. rufinus, P. sejugis (SD), P. sejugis(SC), P. k. interdictus, P. k. oreas, P. slev-ini, P. polionotis, and P. melanotis are onfile with GenBank. GenBank accessionnumbers for each sequence are listed in Ap-pendix I. Of the 1,439 nucleotide positionsexamined, 489 (34%) were variable and277 (19% of total, 57% of variable sites)were phylogenetically informative. Amongthe protein coding regions, the frequenciesof variable and phylogenetically informa-tive sites were relatively consistent. Of the348 bp of ND3, 125 (35.9%) were variableand 75 (21.6% of total, 60.0% of variablesites) were phylogenetically informative. Inthe ND4L gene, 109 sites (36.7%) werevariable and 63 (21.2% of total, 57.8% ofvariable sites) were informative. Of the 672bp sequenced in ND4, 232 sites (34.5%)were variable and 129 (19.2% of total,55.6% of variable sites) were phylogeneti-cally informative. The average frequency ofeach type of substitution observed for thesevariable sites, based on 100 randomlyjoined trees, is given in Table 1; a general4:1 bias in favor of substitution involvingtransitions over transversions was observed.Additionally, a two-fold bias in the fre-quency of transitions from A to G relativeto transitions from G to A was observed. Acorresponding bias was not detected in tran-sitional substitutions of pyrimidines. Per-centage sequence divergence (Kimura,1980) among taxa is given in Table 2.Results of the phylogenetic analysis withall characters weighted equally generated asingle most-parsimonious tree (RetentionIndex, RI = 0.633; Farris, 1989) presented

TABLE.-Average numberof base changesfor the entire 1,439 base-pairfragment (aboveline) and the ND3, ND4L, and 672 base pairsof the ND4 genes (below line). Analysis basedon 100 randomly oined trees of the taxa ex-amined in this study.Substitu-tions: A C G TFrom: A 65.7 132.3 71.5ND3 15.1 38.9 14.5ND4L 19.7 31.0 18.2ND4 28.6 53.6 29.7

C 72.1 7.3 300.3ND3 18.9 2.3 82.6ND4L 19.1 2.0 73.6ND4 31.0 2.9 138.3

G 72.4 2.9 5.6ND3 17.8 1.0 3.3ND4L 19.9 0.8 2.1ND4 33.8 1.1 0.2

T 49.1 310.5 14.6ND3 16.7 88.6 3.7ND4L 15.5 53.1 7.9ND4 11.0 157.3 2.9

in Fig. la. The topology of this tree did notchange when nucleotide positions wereweighted in favor of transversions overtransitions by the observed frequency of 4to 1. However, total length of tree increasedfrom 808 to 1,432 steps. The g&-values,forboth the equally weighted and weightedanalyses in Fig. la were -1.18 and -1.37,respectively. Subsequent analysis using dis-tance data resulted in a topology that wasidentical to the tree derived from the par-simony analysis (Fig. lb).The four enzymes used in this study re-vealed a total of 27 restriction sites ofwhich three were found in all taxa exam-ined. The remaining 24 restriction siteswere phylogenetically informative, and re-sulted in eight composite mtDNA haplo-types (Table 3). Of the 76 individuals ex-amined, intraspecific variation was ob-served only in P. maniculatus. Of the twohaplotypes observed in P. maniculatus (Gand H; Table 3), one occurred in specimensof P. m. coolidgei from Baja California delSur, Mexico (locality 11). The second hap-



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August 1997 HOGAN ET AL.-MITOCHONDRIAL OF PEROMYSCUS DNA 737

TABLE2.-Percentage sequence divergence (Kimura, 1980) for the 1,439 base-pair fragment ofmtDNAamong taxa.OTU 1 2 3 4 5 6 7 8 9 10 11 12 13

1. R. megalotis 0.23 0.22 0.22 0.22 0.22 0.23 0.22 0.22 0.23 0.23 0.22 0.232. P. leucopus 0.07 0.20 0.15 0.14 0.15 0.15 0.15 0.15 0.16 0.15 0.153. P. gossypinus 0.19 0.14 0.41 0.14 0.15 0.14 0.13 0.14 0.13 0.144. P. slevini 0.19 0.18 0.18 0.18 0.18 0.18 0.19 0.18 0.195. P. melanotis 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.096. P. polionotus 0.04 0.05 0.05 0.05 0.05 0.05 0.057. P. m. rufinus 0.01 0.04 0.04 0.05 0.04 0.058. P. m. austerus 0.05 0.04 0.05 0.05 0.059. P. m. coolidgei 0.04 0.04 0.02 0.0210. P. k. interdictus 0.02 0.04 0.0411. P. k. oreas 0.04 0.0412. P. sejugis (SD) 0.0113. P. sejugis (SC)

lotype characterized specimens of P. m. ar-temisiae from Washington state (locality 9)and P. m. rufinus from Colorado (locality12).The remaining haplotypes (A, B, C, D,E, and H) were species-specific and char-acterized the taxa P. slevini, P. sejugis, P.polionotus, P. melanotis, P. keeni, and P.leucopus, respectively. Of the eight locali-ties sampled for P. keeni, three (localities5, 6, and 7) represented P. k. macrorhinus,whereas the remainder represented one lo-cality each of P. k. algidus (locality 1), P.k. hylaeus (locality 2), P. k. interdictus (lo-cality 3), P. k. isolatus (locality 4), and P.k. oreas (locality 8). In addition, intraspe-cific variation was not observed betweenthe two island populations, localities 15 and16, of P. sejugis or the five localities of P.leucopus sampled (localities 18-22).

DIscusSIONThe phylogenetic analysis of mtDNA se-quence data clearly indicates that P. slevinishould not be included in the P. manicu-latus species group (Fig. 1). Although theexact affiliation of P. slevini within the ge-

nus was beyond the scope of this research,its exclusion from the P. maniculatus spe-cies group prompts two avenues for furtherstudy. First, the branch length observed inP. slevini, relative to the other taxa in this

study, suggests that P. slevini is extremelydivergent from both the P. leucopus and P.maniculatus species groups. Further analy-sis of the taxonomic position of P. sleviniwould elucidate the taxonomic scope of thesubgenus Peromyscus. Second, the biogeo-graphic origin of P. slevini is ambiguouswhen the extant population of P. manicu-latus on Baja California del Sur is ruled outas the ancestral stock for this insular spe-cies. The prevailing view of island bioge-ography in the Sea of Cortez assumes thatisland populations of mammals are derivedfrom the nearest mainland populations, andhave subsequently become isolated by ris-ing sea levels during the Pleistocene (Law-lor, 1971, 1983). Because P. slevini has his-torically been aligned with the P. manicu-latus group, the current distribution of P.m. coolidgei on the Baja California penin-sula supported this model. Ruling out aclose systematic relationship between P.slevini and P. maniculatus raises questionsas to the general applicability of this modelof island biogeography. However, the originand potential reinterpretation of the modelof island biogeography in the Sea of Cortezmust await a detailed treatment of the sys-tematic affinities of P. slevini within the ge-nus.

As stated previously, inclusion of P. se-jugis within the P. maniculatus group is not



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10092 7) . k. interdictusao 13 ( P. k.oreas(12)

98(13) E .m.coolidgei

1 0 ) j u g i s S D )(6) E. ejugisSC)

99100 100 (6) P. m.rufinus(10) P. m.austerus

(3)6) P. olionotus100(45) (38) P. melanotis100 (28) P. gOSSVyinUS

(43) P. leucopus(85) E. sleviniR_.megalotis

P. seiugis SD)P. sejugis SC)P. m.oolid iP. .nterdictusP.m.rufinus

m a a .a u s t e r m-. polionotus

Smel.anotisP gossvpinusP. leucoous

R.megalotis0.01FIG. 1.-Inferred phylogeneticrelationshipsamong the taxa included in this study based on aparsimonyanalysis (a) and distance data (Kimura,1980) employingthe neighbor-joining lgorithm(b) of all 1,439 base pairs of mtDNA. Bootstrapvalues for each node are listed for the equallyweightedandweightedanalyses,respectively.Single bootstrapvalues at each node indicate denticalvalues for each weighting scheme. Numbers in parentheses ndicate the number of unambiguoussubstitutionsoccurringon each branch.

as problematic as the inclusion of P. slevini.Results of this study are consistent with theallozymic (Avise et al., 1979) and morpho-logic (Hooper and Musser, 1964) data insupporting the inclusion of P. sejugis within

the P. maniculatus species group. The re-lationship of P. sejugis to other specieswithin the P. maniculatus species groupraises a question regarding the taxonomicaffinities of species of Peromyscus inhab-



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August1997 HOGANET AL.--MITOCHONDRIALOF PEROMYSCUSDNA 739TABLE .-Composite mtDNA haplotypes resulting from an analysis of restriction sites in the taxaexamined.

HaplotypeDde I Dpn II Rsa I Taq I

Clone Taxon 123456789 123456 123456 123456A P. slevini 100010010 110101 100011 100000B P. sejugis 000001000 100100 100000 111000C P. polionotus 001001000 101100 110000 110010D P. melanotis 101001000 110111 101110 100100E P. keeni 001000000 101100 100010 111000F P. leucopus 000001000 101100 100000 111000G P. maniculatus 011001000 101100 100000 111000H P. maniculatus 001110111 110101 100000 100001

Dde I Dpn II Rsa I Taq I1) 9,752-9,756 1) 9,433-9,436 1) 9,409-9,412 1) 9,835-9,8382) 9,758-9,762 2) 9,579-9,582 2) 9,563-9,566 2) 9,939-9,9423) 9,796-9,800 3) 9,941-9,944 3) 9,744-9,747 3) 9,955-9,9584) 9,921-9,925 4) 10,627-10,630 4) 9,774-9,777 4) 9,971-9,9745) 10,060-10,064 5) 10,719-10,722 5) 10,138-10,141 5) 10,428-10,4316) 10,092-10,096 6) 10,729-10,732 6) 10,794-10,797 6) 10,525-10,5287) 10,271-10,2758) 10,391-10,3959) 10,565-10,569

Mappedrestriction-siteocationsare relative o the Mus sequence(Bibbet al., 1981).

iting the Pacific coast. In this investigation,P. sejugis clustered with P. m. coolidgei.This clade in turn is sister to P. keeni fromthe Pacific Northwest (Fig. 1). This ar-rangement suggests that P. maniculatus, ascurrently recognized, is paraphyletic. Twoscenarios can be invoked to explain this re-lationship. First, the paraphyly within P.maniculatus may be an artifact of lineagesorting (Avise, 1994) within P. maniculatusas a result of recent divergence within thespecies. Alternatively, the grouping of thesetaxa may represent multiple species.Although neither alternative can be elim-inated based on available data, the first pos-sibility seems least likely because of the ob-served variation in haplotypes. With the ex-ception of two haplotypes observed in sam-ples of P. maniculatus, restriction-enzymeanalysis revealed no intraspecific variationin the taxa examined. In P. maniculatus, allindividuals of P. m. rufinus examined fromColorado and P. m. artemisiae from Wash-ington shared the same haplotype (Table 3,

clone G), whereas P. m. coolidgei fromBaja California differed by having an ad-ditional restriction site (Table 3, clone F).This observation differs from the situationfound in P. keeni in which a single haplo-type represented populations as disjunct asVancouver and Graham islands, as well asthe state of Washington (Table 3, clone C).In addition, only a single restriction-frag-ment pattern was observed (Table 3, cloneH) in the five populations of P. leucopussurveyed.The lack of variation within species mayindicate that the restriction sites surveyedare highly conserved among species ofPeromyscus. Alternatively, this lack of in-traspecific variation could have resultedfrom a failure to sample low-frequencyhaplotypes in the various populations.While the latter scenario cannot be exclud-ed, the former is more likely, particularlyconsidering the results of our phylogeneticanalyses and when compared to the pub-lished studies discussed below.



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In an analysis of variation of mtDNA inP. maniculatus, Lansman et al. (1983) con-cluded that the geographically variable P.maniculatus could be divided into five ma-jor assemblages, each separable at ca. 3%sequence divergence. These five assem-blages corresponded to specimens from thecentral United States, the eastern UnitedStates, northern Michigan, Texas-Mexico,and southern California (Lansman et al.,1983:9; figures 7 and 8). Although Lans-man et al. (1983) did not examine P. m.coolidgei, they did include P. m. gambelii,a sister-taxon of P. m. coolidgei (Calhounet al., 1988). The taxa examined in thisstudy appear to represent the central UnitedStates (P. m. rufinus and P. m. artemisiae)and southern California (P. m. coolidgei)assemblages as depicted in Lansman et al.(1983).In view of the apparent consistency ingeographic variation between our study andthat of Lansman et al. (1983), we suggestthat the assemblages previously recognizedby Lansman et al. (1983) warrant furtherinvestigation as to their taxonomic status.Future studies should resolve the relation-ship between P. keeni and P. sejugis-P. m.coolidgei to determine whether these taxaconstitute a single lineage along the Pacificcoast or multiple species.

ACKNOWLEDGMENTSFor assistance n the laboratorywe gratefully

acknowledgethe help of T. Guerra,D. Starky,M. Forstner,S. Engel, E. Louis, and C. Young.For readingearlier drafts of the manuscriptwethank S. Engel, S. A. Berend,J. G. Gable, M.Wike,R. Honeycutt,andJ. Bickham.The manu-script also benefitted from two anonymousre-viewers. Financialsupportfor this projectwasprovided by National Institutesof HealthgrantGM 27014 to I. E Greenbaum nd NationalSci-ence FoundationgrantBSR 88-45298 to S. K.Davis, I. E Greenbaum nd J. Sites.LITERATURE CITED

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APPENDIXSpecimens examined.--Collection localities for

specimens of Peromyscus. Locality numbers cor-respond to those discussed in text. Number of in-dividuals for each taxon examined in each portionof this study are given in the text. Taxonomic iden-tity of specimens based on data from Hall (1981),Gunn and Greenbaum (1986), Allard and Green-baum (1988), and Hogan et al. (1993). Numbersin parentheses are catalog numbers correspondingto voucher specimen sequenced in this study (GK= Greenbaum karyotype number; NK = NewMexico karyotype number;KMH = Kelly M. Ho-gan catalog number). Voucher specimens are de-posited in the Texas Cooperative Wildlife Collec-tion, Texas A&M University, College Station, andthe Museum of Southwestern Biology, Universityof New Mexico, Albuquerque.

Peromyscus keeni algidus. USA: Alaska: Lo-cality 1-2 km N Skagway, Liarsville Camp-ground.

Peromyscus keeni hylaeus. USA: Alaska: Lo-cality 2-21.2 km NW Auke Bay Ferry Termi-nal, Herbert River.

Peromyscus keeni isolatus. Canada; BritishColumbia: Locality 3-Malcolm Island, 4.8 kmE Sointula.Peromyscus keeni interdictus. Canada: BritishColumbia: Locality 4-Vancouver Island,Mount Washington Ski Area (GK 2448; Gen-Bank Accession U40063).Peromyscus keeni macrorhinus. Canada: Brit-ish Columbia: Locality 5-32.4 km E Prince Ru-

pert. Locality 6--4.6 km W Exchamsiks RiverProvincial Park. Locality 7-Kleanza Creek,Highway 16.

Peromyscus keeni oreas. USA: WashingtonGrays Harbor, Locality 8-Satsop Workcamp(GK 5905; GenBank Accession U40062).Peromyscus maniculatus artemisiae. USA:

Washington: Locality 9-2.8 km W Mazama,Early Winters Campground.Peromyscus maniculatus austerus. Canada:British Columbia: Locality 0--Vancouver Is-land, 35.7 km W Port Alberni, Sproat Lake (GK5604; GenBank Accession U40249).

Peromyscus maniculatus coolidgei. Mexico:Baja California del Sur: Locality 11---25 km SEGuerrero Negro (NK 5166; GenBank AccessionU40251).

Peromyscus maniculatus rufinus. USA: Col-orado: Locality 12-7.2 km N, 8.8 km W Cen-

Submitted16 November1995.Accepted20 November1996.Associate Editorwas JamesR. Purdue.



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tral City, Elk Park (KMH 1038; GenBank Ac-cession U40250).Peromyscus melanotis. Mexico: Hidalgo: Lo-cality 13-Lab colony from the PeromyscusStock Center, originally from Mexico (KMH

1024; GenBank Accession U40247).Peromyscus polionotus. USA: South Carolina:Lexington Co.: Locality 14--0.6 km S EdmundBethel Church Field (GK 5904; GenBank Ac-cession U40254).Peromyscus sejugis. Mexico: Baja Californiadel Sur, Locality 15-San Diego Island(SD)(GK 5893; GenBank Accession U40255);

Locality 16-Santa Cruz Island (SC)(GK 5888;GenBank Accession U40253).Peromyscus slevini. Mexico: Baja California

del Sur, Locality 17-Santa Catalina Island (GK5880; GenBank Accession U40248).

Peromyscus leucopus. USA: Texas: Locality18-Robertson Co. (GK 5848; GenBank Ac-cession U40252). Locality 19-Hunt Co., vi-cinity of Clest. Locality 20-Wichita Co., 13.8km N, 0.6 km W Iowa Park. Locality 21-Michigan (laboratory colony). Locality 22-Maine, Hancock Co., 3.0 km S, 0.6 km E BarHarbor.

Peromyscus gossypinus. USA: Texas: Ander-son Co., Locality 23-3.2 km S, 6.4 km E Ca-yuga, Gus A. Engeling Wildlife ManagementArea (KMH 1041; GenBank AccessionU40246).

Reithrodontomys megalotis. USA: Texas: JeffDavis Co., Locality 24--6.1 km N, 11.2 km WFort Davis (KMH 1060; GenBank AccessionU40031).

APPENDIXI.List of PCR and sequencing primers used to amplify 1,439 base pairs of mtDNA. All primer con-centrations were 10 ng/pIL.Nucleotides in parentheses denote nucleotides at degenerative positionsin primer.

ReferencePrimer positionsa Sequenceb

PI'c 9,403-9,524 cga act agt aca gct gac ttc cND3M 9,560-9,580 cc(acgt) ta(tc) gaa tgc gg(ag) ttt gaND3M2 9,616-9,637 aat ttt t(tc)c tag tag caa t(tc)a cMarg 9,777-9,794 caa aaa gga (tc)ta gaa tgaMargRev 9,783-9,799 gga (tc)ta gaa tga ac(agc) gaND4L 10,171-10,191 gca tgt gaa gca gc(tc) at(tc) ggRataway 10,467-10,483 caa at(ct) ct(cat) cta atc atMouseMerl 10,615-10,634 ata c(act)(ct) taa ttg g(ag)t caa tcNap2c 10,812-10,832 aaa gc(tc) cac gta gaa gct cca

a Referencepositionsare relative o the Mussequence(Bibbet al., 1981).bPrimer equencesare listed left to rightfrom 5' to 3' correspondingo the lightstrandof mtDNA.SFromAr6valoet al. (1994).

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