American Society of Mammalogists

Mitochondrial-DNA Analysis of the Systematic Relationships within the Peromyscus maniculatus Species Group Author(s): Kelly M. Hogan, Scott K. Davis and Ira F. Greenbaum Source: Journal of Mammalogy, Vol. 78, No. 3 (Aug., 1997), pp. 733-743 Published by: American Society of Mammalogists Stable URL: http://www.jstor.org/stable/1382932 . Accessed: 27/11/2013 18:39
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MITOCHONDRIAL-DNA ANALYSIS OF THE SYSTEMATIC RELATIONSHIPS WITHIN THE PEROMYSCUS MANICULATUS SPECIES GROUP
AND IRAE GREENBAUM KELLY M. HOGAN, ScoTTK. DAVIS, Departmentof Biology, Texas A&M University,College Station, IX 77843 (KMH,IFG) Departmentof Animal Sciences, TexasA&M University,College Station, TX 77843 (SKD). Present address of KMH:Departmentof Biology, South Texas Community College, 3201 WestPecan Boulevard,McAllen, TX 78501 Both parsimony and distance-based phylogenetic analyses of sequence data from three mitochondrial DNA (mtDNA) genes (ND3, ND4L, and ND4) revealed that, as currently recognized, the Peromyscus maniculatus species group is polyphyletic. This apparent polyphyly is resolved when P. slevini is removed from the species-group, thus limiting the group 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 the other subspecies of P. maniculatus examined. The systematic integrity of P. maniculatus is 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 relationship and pattern of descent" (Carleton, 1989:54). With the recent systematic realignments in the P. maniculatus species group under the specific epithet keeni (Hogan et al., 1993), the systematic scope of the species-group warrants further investigation. The P. maniculatus species group initially consisted of P. maniculatus, P. sitkensis, P. polionotus, and P. melanotis, ranging from the highlands of central Mexico north to southern Alaska and the Canadian taiga (Osgood, 1909). Subsequent to Osgood's (1909) revision, the number of species in the P maniculatus species group increased to seven, with the recognition of P. slevini and P. sejugis from the islands in the Gulf of California, Mexico, and P. oreas from the Pacific Northwest. Peromyscus slevini was described by Maillaird (1924) as endemic to Santa Catalina Island, Baja California del Sur, Mexico. Based on body size and molar morphology, Maillaird (1924) aligned P. slevini
733

The attributes of mitochondrial DNA (mtDNA), particularly its rapid rate of evolution and simple clonal mode of inheritance (Brown, 1983, 1985; Brown et al., 1979; Honeycutt and Wheeler, 1990; Moritz et al., 1987), make it a popular choice for studies involving the systematics of rodents, including Peromyscus (Avise et al., 1983). These attributes are particularly relevant to Peromyscus given the diversity (Hall, 1981) and recent divergence (Hibbard, 1968) of taxa in the genus. To communicate the diversity within, and general pattern of relationships among taxa of Peromyscus, Osgood (1909) employed the species-group concept. Of the eight speciesgroups recognized (Osgood, 1909), the Peromyscus maniculatus group has the widest distribution and serves as a natural model for numerous theories in the fields of systematics, ecology, behavior, and evolution (Kirkland and Layne, 1989). However, "Our confidence in such theories demands, if not presupposes, a sound framework of
Journal of Mammalogy, 78(3):733-743, 1997

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with the P. californicus group of the subgenus Haplomylomys. Burt (1934) disputed this arrangement, citing that the cranial morphology of P. slevini was similar to that of P. maniculatus, and that P. slevini belonged in the subgenus Peromyscus. The inclusion of P. slevini within the P. maniculatus species group, although not specifically addressed by Burt (1934), appears to have resulted from the mention of its similarity with P. maniculatus. Hooper (1968) considered the placement of P. slevini within the P. maniculatus species group as incertae sedis, noting that the supraorbital shelf is well expressed in P. slevini and, thus, is similar to species in the P. mexicanus species group. The affiliation of P. sejugis within the P. maniculatus species group is less problematic. Originally described by Burt (1932), P. sejugis is endemic to the islands of San Diego and Santa Cruz, in the Sea of Cortez, Mexico. The inclusion of P. sejugis within the P. maniculatus species group is supported by an analysis of both phallic morphology (Hooper and Musser, 1964) and allozymes (Avise et al., 1974, 1979). In their analysis of allozymic variation, Avise et al. (1979) supported the cohesiveness (and inferred monophyly) of P. maniculatus, P. melanotis, P. polionotus, and P. sejugis relative to 20 other species of peromyscine rodents. Cytological data, particularly C- and G-banding, have further supported the monophyletic relationships of P. maniculatus, P. melanotis, and P. polionotus (Greenbaum and Baker, 1978; Greenbaum et al., 1978; Robbins and Baker, 1981; Rogers et al., 1984; Stangl and Baker, 1984; Yates et al., 1979). Cytological data are not available for P. sejugis or P. slevini. Past cytological (Gunn, 1988; Gunn and Greenbaum, 1986; Hedin, 1989; Pengilly et al., 1983; Thomas, 1973), ecological (Dice, 1949; Liu, 1954; Sheppe, 1961), morphologic (Allard and Greenbaum, 1988; Allard et al., 1987; Gunn and Greenbaum, 1986; Sheppe, 1961; Sullivan et al., 1990), allozymic (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 a dichotomy consistent with the recognition of two species in the Pacific Northwest. As a result of these studies, Hogan et al. (1993) recognized those mice with a karyotype characterized by a large proportion of biarmed chromosomes as P. keeni, whereas those with a reduced number of autosomal arms corresponded to P. maniculatus. This change places the species P. oreas and P. sitkensis in synonymy under P. keeni, thereby reducing the number of species in the P. maniculatus species group to six. In this paper, we examine systematic relationships within the P. maniculatus species group using sequence data from a 1,439 base-pair (bp) region of mtDNA. We seek to resolve the uncertainty regarding the systematic position of P. slevini, as well as elucidate the phylogenetic relationship of P. keeni within the species-group. In addition, we provide insight into the degree of intraspecific variation in the nucleotide sequences examined by an analysis of restriction fragment length polymorphisms (RFLP) generated from a restriction-enzyme analysis of the amplified fragment.
METHODSAND MATERIALS

DNA was isolated from all species, and several subspecies, in the P. maniculatus species group, as well as the outgrouptaxa P. leucopus,
P. gossypinus, and Reithrodontomys megalotis.

Correspondingcollecting localities, and specimen numbersare listed in AppendixI. DNA was extractedfrom heartmuscle, liver, or kidney tissue using the methodsof Maniatiset al. (1982). A 1,439 base-pair(bp) region of mtDNA extending from the 3' end of the glycine tRNA (glYtRNA)through672 bp of the 5' end of ND4 was amplifiedusing the polymerasechain reaction (PCR)with Amplitaq(PerkinElmer/Cetus). are listed in Primersused in these amplifications AppendixII. Amplificationof templatemtDNA of Ar6valoet al. (1994). followed the procedures The amplifiedmtDNA fragmentwas then ligated into an Eco RV digested BluescriptSK plasmid modified by addition of dT overhangsby

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the procedure of Marchuk et al. (1991), transformed into Escherichia coli DH5 (Gibco BRL), and grown for 16 h at 370C. Procedures for recovering the amplified DNA, and denaturation prior to sequencing, followed Kraft et al. (1988) and Ar6valo et al. (1994). Dideoxy-DNA chain-termination sequencing (Sanger et al., 1977) with [35S]ATPlabelling followed protocols included with the Sequenase 2.0 sequencing kit (United States Biochemical Corporation), which employs the bacteriophage T7 DNA polymerase method of Tabor and Richardson (1987). With the exception of the universal M13 primers (-40: gtt ttc cca gtc acg and Reverse: ttc aca cag gaa a, Sequenase Version 2.0, United States Biochemical Company) used for the initial sequencing of the 5' and 3' ends of the fragment, the position and orientation of the sequencing primers used in this study are listed in Appendix II. These primers allowed for complete sequencing of both strands of the 1,439 bp fragment. Sequencing gels were run on an IBI model STS60 sequencer, dried under vacuum for 30 min and exposed to Kodak Diagnostic Film SB 100 for ca. 24 h at -800C. Sequences were read manually from the exposed film and entered directly into the computer program MacVector 4.17 (IBI-Kodak). Complete sequences were aligned manually using the Mus sequence (Bibb et al., 1981) as a guide. Phylogenetic reconstructions were performed under the principle of maximum parsimony with the aid of the Phylogenetic Analysis Using Parsimony (PAUP) computer program of Swofford (1993, version 3.1.1). Because transitions and changes at third-base positions in codons tend to accumulate rapidly relative to transversions and changes at first- and second-base positions (Brown, 1983; Brown et al., 1982; Honeycutt and Wheeler, 1990), several approaches to character weighting were employed. The first approach weighted all substitutions equally, regardless of type of substitution or nucleotide position. A second analysis in which transversions were weighted over transitions according to observed frequency difference between the two types of substitutions was conducted using the computer program McClade 3.01 (Maddison and Maddison, 1992). Additionally, substitutions at third positions were weighted to avoid the affect of positional bias. Gaps observed in the sequence were treated as missing data. The number of taxa examined (13) rendered

an exact search for the shortest topology computationally unfeasible. Instead, the method of Hendy and Penny (1982) was employed using the branch-and-bound option in PAUP (Swofford, 1993) to generate the most parsimonious phylogenetic tree(s). A strict consensus tree was produced if two or more equally parsimonious trees were generated in either analysis. Robustness of the inferred phylogeny was evaluated by bootstrap resampling (Felsenstein, 1985) using the bootstrap option in PAUP (Swofford, 1993) with 1,000 replications. In addition, the distribution (g, statistic-Hillis, 1991; Hillis and Huelsenbeck, 1992; Huelsenbeck, 1991) of the most parsimonious tree relative to the distribution of randomly produced topologies also was calculated. Relationships among taxa also were investigated using an analysis of distance data derived from nucleotide substitutions among taxa employing the two-parameter model (Kimura, 1980). Distance data were analyzed using the neighbor-joining algorithm (Saitou and Nei, 1987) to derive the shortest phylogenetic tree. These analyses were conducted using the Molecular Evolutionary Genetics Analysis (MEGA) computer program (Kumar et al., 1993). To assess potential intraspecific variation in the fragment used for phylogenetic analyses, and thereby establish reliability of the fragment in representing taxa, an analysis of restriction-site polymorphism was conducted. The number of individuals 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. polionotus (locality 14, n = 12); P. sejugis (localities 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 for each of the taxa are listed in Appendix I. A comparative analysis (using the MacVector 4.17 program) of sequence data for each species in the P. maniculatus species group revealed four restriction enzymes, Dde I, Dpn II, Rsa I, and Taq I, with restriction sites that were polymorphic among species. After PCR amplification, the 1,439 bp fragment was digested with each restriction enzyme following the manufacturer's recommendations (New England Biolabs). The digests were separated by electrophoresis on 2.0% agarose gels for 10-15 h, stained with ethidium bromide, and photographed under

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UV light (Davis, 1986; Hillis and Davis, 1986). of lambdaphage DNA, cut with Dra A standard I, was included on each gel as a size marker. The resultingrestriction-fragment patternswere sites to restriction using the sequence mapped for analysis. data and coded as binarycharacters
RESULTS

TABLE 1.-Average numberof base changes for the entire 1,439 base-pairfragment (above line) and the ND3, ND4L, and 672 base pairs of the ND4 genes (below line). Analysis based on 100 randomlyjoined trees of the taxa examined in this study.
Substitutions: From: A ND3 ND4L ND4 C ND3 ND4L ND4 G ND3 ND4L ND4 T ND3 ND4L ND4 A C 65.7 15.1 19.7 28.6 72.1 18.9 19.1 31.0 72.4 17.8 19.9 33.8 49.1 16.7 15.5 11.0 G 132.3 38.9 31.0 53.6 7.3 2.3 2.0 2.9 T 71.5 14.5 18.2 29.7 300.3 82.6 73.6 138.3 5.6 3.3 2.1 0.2

The sequences of mtDNA examined in this 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. slevini, P. polionotis, and P. melanotis are on file with GenBank. GenBank accession numbers for each sequence are listed in Appendix I. Of the 1,439 nucleotide positions examined, 489 (34%) were variable and 277 (19% of total, 57% of variable sites) were phylogenetically informative. Among the protein coding regions, the frequencies of variable and phylogenetically informative sites were relatively consistent. Of the 348 bp of ND3, 125 (35.9%) were variable and 75 (21.6% of total, 60.0% of variable sites) were phylogenetically informative. In the ND4L gene, 109 sites (36.7%) were variable and 63 (21.2% of total, 57.8% of variable sites) were informative. Of the 672 bp sequenced in ND4, 232 sites (34.5%) were variable and 129 (19.2% of total, 55.6% of variable sites) were phylogenetically informative. The average frequency of each type of substitution observed for these variable sites, based on 100 randomly joined trees, is given in Table 1; a general 4:1 bias in favor of substitution involving transitions over transversions was observed. Additionally, a two-fold bias in the frequency of transitions from A to G relative to transitions from G to A was observed. A corresponding bias was not detected in transitional substitutions of pyrimidines. Percentage sequence divergence (Kimura, 1980) among taxa is given in Table 2. Results of the phylogenetic analysis with all characters weighted equally generated a single most-parsimonious tree (Retention Index, RI = 0.633; Farris, 1989) presented

2.9 1.0 0.8 1.1 310.5 88.6 53.1 157.3

14.6 3.7 7.9 2.9

in Fig. la. The topology of this tree did not change when nucleotide positions were weighted in favor of transversions over transitions by the observed frequency of 4 to 1. However, total length of tree increased from 808 to 1,432 steps. The g&-values, for both the equally weighted and weighted analyses in Fig. la were -1.18 and -1.37, respectively. Subsequent analysis using distance data resulted in a topology that was identical to the tree derived from the parsimony analysis (Fig. lb). The four enzymes used in this study revealed a total of 27 restriction sites of which three were found in all taxa examined. The remaining 24 restriction sites were phylogenetically informative, and resulted in eight composite mtDNA haplotypes (Table 3). Of the 76 individuals examined, intraspecific variation was observed only in P. maniculatus. Of the two haplotypes observed in P. maniculatus (G and H; Table 3), one occurred in specimens of P. m. coolidgei from Baja California del Sur, Mexico (locality 11). The second hap-

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TABLE2.-Percentage

mtDNAamong taxa.
OTU 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. R. megalotis P. leucopus P. gossypinus P. slevini P. melanotis P. polionotus P. m. rufinus P. m. austerus P. m. coolidgei P. k. interdictus P. k. oreas P. sejugis (SD) P. sejugis (SC)

sequence divergence (Kimura, 1980) for the 1,439 base-pair fragment of

1 2 0.23 3 0.22 0.07 4 0.22 0.20 0.19 5 0.22 0.15 0.14 0.19 6 0.22 0.14 0.41 0.18 0.08 7 0.23 0.15 0.14 0.18 0.08 0.04 8 0.22 0.15 0.15 0.18 0.09 0.05 0.01 9 0.22 0.15 0.14 0.18 0.08 0.05 0.04 0.05 10 0.23 0.15 0.13 0.18 0.09 0.05 0.04 0.04 0.04 11 0.23 0.16 0.14 0.19 0.08 0.05 0.05 0.05 0.04 0.02 12 0.22 0.15 0.13 0.18 0.08 0.05 0.04 0.05 0.02 0.04 0.04 13 0.23 0.15 0.14 0.19 0.09 0.05 0.05 0.05 0.02 0.04 0.04 0.01

lotype characterized specimens of P. m. artemisiae from Washington state (locality 9) and P. m. rufinus from Colorado (locality 12). The remaining haplotypes (A, B, C, D, E, and H) were species-specific and characterized the taxa P. slevini, P. sejugis, P. polionotus, P. melanotis, P. keeni, and P. leucopus, respectively. Of the eight localities sampled for P. keeni, three (localities 5, 6, and 7) represented P. k. macrorhinus, whereas the remainder represented one locality each of P. k. algidus (locality 1), P. k. hylaeus (locality 2), P. k. interdictus (locality 3), P. k. isolatus (locality 4), and P. k. oreas (locality 8). In addition, intraspecific variation was not observed between the two island populations, localities 15 and 16, of P. sejugis or the five localities of P. leucopus sampled (localities 18-22).
DIscusSION

The phylogenetic analysis of mtDNA sequence data clearly indicates that P. slevini should not be included in the P. maniculatus species group (Fig. 1). Although the exact affiliation of P. slevini within the genus was beyond the scope of this research, its exclusion from the P. maniculatus species group prompts two avenues for further study. First, the branch length observed in P. slevini, relative to the other taxa in this

study, suggests that P. slevini is extremely divergent from both the P. leucopus and P. maniculatus species groups. Further analysis of the taxonomic position of P. slevini would elucidate the taxonomic scope of the subgenus Peromyscus. Second, the biogeographic origin of P. slevini is ambiguous when the extant population of P. maniculatus on Baja California del Sur is ruled out as the ancestral stock for this insular species. The prevailing view of island biogeography in the Sea of Cortez assumes that island populations of mammals are derived from the nearest mainland populations, and have subsequently become isolated by rising sea levels during the Pleistocene (Lawlor, 1971, 1983). Because P. slevini has historically been aligned with the P. maniculatus group, the current distribution of P. m. coolidgei on the Baja California peninsula supported this model. Ruling out a close systematic relationship between P. slevini and P. maniculatus raises questions as to the general applicability of this model of island biogeography. However, the origin and potential reinterpretation of the model of island biogeography in the Sea of Cortez must await a detailed treatment of the systematic affinities of P. slevini within the genus. As stated previously, inclusion of P. sejugis within the P. maniculatus group is not

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10092

7)
(

. k. interdictus

ao
(12) 98
(10)

13

P. k. oreas

(13)
9

E m. . coolidgei
(1)

93

/ 92

I 10

P.

Wjugis

(SD)

(6) E. ejugis (SC)

99100 100 (6)

P. m. rufinus

(10) P. m. austerus
16) (3) 100

P. olionotus

(45)

(38)

P. melanotis
100 (28) P. gOSSVyinUS

(85)

(43) P. leucopus E. slevini megalotis R_. P. seiugis(SD) P. sejugis(SC)

P. m. ei coolid
b

interdictus P. k.
P. m.
.

rufinus
austerm

ma . -. polionotus

Smel.anotis
P gossvpinus P. leucoous
0.01

R. megalotis

parsimonyanalysis (a) and distance data (Kimura,1980) employing the neighbor-joining algorithm (b) of all 1,439 base pairs of mtDNA. Bootstrap values for each node are listed for the equally weighted and weighted analyses, respectively.Single bootstrapvalues at each node indicateidentical values for each weighting scheme. Numbers in parenthesesindicate the number of unambiguous substitutionsoccurringon each branch.

FIG. 1.-Inferred phylogenetic relationshipsamong the taxa included in this study based on a

as problematic as the inclusion of P. slevini. Results of this study are consistent with the allozymic (Avise et al., 1979) and morphologic (Hooper and Musser, 1964) data in supporting the inclusion of P. sejugis within

the P. maniculatus species group. The relationship of P. sejugis to other species within the P. maniculatus species group raises a question regarding the taxonomic affinities of species of Peromyscus inhab-

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TABLE 3.-Composite examined.

mtDNA haplotypes resulting from an analysis of restriction sites in the taxa

Haplotype Dde I Dpn II 123456 110101 100100 Rsa I 123456 100011 100000 Taq I 123456 100000 111000

Clone A B

Taxon P. slevini P. sejugis

123456789 100010010 000001000

C D E F G H

P. polionotus P. melanotis P. keeni P. leucopus P. maniculatus P. maniculatus

Dde I

001001000 101001000 001000000 000001000 011001000 001110111

Dpn II 1) 9,433-9,436 2) 9,579-9,582 3) 9,941-9,944 4) 10,627-10,630 5) 10,719-10,722 6) 10,729-10,732

101100 110111 101100 101100 101100 110101

Rsa I 1) 9,409-9,412 2) 9,563-9,566 3) 9,744-9,747 4) 9,774-9,777 5) 10,138-10,141 6) 10,794-10,797

110000 101110 100010 100000 100000 100000

110010 100100 111000 111000 111000 100001

Taq I

1) 2) 3) 4) 5) 6) 7) 8)

9,752-9,756 9,758-9,762 9,796-9,800 9,921-9,925 10,060-10,064 10,092-10,096 10,271-10,275 10,391-10,395

1) 9,835-9,838 2) 9,939-9,942 3) 9,955-9,958 4) 9,971-9,974 5) 10,428-10,431 6) 10,525-10,528

9) 10,565-10,569
locationsare relativeto the Mus sequence(Bibb et al., 1981). Mappedrestriction-site

iting the Pacific coast. In this investigation, P. sejugis clustered with P. m. coolidgei. This clade in turn is sister to P. keeni from the Pacific Northwest (Fig. 1). This arrangement suggests that P. maniculatus, as currently recognized, is paraphyletic. Two scenarios can be invoked to explain this relationship. First, the paraphyly within P. maniculatus may be an artifact of lineage sorting (Avise, 1994) within P. maniculatus as a result of recent divergence within the species. Alternatively, the grouping of these taxa may represent multiple species. Although neither alternative can be eliminated based on available data, the first possibility seems least likely because of the observed variation in haplotypes. With the exception of two haplotypes observed in samples of P. maniculatus, restriction-enzyme analysis revealed no intraspecific variation in the taxa examined. In P. maniculatus, all individuals of P. m. rufinus examined from Colorado and P. m. artemisiae from Washington shared the same haplotype (Table 3,

clone G), whereas P. m. coolidgei from Baja California differed by having an additional restriction site (Table 3, clone F). This observation differs from the situation found in P. keeni in which a single haplotype represented populations as disjunct as Vancouver and Graham islands, as well as the state of Washington (Table 3, clone C). In addition, only a single restriction-fragment pattern was observed (Table 3, clone H) in the five populations of P. leucopus surveyed. The lack of variation within species may indicate that the restriction sites surveyed are highly conserved among species of Peromyscus. Alternatively, this lack of intraspecific variation could have resulted from a failure to sample low-frequency haplotypes in the various populations. While the latter scenario cannot be excluded, the former is more likely, particularly considering the results of our phylogenetic analyses and when compared to the published studies discussed below.

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In an analysis of variation of mtDNA in P. maniculatus, Lansman et al. (1983) concluded that the geographically variable P. maniculatus could be divided into five major assemblages, each separable at ca. 3% sequence divergence. These five assemblages corresponded to specimens from the central United States, the eastern United States, northern Michigan, Texas-Mexico, and southern California (Lansman et al., 1983:9; figures 7 and 8). Although Lansman et al. (1983) did not examine P. m. coolidgei, they did include P. m. gambelii, a sister-taxon of P. m. coolidgei (Calhoun et al., 1988). The taxa examined in this study appear to represent the central United States (P. m. rufinus and P. m. artemisiae) and southern California (P. m. coolidgei) assemblages as depicted in Lansman et al.

Northwest. Canadian Journal of Zoology, 66:27342739. M. W., S. J. GUNN, AND I. E GREENBAUM. ALLARD, 1987. Mensural discrimination of chromosomally characterized Peromyscus oreas and P. maniculatus. Journal of Mammalogy, 68:402-406. ARtVALO, E., S. K. DAVIS,ANDJ. W. SITES.1994. Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosome races of the Sceloporus grammicus complex (Phrynosomatidae) in Central Mexico. Systematic Biology, 43: 387-418. AVISE,J. C. 1994. Molecular markers, natural history and evolution. Chapman and Hall, Inc., New York, 511 pp. ANDR. K. SELANDER. 1979. AVISE,J. C., M. H. SMITH, Biochemical polymorphism and systematics in the genus Peromyscus. VII. Geographic differentiation in members of the truei and maniculatus species groups. Journal of Mammalogy, 60:177-192. S. W. DANIEL, C. E AQUADAVISE,J. C., J. E SHAPIRA, 1983. Mitochondrial DNA RO,ANDR. A. LANSMAN. differentiation during the speciation process in Peromyscus. Molecular Biology and Evolution, 1:38-56. R. K. SELANDER, T. E. LAWAvISE, J. C., M. H. SMITH,
LOR, AND P. R. RAMSEY. 1974. Biochemical poly-

(1983).
In view of the apparent consistency in geographic variation between our study and that of Lansman et al. (1983), we suggest that the assemblages previously recognized by Lansman et al. (1983) warrant further investigation as to their taxonomic status. Future studies should resolve the relationship between P. keeni and P. sejugis-P. m. coolidgei to determine whether these taxa constitute a single lineage along the Pacific coast or multiple species.
ACKNOWLEDGMENTS

morphism and systematics in the genus Peromyscus. V. Insular and mainland species of the subgenus Haplomylomys. Systematic Zoology, 23:226-238. C. T. WRIGHT, M. W. BIBB, M. J., R. A. VAN ETTEN,
WALBERG,AND D. A. CLAYTON. 1981. Sequence and

For assistancein the laboratorywe gratefully acknowledge the help of T. Guerra,D. Starky, M. Forstner,S. Engel, E. Louis, and C. Young. For reading earlier drafts of the manuscriptwe thank S. Engel, S. A. Berend, J. G. Gable, M. Wike, R. Honeycutt,and J. Bickham.The manuscript also benefitted from two anonymous reviewers. Financial supportfor this project was provided by National Institutesof Health grant GM 27014 to I. E Greenbaum and NationalScience Foundationgrant BSR 88-45298 to S. K. and J. Sites. Davis, I. E Greenbaum
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from the Pacific Northwest. Journal of Mammalogy, 74:819-831. R. L., AND W. C. WHEELER. 1990. MiHoNEYCUTr, tochondrial DNA: variation in humans and higher primates. Pp. 91-129, in DNA systematics: humans and higher primates (S. K. Dutta and W. P. Winter, eds.). CRC Press, Inc., Boca Raton, Florida, 3:1205. E. T. 1968. Classification. Pp. 27-74, in BiHOOPER, ology of Peromyscus (Rodentia) (J. A. King, ed.). Special Publication, The American Society of Mammalogists, 2:1-593. E. T., ANDG. G. MUSSER.1964. Notes on the HOOPER, classification of the rodent genus Peromyscus. Occasional Papers of the Museum of Zoology, University of Michigan, 635:1-13. J. P. 1991. Tree-length distribution HUELSENBECK, skewness: an indicator of phylogenetic information. Systematic Zoology, 40:257-270. M. 1980. A simple method for estimating KIMURA, evolutionary rate of base substitution through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16:111-120. G. L., ANDJ. N. LAYNE. 1989. IntroducKIRKLAND, tion. Pp. 1-5, in Advances in the study of Peromyscus (Rodentia) (G. L. Kirkland, Jr. and J. N. Layne, eds.). Texas Tech University Press, Lubbock, 366 PP. K. S. KRAUTER, ANDL. A. LEINR., J. TARDIFF, KRAFT, WAND.1988. Using mini-prep plasmid DNA for sequencing double-stranded templates with sequenase. BioTechniques, 6:544-547.
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molecular evolutionary genetics analysis, version 1.01. The Pennsylvania State University, University Park, 139 pp. J. E R. A., J. C. AVISE, C. E AQUADRO, LANSMAN, AND S. W. DANIEL. 1983. Extensive geSHAPIRA, netic variation in mitochondrial DNA's among geographic populations of the deer mouse, Peromyscus maniculatus. Evolution, 37:1-16. T E. 1971. Evolution of Peromyscus on LAWLOR, northern islands in the Gulf of California, Mexico. Transactions of the San Diego Society of Natural History, 16:91-124. . 1983. The mammals. Pp. 265-289, in Island biogeography in the Sea of Cortez (T J. Case and M. L. Cody, eds.). University of California Press, Berkeley, 508 pp. Liu, T. T 1954. Hybridization between Peromyscus maniculatus, P. oreas and P. m. gracilis. Journal of Mammalogy, 35:448-449. MADDISON,W. P., AND D. R. MADDISON. 1992. MacClade: analysis of phylogeny and character evolution. Version 3.0. Sinauer Associates, Inc., Publishers, Sunderland, Massachusetts, 398 pp. J. 1924. A new mouse (Peromyscus slevMAILLAIRD, ini) from the Gulf of California. Proceedings of the California Academy of Science, Series 4, 12:12191222. ANDJ. SAMBROOK. 1982. MANIATIS, T., E. E FRITSCH, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, 545 pp. ANDE S. COLA. SAULINO, MARCHUK, D., M. DRUMM, LINS. 1991. Construction of T-vectors, a rapid and

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general system for direct cloning of unmodified PCR products. Nucleic Acid Research, 19:1154. ANDW. M. BROWN.1987. MORITZ, C., T. E. DOWLING, Evolution of animal mitochondrial DNA: relevance for population biology and systematics. Annual Review of Ecology and Systematics, 18:269-292. W. H. 1909. Revision of the mice of the OSGOOD, American genus Peromyscus. North American Fauna, 28:1-285.
PENGILLY,D., G. H. JARRELL,AND S. O. MACDONALD.

APPENDIX I
Specimens examined.--Collection localities for specimens of Peromyscus. Locality numbers correspond to those discussed in text. Number of individuals for each taxon examined in each portion of this study are given in the text. Taxonomic identity of specimens based on data from Hall (1981), Gunn and Greenbaum (1986), Allard and Greenbaum (1988), and Hogan et al. (1993). Numbers in parentheses are catalog numbers corresponding to voucher specimen sequenced in this study (GK
= Greenbaum karyotype number; NK = New

1983. Banded karyotypes of Peromyscus sitkensis from Baranof Island, Alaska. Journal of Mammalo-

gy, 64:682-685.
ROBBINS, L. W., AND R. J. BAKER. 1981. An assess-

ment of the nature of chromosomal rearrangements in 18 species of Peromyscus (Rodentia: Cricetidae). Cytogenetics and Cell Genetics, 31:194-202. ROGERS, D. S., I. E GREENBAUM,S. J. GUNN, AND M. D. ENGSTROM. 1984. Cytosystematic value of chromosomal inversion data in the genus Peromyscus (Rodentia: Cricetidae). Journal of Mammalogy, 65:

457-465.

SAITOU, N., ANDM. NEI. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406-425.
SANGER, E, S. NICKLEN, AND A. R. COULSON. 1977.

Mexico karyotype number; KMH = Kelly M. Hogan catalog number). Voucher specimens are deposited in the Texas Cooperative Wildlife Collection, Texas A&M University, College Station, and the Museum of Southwestern Biology, University of New Mexico, Albuquerque. Peromyscus keeni algidus. USA: Alaska: Locality 1-2 km N Skagway, Liarsville Campground. Peromyscus keeni hylaeus. USA: Alaska: Locality 2-21.2 km NW Auke Bay Ferry Termi-

DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Science,

74:5463-5467.
SHEPPE, W., JR. 1961. Systematic and ecological re-

lations of Peromyscus oreas and P. maniculatus.

Proceedings of the American Philosophical Society, 105:421-446. AND I. E GREENR. M., S. W. CALHOUN, SULLIVAN, BAUM. 1990. Geographic variation in genital morphology among insular and mainland populations of

Peromyscus maniculatus and Peromyscus oreas.

Journal of Mammalogy, 71:48-58. STANGL, E B., ANDR. J. BAKER. 1984. Evolutionary relationships in Peromyscus: congruence in chromosomal, genic, and classical data sets. Journal of Mammalogy, 65:643-654. D. L. 1993. PAUP: phylogenetic analysis SWOFFORD, using parsimony, version 3.1.1. Illinois Natural History Survey, Champaign, 98 pp.
TABOR, S., AND C. C. RICHARDSON. 1987. DNA se-

quence analysis with a modified bacteriophage T7 DNA polymerase. Proceedings of the National Academy of Sciences, 84:4767-4771. B. 1973. Evolutionary implications of karyoTHOMAS, typic variation in some insular Peromyscus from British Columbia, Canada. Cytologia, 38:483-493. ANDR. K. BARNETr.1979. YATES,T. L., R. J. BAKER, Phylogenetic analysis of karyological variation in three species of peromyscine rodents. Systematic Zoology, 28:40-48.

Submitted16 November1995. Accepted20 November 1996. Associate Editor was James R. Purdue.

nal, Herbert River. Peromyscus keeni isolatus. Canada; British Columbia: Locality 3-Malcolm Island, 4.8 km E Sointula. Peromyscus keeni interdictus. Canada: British Columbia: Locality 4-Vancouver Island, Mount Washington Ski Area (GK 2448; GenBank Accession U40063). Peromyscus keeni macrorhinus. Canada: British Columbia: Locality 5-32.4 km E Prince Rupert. Locality 6--4.6 km W Exchamsiks River Provincial Park. Locality 7-Kleanza Creek, Highway 16. Peromyscus keeni oreas. USA: Washington Grays 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 Island, 35.7 km W Port Alberni, Sproat Lake (GK 5604; GenBank Accession U40249). Peromyscus maniculatus coolidgei. Mexico: Baja California del Sur: Locality 11---25 km SE Guerrero Negro (NK 5166; GenBank Accession U40251). Peromyscus maniculatus rufinus. USA: Colorado: Locality 12-7.2 km N, 8.8 km W Cen-

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tral City, Elk Park (KMH 1038; GenBank Accession U40250). Peromyscus melanotis. Mexico: Hidalgo: Locality 13-Lab colony from the Peromyscus Stock Center, originally from Mexico (KMH 1024; GenBank Accession U40247). Peromyscus polionotus. USA: South Carolina: Lexington Co.: Locality 14--0.6 km S Edmund Bethel Church Field (GK 5904; GenBank Accession U40254). Peromyscus sejugis. Mexico: Baja California del 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 (GK 5880; GenBank Accession U40248).

Peromyscus leucopus. USA: Texas: Locality Co. (GK 5848; GenBank Ac18-Robertson cession U40252). Locality 19-Hunt Co., vicinity of Clest. Locality 20-Wichita Co., 13.8 km N, 0.6 km W Iowa Park. Locality 21Michigan (laboratory colony). Locality 22Maine, Hancock Co., 3.0 km S, 0.6 km E Bar Harbor. Peromyscus gossypinus. USA: Texas: Anderson Co., Locality 23-3.2 km S, 6.4 km E Cayuga, Gus A. Engeling Wildlife Management Area (KMH 1041; GenBank Accession U40246). Reithrodontomys megalotis. USA: Texas: Jeff Davis Co., Locality 24--6.1 km N, 11.2 km W Fort Davis (KMH 1060; GenBank Accession U40031).

APPENDIX II.
List of PCR and sequencing primers used to amplify 1,439 base pairs of mtDNA. All primer concentrations were 10 ng/pIL. Nucleotides in parentheses denote nucleotides at degenerative positions in primer.
Primer
PI'c

Reference positionsa 9,403-9,524 9,560-9,580 9,616-9,637 9,777-9,794 9,783-9,799 10,171-10,191 10,467-10,483 10,615-10,634 10,812-10,832

Sequenceb cga act agt aca gct gac ttc c cc(acgt) ta(tc) gaa tgc gg(ag) ttt ga aat ttt t(tc)c tag tag caa t(tc)a c caa aaa gga (tc)ta gaa tga gga (tc)ta gaa tga ac(agc) ga gca tgt gaa gca gc(tc) at(tc) gg caa at(ct) ct(cat) cta atc at ata c(act)(ct) taa ttg g(ag)t caa tc aaa gc(tc) cac gta gaa gct cca

ND3M ND3M2 Marg MargRev ND4L Rataway MouseMerl Nap2c

a

Referencepositionsare relativeto the Mus sequence(Bibb et al., 1981). to the light strandof mtDNA. rightfrom 5' to 3' corresponding SFromAr6valoet al. (1994).
b Primersequencesare listed left to

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