Molecular Phylogenetics and Evolution
Vol. 16, No. 3, September, pp. 366 –378, 2000
doi:10.1006/mpev.2000.0806, available online at http://www.idealibrary.com on
Molecular Phylogenetics of the Peromyscus boylii Species Group
(Rodentia: Muridae) Based on Mitochondrial
Cytochrome b Sequences
Irene Tiemann-Boege,* C. William Kilpatrick,† David J. Schmidly,* ,‡
and Robert D. Bradley* ,‡
*Department of Biological Sciences, Texas Tech University, Lubbock, Texas 79409-3131; †Department of Biology, University of
Vermont, Burlington, Vermont 05405-0086; and ‡Museum of Texas Tech University, Lubbock, Texas 79409-3191
Received September 21, 1999; revised January 25, 2000
Variation in the mitochondrial cytochrome b gene
(1143 bp) was examined to estimate the phylogenetic
relationships of taxa within the Peromyscus boylii species group. In addition, phylogenetic relationships
among the aztecus, boylii, and truei species groups
were addressed. Maximum-likelihood, neighbor-joining, and maximum-parsimony (weighted and equally
weighted) analyses produced similar topologies with
P. boylii, P. beatae, P. simulus, P. stephani, P. madrensis, P. levipes, and three undescribed taxa from western Mexico forming a monophyletic unit. At least two
of the undescribed taxa from western Mexico potentially represent species. Members of the P. aztecus
species group formed a clade separate from the P.
boylii group and should be recognized as a distinct
species group. P. sagax, P. polius, and P. pectoralis,
formerly placed in the P. boylii species group, generally formed an unresolved polytomy with the P. truei,
P. aztecus, and P. boylii species groups. P. attwateri
formed a sister taxon relationship with members of
the P. truei species group (P. difficilis and P. nasutus)
and should be considered a member of this group.
Members of the P. truei species group did not form a
monophyletic unit, indicating that this species group
is not monophyletic and may be composed of two assemblages. © 2000 Academic Press
INTRODUCTION
The Peromyscus boylii species group is distributed
throughout the southwestern United States and Mexican highlands, extending south into Central America
(Hall, 1981). Members of this group occupy a variety of
habitats, ranging from arid scrublands to pine– oak
and cloud forests (Schmidly et al., 1988), but typically
occur in pine– oak forests of montane regions at elevations higher than 1500 m. Currently 10 species are
contained within the boylii species group (Osgood,
1055-7903/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
1909; Hooper, 1968; Carleton, 1977, 1989; Rennert and
Kilpatrick, 1986; Houseal et al., 1987; Schmidly et al.,
1988; Bradley et al., 1996) P. attwateri, P. boylii, P.
beatae, P. levipes, P. madrensis, P. pectoralis, P. polius,
P. sagax, P. simulus, and P. stephani. Hereafter these
taxa are referred to by specific or subspecific epithets.
Although much attention has been given to understanding the taxonomy and systematics of this complex
group, many significant questions remain untested or
unresolved.
First, the relationship of the boylii species group to
the P. aztecus and P. truei species groups is unclear.
Historically, members of the aztecus and boylii groups
were classified within the same species group (Osgood,
1909; Hooper, 1968). However, more recent studies
have provided morphological (Carleton, 1977; Carleton
et al., 1982; Bradley and Schmidly, 1987) and chromosomal (Schmidly and Schroeter, 1974; Houseal et al.,
1987; Smith et al., 1989) evidence that the boylii and
aztecus groups are two distinct moieties. Carleton
(1989) summarized this evidence and elevated the aztecus assemblage to the rank of species group. However, Smith (1990) and Sullivan et al. (1991) have
argued against separation into two species groups
based on the lack of allozymic and karyotypic resolution between the two groups. However, neither Smith
(1990) nor Sullivan et al. (1991) provided data to support the monophyly of the P. boylii species group. This
question is further complicated, as many studies addressing this issue either included multiple species
groups with few representatives of the boylii species
group (Robbins and Baker, 1981; Rogers et al., 1984;
Stangl and Baker, 1984; Smith, 1990; Sullivan et al.,
1991; DeWalt et al., 1993a) or included only members
of the boylii species group (Carleton et al., 1982; Rennert and Kilpatrick, 1987; Bradley and Schmidly,
1987) or the aztecus species group (Sullivan and Kilpatrick, 1991).
Second, several recent studies have questioned the
366
MOLECULAR PHYLOGENETICS OF Peromyscus boylii
validity of the boylii group and its composition. For
example, attwateri and pectoralis have been aligned to
the truei group and to P. eremicus (Avise et al., 1974a,
1979; Zimmerman et al., 1975; Stangl and Baker, 1984;
Janecek, 1990; Sullivan et al., 1991; DeWalt et al.,
1993a). Similarly, polius and sagax were placed tentatively in the boylii group (Hoffmeister, 1951; Bradley et
al., 1996); however, comparisons of allozymic and
karyotypic data resulted in weak or no association of
these taxa to the boylii group (Kilpatrick and Zimmerman, 1975; Zimmerman et al., 1975; Schmidly et al.,
1985; Houseal et al., 1987; Bradley et al., 1996).
Third, although the systematics of taxa occurring in
eastern Mexico have been resolved relatively well
(Rennert and Kilpatrick, 1986; Bradley and Schmidly,
1987; Houseal et al., 1987; Schmidly et al., 1988; Castro-Campillo et al., 1999; Bradley et al., 2000), those
taxa occurring in western Mexico present several unresolved issues. For example, specimens occurring in
Nayarit, northeastern Michoacán, and Jalisco, with
atypical fundamental numbers of 65, 66, and 68, are
morphologically and karyotypically distinct from levipes, beatae, or boylii (Schmidly and Schroeter, 1974;
Carleton et al., 1982; Houseal et al., 1987; Bradley et
al., 1989; Smith et al., 1989). A second form with fundamental numbers ranging from 54 to 56 occurs in
central and southwestern Durango, inhabiting desert
and montane oak forests (Lee et al., 1972; Schmidly
and Schroeter, 1974; Kilpatrick and Zimmerman,
1975; Houseal et al., 1987). A third variant from southwestern Michoacán was characterized by fundamental
numbers ranging from 65 to 68 and is karyotypically
and morphologically distinct from other species of boylii (Houseal et al., 1987; Bradley et al., 1989). The
status of these taxa is unclear due to the lack of information about their phylogenetic affinity and level of
divergence to other members of the boylii group
(Schmidly and Schroeter, 1974; Carleton et al., 1982;
Houseal et al., 1987; Bradley et al., 1989).
The primary objective of this study was to examine
the mitochondrial cytochrome b gene of all taxa that
have been classified under the boylii epithet in combination with members of closely related species groups
(aztecus and truei). Specifically, this study addresses
(1) the relationship of the boylii species group to the
aztecus and truei species groups, (2) which taxa form a
monophyletic unit that defines the boylii group, (3) the
phylogenetic relationships among members of the boylii species group, and (4) the status of the undescribed
forms from western Mexico.
MATERIALS AND METHODS
Specimens Examined
The nucleotide sequence of the mitochondrial cytochrome b gene was analyzed for 29 individuals repre-
367
senting eight species, six subspecies, and three undescribed forms of the boylii and four species of the truei
species group. For taxa with a broad geographic distribution, more than one specimen was examined, insuring that variants from the extremes of the ranges were
represented. In addition, for polytypic species, multiple
subspecies were examined. Specimens examined are
listed with collecting locality (Fig. 1), assigned sample
number for cross-referencing purposes, museum acronyms (Hafner et al., 1997), specific identification numbers, collector numbers or catalogue numbers, and
GenBank Accession numbers. Osgoodomys banderanus—Mexico. Jalisco: 6 km SE Chamela (TTU: TK
11796; AF155383). Peromyscus attwateri—USA. Oklahoma (Sample 1): McIntosh Co., 3.1 mi E Dustin (TTU:
TK 23396; AF155384); Texas (Sample 2): Knox Co., 3
mi E Benjamin (ZTNHC: DNA 27; AF155385). Peromyscus boylii boylii—USA. California (Sample 3):
Monterey Co., Hastings Natural History Reservation
(MVZ: K. Nutt 120; AF155386). Peromyscus boylii glasselli—Mexico. Sonora (Sample 4): Isla San Pedro Nolasco (UMMZ: 117347; AF155387). Peromyscus boylii
rowleyi—Mexico. Durango (Sample 5): 29.2 mi W Cd.
Durango (ZTNHC: CWK 1965; AF155413); Jalisco
(Sample 6): 30 km W Huejuquilla del Alto (TTU: TK
48636; AF155388); USA. Texas (Sample 7): Jeff Davis
Co., Mt. Livermore Preserve (TTU: TK 78820;
AF155389); Texas (Sample 8): Culberson Co., Sierra
Diablo Wildlife Management Area UTM: 345915-13508596 (TTU: TK 54095; AF155390); California (Sample 9): Orange Co., Santa Ana Mountains, Holy Jim
Canyon (Trabuco Creek) (TTU: TK 83537; AF155391).
Peromyscus boylii utahensis—USA. Utah (Sample 10):
Garfield Co., Henry Mountains, Mt. Pennell, Sidehill
Spring, 8220 ft (MSB: NK 39457; AF155392). Peromyscus californicus—USA. California: San Diego Co., San
Onofre State Beach, 3.5 mi NNE on Christmas Road
(TTU: TK 83632; AF155393). Peromyscus difficilis—
Mexico. Hidalgo: 5.4 mi SE, 3.2 mi S Ixmiquilpan
(TCWC: GK 2642; AF155394). Peromyscus gratus—
Mexico. Durango: 2.2 km S, 2.5 km E Vicente Guerrero
(TTU: TK 48800; AF155395). Peromyscus levipes levipes—Mexico. Querétaro (Sample 11): 8.2 mi N, 1.8 mi
W Amealco (TCWC: GK 3027; AF155396). Peromyscus
madrensis—Mexico. Nayarit (Sample 12): Isla Marı́a
Madre (USNM: 512599; AF155397). Peromyscus melanotis—Mexico. Durango: 12 km Ojitos (TTU: TK
70997; AF155398). Peromyscus nasutus—USA. New
Mexico: Lincoln Co., 4 mi S Carrizozo (TTU: TK 77922;
AF155399). Peromyscus pectoralis laceianus—USA.
Texas (Sample 13): Kimble Co., Walter Buck Wildlife
Management Area, UTM: 3366232-14-423359 (TTU:
TK 52050; AF155400). Peromyscus pectoralis pectoralis—Mexico. Durango (Sample 14): 1.5 km SE Las Herreras, 1694 m (TTU: TK 48567; AF155401). Peromyscus polius—Mexico. Chihuahua (Sample 15): 3 mi SW
Santa Bárbara (TCWC: 47255; AF155403). Peromyscus
368
TIEMANN-BOEGE ET AL.
FIG. 1. Map depicting populations of the Peromyscus boylii species group sampled in this study. Samples of the truei and aztecus species
groups and the outgroups are not plotted here.
sagax—Mexico. Michoacán (Sample 16): Puerto Garnica, 9200 ft. (ZTNHC: CWK 2032; AF155404). Peromyscus sp. (FN ⫽ 54 –56 —Mexico. Durango (Sample
17): 12 km E Ojitos (TTU: TK 70966; AF155405); Durango (Samples 18 –20): 3.8 mi Hacienda Coyotes
(TTU: TK 72380; AF155406; ZTNHC: CWK 1997;
AF155407; ZTNHC: CWK1993; AF155408). Peromyscus sp. (FN ⫽ 65– 68)—Mexico. Michoacán (Sample
21): 7.4 mi WSW Dos Aguas (TCWC: GK 3282;
AF155409). Peromyscus sp. (FN ⫽ 65, 66, 68)—Mexico.
Nayarit (Sample 22): Ocota, 21°5⬘ N, 104°30⬘ W
(TCWC: GK 3264; AF155410). Peromyscus stephani—
Mexico. Sonora (Sample 23): Isla San Esteban (UMMZ:
117385; AF155411). Peromyscus truei—USA. Arizona:
Navajo Co., 3 mi S Woodruff (TTU: TK 77921;
AF155412).
Sequences for P. simulus (Sample 24: AF131927), P.
boylii rowleyi (Sample 25: AF131924; Sample 26:
AF131925), P. beatae beatae (Sample 27: AF131921), P.
beatae sacarensis (Sample 28: AF131915), P. levipes
levipes (Sample 29; AF131929), P. levipes ambiguus
(Sample 30; AF131928), and P. leucopus (AF131926)
were obtained from R. D. Bradley et al. (unpublished).
Partial sequences (approximately 750 bp) published by
Sullivan et al. (1997) were obtained for the aztecus
species group (P. aztecus aztecus, U89968; P. a. evides,
U89970; P. a. oaxacensis, U89972; P. hylocetes,
U89976; P. spicilegus, U89980; and P. winkelmanni,
U89983).
Data Collection
Mitochondrial DNA was extracted from liver samples and purified using the Wizard Miniprep kit (Promega, Madison, WI). For most taxa, the complete cytochrome b gene was amplified via the polymerase
chain reaction (Saiki et al., 1988) using the following
parameters: 39 cycles of 92°C (15 s) denaturing, 50°C
(1 min) annealing, and 72°C (1 min, 10 s) extension;
followed by 1 cycle of 72°C (4 min). Amplification reactions were performed in 50-␮l volumes, 10 mM Tris–Cl,
pH 8.3, 50 mM KCl, 2 mM MgCl 2, 1 ␮M primer concentration, and 1.25 U of Taq (Fisher Scientific, Pittsburgh, PA). For rare specimens with no available tissues (glasselli, madrensis, polius, and stephani), DNA
was obtained from skin clips which were homogenized
and predigested with 0.4% collagenase (Pääbo et al.,
1988) followed by a treatment of 2% (w/v) cetyltrimethylammonium bromide (CTAB) as described in Yang et
al. (1997). The sample was purified by phenol:chloroform extractions, ethanol precipitated, and resus-
369
MOLECULAR PHYLOGENETICS OF Peromyscus boylii
TABLE 1
Primers Used for Amplification and Sequencing of the Cytochrome b Gene for Samples
of the Peromyscus boylii Species Group
Primer name
Primer sequence
Gene/tRNA
L14724*
WDRAT400F
WDRAT400R
752R
NEO700L
PERO3⬘
H15915*
5⬘-CGAAGCTTGATATGAAAAACCATCGTTG
5⬘-CCATGAGGACAAATATCCTTCTGAGGG
5⬘-GCCCTCAGAAGGATATTGTCCTCATGG
5⬘-GCAGGAGTGTAATTATCGGGGTCTC
5⬘-CCCCAGCACATATTAAACCAGAATG
5⬘-TCTCTCCGGTTTCAAGACCAAAGT
5⬘-AACTGCAGTCATCTCCGGTTTACAAGAC
NADH 6
Cyt b
Cyt b
Cyt b
Cyt b
tRNA thr
tRNA thr
Annealing position
L14087–L14114
L14537–L14563
H14564–H14537
H14889–H14913
L14928–L14952
H15329–H15303
H15337–H15309
Note. The third and fourth columns indicate annealing position of the primers based on the sequence of Mus domesticus (Bibb et al., 1981).
An asterisk indicates the primers published by Irwin et al. (1991).
pended in 10 ␮l of TE (10 mM Tris–Cl, 1 mM EDTA,
pH 8.0). PCR products were amplified in fragments
using various combinations of primers. Primers (Table
1) were either designed or were those of Irwin et al.
(1991). The amplified products were purified with silica
gel using QIAquick PCR Purification Kit (Qiagen, Valencia, CA).
Amplicons were sequenced with dye-labeled terminators, the aforementioned primers, and approximately 60 to 80 ng of DNA using cycle sequencing
conditions of 95°C for 30 s of denaturing, 50°C for 20 s
of annealing, and 60°C for 3 min of extension. The
reaction was then ethanol precipitated after 25 to 29
cycles. Sequences for the heavy and the light strand
were analyzed with the ABI-Prism 310 automated sequencer (PE Applied Biosystems, Foster City, CA).
Data Analyses
Maximum-parsimony analyses were conducted using PAUP (Phylogenetic Analysis Using Parsimony
and other Methods, version 4.0b2a; Swofford, 1999).
Robustness and nodal support was evaluated using
1000 bootstrap iterations (Felsenstein, 1985) and Bremer support indices (Bremer, 1994) calculated with the
Autodecay Analysis software (Eriksson, 1997). Variable nucleotide positions were treated as unordered,
discrete characters with four possible character states:
A, C, G, and T. Incomplete sequences for madrensis
and polius at the 5⬘ or 3⬘ end of the cytochrome b gene
were represented by dashes. Following the functional
ingroup/functional outgroup approach of Watrous and
Wheeler (1981), an initial analysis utilizing Osgoodomys banderanus and P. californicus as outgroups
was conducted to determine the most appropriate outgroup. Subsequent analyses utilized P. leucopus and P.
melanotis to define the character polarity of the ingroup formed by taxa of the aztecus, truei, and boylii
species groups.
Several weighting schemes were employed in the
parsimony analyses including equal weighting and
downweighting of transitions by factors of 2 and 5.
The weight of 5 was inherent to the ingroup and was
calculated from the average ratio of pairwise comparisons. First, second, and third position substitutions were differentially weighted employing the inherent ratios calculated for the ingroup of 4:16:1 (all
taxa) and 6:58:1 (boylii group). Combinations of the
inherent transition/transversion (ti/tv) ratios and
positional weighting schemes also were performed.
Nucleotide sequence data were weighted using MacClade (version 3.04; Maddison and Maddison, 1992)
and then analyzed using the maximum-parsimony
option of PAUP.
Neighbor-joining trees, optimized for the minimalevolution criterion, were constructed based on the pairwise distance comparisons calculated for the Tamura
and Nei (1993) model of evolution. In addition, the
gamma version for the Tamura and Nei model was
employed to assess the effect of among-site rate variation on the tree topology. Nodal support of the neighbor-joining tree was evaluated with 1000 bootstrap
replicates.
The analyses performed with maximum-likelihood
included the estimation of parameters (ti/tv ratios
and gamma shape parameters) for the HKY85-⌫
model of evolution (Hasegawa et al., 1985). This
model was chosen based on the findings of Yang et al.
(1994), who obtained superior likelihood scores compared to simpler models of substitution. Additionally, this model was identified by Sullivan et al.
(1997) as being the most appropriate model for maximum-likelihood analysis of the aztecus species
group. The model parameters estimated for the
most-parsimonious tree were fixed for subsequent
maximum-likelihood searches following Sullivan et
al. (1997). Analyses employed empirical base compositional biases, 10 random input orders, and TBR
branch swapping. Nodal support was estimated via
bootstrap analysis using 100 iterations.
370
TIEMANN-BOEGE ET AL.
RESULTS
Nucleotide Composition
The cytochrome b gene sequences coded for 381
amino acids, lacked a stop codon, and terminated with
a single thiamine base comparable to that in Mus domesticus (Bibb et al., 1981) and Rattus norvegicus
(Gadaleta et al., 1989). Analysis of the base content
depicted equal base frequencies (approximately 25%)
at the first codon position, a high thymine content at
the second codon position (⬎41%), and a depauperate
guanidine content at the third codon position (⬍3%).
The compositional bias for this data set (0.066, 0.226,
and 0.378 for the first, second, and third codon position,
respectively) was similar to that of Irwin et al. (1991),
with the highest bias at the third position.
Transition/transversion ratios ranged from 36:1 for
comparisons among geographic variants of the same
taxon to 1.6:1 for comparisons of several ingroup taxa
(sacarensis, levipes, difficilis, and P. sp. Sample 22)
with P. leucopus. Transitions at the third codon position occurred most frequently (81.8%), followed by
transitions at the first position (14.8%), and the second
position (2.34%). In contrast, transversions occurred at
a lower frequency of 0.2, 0.05, and 0.9% at first, second,
and third codon position, respectively.
Relationships among Species Groups
Relationships among species groups (boylii, truei,
and aztecus) were examined utilizing a reduced data
set that included a single representative of each taxon.
To test the position of P. leucopus and P. melanotis
relative to the ingroup, O. banderanus and P. californicus were included in an initial analysis (data not
shown). In this analysis, P. leucopus and P. melanotis
formed a sister group to the boylii/aztecus/truei clade,
supporting the appropriate use of these taxa as outgroups in subsequent analyses, following Watrous and
Wheeler (1981).
A parsimony analysis was performed using a heuristic search based on 285 informative characters that
were equally weighted. This analysis produced a single
most-parsimonious tree of 1055 steps, consistency index (CI) of 0.369, and retention index (RI) of 0.601.
Bootstrap analysis (1000 iterations) provided support
for six discrete monophyletic groups (Fig. 2). The first
clade was formed by attwateri/difficilis/nasutus, the
second contained taxa of the boylii group (simulus,
boylii, utahensis, rowleyi, glasselli, stephani, madrensis, beatae, sacarensis, levipes, ambiguus, and three
undescribed taxa from western Mexico), the third clade
contained gratus, the fourth contained truei and polius,
the fifth contained subspecies of pectoralis, and the
sixth was composed of the aztecus group (winkelmanni,
aztecus, evides, oaxacensis, hylocetes, and spicilegus).
The phylogenetic arrangement among these six clades
was not resolved. A single bipartition split sagax from
FIG. 2. Single most-parsimonious tree of members of the Peromyscus boylii, P. aztecus, and P. truei species groups using P. leucopus and P. melanotis as outgroups. Characters were equally
weighted. Bootstrap values are above branches and Bremer support
indices are below.
the rest of the ingroup. The clade containing attwateri,
difficilis, and nasutus had the highest nodal support
(bootstrap of 99 and Bremer support index of 16). The
boylii-group clade had a bootstrap value of 96 and a
Bremer support index of 9, and the aztecus complex
possessed a bootstrap value and a Bremer decay index
of 71 and 1, respectively. The sister taxon relationship
of truei and polius was only weakly supported (bootstrap of 57 and Bremer support of 4).
Downweighting of transitions by factors of 2 and 5
depicted a topology (1256 and 1831 steps; CI ⫽ 0.393
and 0.431; RI ⫽ 0.614 and 0.641, respectively) similar
to that of the equally weighted analysis (Fig. 2), with
the exception of the boylii/aztecus clade, which formed
a sister taxon relationship (bootstrap values of 57 and
60, respectively). Additionally, the clade joining polius
and truei collapsed in both weighting schemes and
truei formed a sister taxon relationship with gratus
(bootstrap 56 and 65). Downweighting of transitions
2:1 resulted in equal or slightly higher bootstrap values; downweighting by a factor of 5 resulted in lower
bootstrap values for the most internal nodes. In gen-
MOLECULAR PHYLOGENETICS OF Peromyscus boylii
eral, Bremer support indices increased when a heavier
weighting scheme was assigned to transversions.
An analysis using the inherent ratio for weighting of
codon positions, estimated from the data set (4:16:1),
produced three most-parsimonious trees (1725 steps;
CI ⫽ 0.379; RI ⫽ 0.616). These trees were similar to the
toplogy depicted in Fig. 2, except for the collapse of the
polius/truei clade and the basal nodes of the aztecus
clade (winkelmanni and spicilegus). Under this weighting scheme, pectoralis and spicilegus were joined to the
attwateri/difficilis/nasutus clade and form an unresolved polytomy. Bootstrap values and Bremer support
indices for most of the internal nodes declined with
increased weighting.
The combined weighting scheme involving the inherent positional bias (4:16:1) and ti/tv ratio (5:1) resulted
in a single most-parsimonious tree with 2667 steps and
consistency and retention indices of 0.431 and 0.641,
respectively. The topology of this tree was similar to
that of Fig. 2, with no change in the resolution of the
phylogenetic relationships among the boylii, aztecus,
and truei groups. Bootstrap values were similar to the
other weighted analyses and Bremer indices increased
for strongly supported nodes.
The most-parsimonious tree generated utilizing
equal weights was evaluated for maximum-likelihood
parameters (⌫, ti/tv, and -ln L) under the HKY85-⌫
model and resulted in a gamma shape parameter of
0.140 and a ti/tv ratio of 5.5. A maximum-likelihood
search assuming the HKY85-⌫ model of evolution
(gamma ⫽ 0.140, ti/tv ⫽ 5.5, and a score of -ln L of
6512.14) produced four trees. The differences in these
four trees were the result of relationships among gratus, pectoralis, and truei. A strict consensus tree (Fig.
3) depicted a topology similar to those of the trees
obtained with maximum-parsimony (Fig. 2), with three
exceptions. First, members of the truei species group
formed a clade along with sagax and pectoralis. Second,
the truei group and the aztecus group formed a sister
taxon relationship. Third, polius was positioned as the
most basal member of the ingroup. The relationships
among taxa in the truei group collapsed in bootstrap
analysis, as did the relationship between the truei and
the aztecus groups.
A neighbor-joining tree was constructed (Fig. 4) with
genetic distances generated using the Tamura and Nei
and Tamura–Nei–⌫ (gamma ⫽ 0.140) models of evolution. The monophyletic clusters and relationships identified in the neighbor-joining tree coincided with those
obtained in the maximum-likelihood analysis (Fig. 3).
This included a sister relationship between the aztecus
and the truei groups, although this relationship and
those within the truei group collapsed during bootstrap
analysis (Fig. 4).
The relationships among species groups (aztecus/
truei, aztecus/boylii, and boylii/truei) were tested by
constraining tree topologies to form monophyletic
371
FIG. 3. Phylogeny obtained for members of the Peromyscus boylii, P. aztecus, and P. truei species groups under the maximumlikelihood framework assuming the HKY85-⌫ model of evolution
using P. leucopus and P. melanotis as outgroups. Model parameters
used were a ti/tv ratio ⫽ 5.5 and gamma ⫽ 0.140.
clades. The Kishino–Hasegawa (1989) test was used to
compare optimality scores for tree length and likelihood scores generated from each comparison of constrained clades (aztecus/truei versus aztecus/boylii, aztecus/truei versus boylii/truei, and aztecus/boylii versus
boylii/truei). Neither tree length or likelihood scores
were significantly different (Table 2) in comparisons of
the constrained clades.
Phylogenetic Relationships within the boylii Species
Group
The phylogenetic arrangement of the boylii species
group was examined utilizing only taxa within the
monophyletic “boylii clade” obtained in previous analyses (parsimony, likelihood, and distance). Consequently, attwateri, polius, sagax, and pectoralis were
not considered ingroup members. Due to the different
phylogenetic relationships among the boylii, aztecus,
and truei groups obtained from parsimony, likelihood,
and distance analyses, the closest relative to the ingroup was not readily apparent. Therefore, four taxa
(pectoralis, attwateri, winkelmanni, and truei) were
372
TIEMANN-BOEGE ET AL.
FIG. 4. Neighbor-joining tree generated from Tamura–Nei–⌫ genetic distances of the cytochrome b sequences examined in this
study. Branch lengths are drawn to scale.
used as outgroups in an initial equally weighted parsimony analysis.
An initial equally weighted heuristic search resulted
in eight most-parsimonious trees (511 steps, CI ⫽
0.481, and RI ⫽ 0.759) which differed in the placement
of populations of rowleyi (Samples 5– 8, 25, and 26) and
the undescribed taxon with a FN ⫽ 54 –56 (Samples
17–20). A 50% majority-rule consensus bootstrap tree
(1000 iterations) depicted two major clades (Fig. 5).
Within the first clade, simulus formed a sister taxon
relationship with a clade containing all subspecies of
boylii, except sacarensis, followed by the stepwise addition of stephani and madrensis. Within the boylii
subspecies clade, boylii, utahensis, and one sample of
rowleyi (Sample 9) formed a smaller unresolved clade
which attached to a clade containing glasselli and Samples 5– 8, 25, and 26 of rowleyi from the Mexican Plateau and Texas. The second major clade was formed by
beatae, sacarensis, levipes, ambiguus, and three undescribed taxa from western Mexico. Within this clade,
beatae and sacarensis formed a sister taxon relationship that was strongly supported (bootstrap of 100 and
Bremer support index of 24). The remaining taxa (undescribed taxa from western Mexico, levipes, and ambiguus) formed a clade that had moderate support
(bootstrap of 67 and Bremer support index of 4).
Maximum-likelihood scores (-ln L) for the eight
equal-weighted parsimony trees ranged from 4384.00
to 4386.66 under the HKY85-⌫ model of evolution; ti/tv
estimates were 7.47 and the gamma value was 0.117. A
heuristic maximum-likelihood search was performed
using the HKY85 model of evolution assuming a
gamma distribution of 0.117 and a ti/tv rate of 7.47.
This analysis depicted the same tree topology as that of
the equally weighted maximum-parsimony analysis
(Fig. 5), with one exception. The likelihood tree depicted a sister relationship between the beatae/sacarensis clade and the levipes (Sample 11)/ P. sp. (Sample
21) clade. This larger clade was then joined by the
levipes (Sample 29)/ambiguus clade. The neighbor-joining analysis based on Tamura–Nei distances
(gamma ⫽ 0.117) resulted in a topology identical to
that of the parsimony analyses (Fig. 5).
DISCUSSION
Identification of Species Groups
The distinctiveness of the boylii species group relative to the aztecus and truei groups was well supported
in the parsimony, distance, and likelihood analyses, as
each group formed a monophyletic clade. However,
similar to the allozyme study of Sullivan et al. (1991),
the weighted parsimony analyses (downweighting of
transitions) depicted a sister taxon relationship of the
TABLE 2
Comparison of Tree Lengths and Maximum-Likelihood Scores (HKY85-⌫ Model) for Tree Topologies
in Which the Different Species Groups Were Constrained to Form a Monophyletic Clade
Constraint type
Tree length
P1
Likelihood score
P2
aztecus/boylii vs boylii/truei
aztecus/boylii vs aztecus/truei
boylii/truei vs aztecus/truei
1140 vs 1151
1140 vs 1146
1151 vs 1146
0.233
0.527
0.059
6527.10 vs 6520.25
6527.10 vs 6514.87
6520.25 vs 6514.87
0.596
0.415
0.238
Note. P 1 and P 2 denote the probability of getting a more extreme T value (Kishino–Hasegawa, 1989; two-tailed test) for no differences in
tree length and likelihood scores (-In L) between the unconstrained and the constrained trees. The truei constraint includes attwateri,
difficilis, gratus, nasutus, pectoralis, polius, sagax, and truei.
373
MOLECULAR PHYLOGENETICS OF Peromyscus boylii
clade was similar to the distance observed between
species groups (13.2 and 13.6%, respectively).
The formation of a basal polytomy among species
groups (truei, boylii, and aztecus) in this study might
be interpreted either as a lack of resolving power of the
cytochrome b gene to identify dichotomous relationships at this level of divergence or as a paraphyly that
requires the additional analysis of taxa from other
closely related species groups to be resolved. An alternative interpretation involves the idea of a burst of
recent speciation occurring in Peromyscus resulting in
the simultaneous diversification of numerous lineages.
The presence of a polytomy at a certain level of divergence with good resolution of branching events above
and below that level favors the interpretation of a rapid
radiation, rather than a lack of resolution of the data
(Lara et al., 1996; Lessa and Cook, 1998). The polytomous nature of the phylogeny of species groups and
TABLE 3
Average Estimates Calculated from Tamura–Nei Genetic Distances of Selected Clades or Taxa Represented by Multiple Samples
Type of comparison
FIG. 5. Phylogeny obtained for members of the Peromyscus boylii
species group utilizing P. pectoralis as an outgroup. Characters were
weighted equally and transitions were downweighted by a factor of 2.
Bootstrap values are above branches and Bremer support indices are
below.
aztecus and boylii group, although the weak nodal support for this relationship obscured any confidence that
can be placed in this association. Support for an aztecus/truei relationship was depicted in the likelihood
and distance analyses (Figs. 3 and 4), although this
relationship collapsed during bootstrap analyses. Additionally, when constraining the aztecus/boylii, aztecus/truei, and boylii/truei clades to form monophyletic
units, none of the constrained trees possessed a significantly better likelihood score (Table 2). Further evidence regarding the differentiation between boylii and
aztecus as two species groups was provided by comparing the average genetic distances (Tamura–Nei) between and within species groups. This approach provided a means of comparison of the degree of genetic
divergence between the boylii and the aztecus groups.
The average sequence divergence within each group
was considerably less (7.0 and 7.8%, respectively) than
the average genetic distance between members of each
species groups (11.1%; Table 3). In comparison, the
average divergence of species within the attwateri/difficilis/nasutus clade was 8.1%, whereas divergence values between this clade and the boylii or the aztecus
Among species groups
boylii vs aztecus
boylii vs attwateri/difficilis/nasutus
aztecus vs attwateri/difficilis/nasutus
Within species group
boylii group
aztecus group
attwateri/difficilis/nasutus
Between species
simulus vs boylii
simulus vs stephani
simulus vs madrensis
boylii vs stephani
boylii vs madrensis
beatae vs boylii
levipes vs boylii
beatae vs stephani
P. sp. FN ⫽ 54–56 (17–20) vs levipes (29)/ambiguus
P. sp. FN ⫽ 54–56 (17–20) vs P. sp. (21)/levipes (11)
P. sp. FN ⫽ 54–56 (17–20) vs P. sp. FN ⫽ 65–68 (22)
P. sp. FN ⫽ 65–68 (21) vs levipes (29)/ambiguus
P. sp. FN ⫽ 65–68 (21) vs levipes (11)
P. sp. FN ⫽ 65–68 (22) vs levipes (29)/ambiguus
P. sp. FN ⫽ 65–68 (22) vs levipes (11)
levipes (11) vs levipes (29)
Between subspecies
levipes (29) vs ambiguus
boylii vs glasselli
boylii vs utahensis
boylii vs rowleyi (9)
boylii vs rowleyi (5–8, 25, 26)
rowleyi (5–8, 25, 26) vs rowleyi (9)
beatae vs sacarensis
Average
(%)
11.1
13.2
13.6
7.0
7.8
8.1
2.7
4.2
3.8
4.2
3.8
8.6
8.8
9.8
3.4
3.4
3.2
2.9
2.2
3.5
3.6
3.2
1.4
1.8
0.5
0.3
1.8
1.5
1.3
Note. Averages within species groups were estimated considering
only pairwise comparisons of genetic distances among species.
374
TIEMANN-BOEGE ET AL.
taxa in the subgenus Peromyscus with resolution at
higher taxonomic ranks was obtained in a comprehensive study of chromosomes (Rogers et al., 1984; Stangl
and Baker, 1984).
Utilizing O. banderanus as an outgroup resulted in
the resolution of taxa at the subgeneric level (P. californicus, P. leucopus, and P. melanotis; data not
shown) and the specific level (members of the boylii
and aztecus group) but not at the intermediate speciesgroup level. These observations favor the interpretation that the observed polytomy might represent a
simultaneous radiation of the truei, aztecus, and boylii
species groups instead of an artifact of the evolution of
the cytochrome b gene. Results from other studies have
shown good resolution among rodent lineages, with
sequence divergence ranging from 3 to 21% (Smith and
Patton, 1991; DeWalt et al., 1993b). Given the 7–14%
divergence among the taxa (truei species group, pectoralis, sagax, and polius) forming this polytomy, adequate variation should exist to resolve the placement of
these taxa.
This and previous studies provide sufficient evidence
that attwateri is more closely related to difficilis and
nasutus than to members of the boylii group (Janecek,
1990; Sullivan et al., 1991; DeWalt et al., 1993a), warranting the removal of attwateri from the boylii species
group and its placement in the truei group. However,
this study did not support the monophyletic association
of attwateri, difficilis, and nasutus with other members
of the truei group (truei and gratus). Instead, the DNA
sequence data support a division of the truei group into
a difficilis and a truei component, a position that is
congruent with allozymic (Janecek, 1990; Sullivan et
al., 1991) and karyotypic (Stangl and Baker, 1984)
data. A more appropriate interpretation of the truei
group is to recognize difficilis and truei assemblages,
analogous to the treatment of the aztecus and boylii
groups by Carleton (1977). However, to satisfactorily
address this question is beyond the scope of this study,
as not all components of the truei group were included
and the relationship among members of the truei group
was polyphyletic.
The tentative placement of pectoralis, polius, and
sagax within the boylii species group as proposed by
Carleton (1989) and Bradley et al. (1996) was not supported in this study. There is some support from likelihood and neighbor-joining analyses for the affiliation
of pectoralis and polius with the truei species group. In
addition, pectoralis, polius, and sagax were genetically
as divergent from the boylii group (12.6, 12.3, and
13.1%, respectively) as the other species groups (aztecus and truei) were from the boylii group (11.1 and
13.2%, respectively; Table 2). Sufficient evidence has
accumulated (Avise et al., 1974b; Kilpatrick and Zimmerman, 1975; Schmidly et al., 1985; Rogers et al.,
1984; Stangl and Baker, 1984; Bradley et al., 1996) to
corroborate that these taxa should not be placed in the
boylii species group. For example, interpretations of
chromosomal inversion patterns placed pectoralis
among species of the P. mexicanus group (Rogers et al.,
1984; Stangl and Baker, 1984), whereas allozymic comparisons affiliated pectoralis with P. eremicus of the
subgenus Haplomylomys (Avise et al., 1974b; Sullivan
et al., 1991) or with representatives of the truei group
(Zimmerman et al., 1975). Similarly, Hoffmeister
(1951) provisionally assigned polius to the boylii group,
although he suggested its possible affiliation with the
melanophrys group, whereas Zimmerman et al. (1975),
using allozyme data, could not determine whether polius was affiliated with either the boylii or the truei
species group. Likewise, comparisons of morphological,
karyotypic, and allozymic data did not resolve the relationship of sagax to the boylii or the truei species
group (Bradley et al., 1996).
Phylogenetic Relationships within the boylii Species
Group
The phylogenies of members of the boylii species
group obtained in this study consistently depicted the
sister taxon relationship between boylii and simulus,
with the stepwise addition of stephani and madrensis.
The relationship of boylii and simulus is supported by
data from DNA sequences of the D-loop (Castro-Campillo et al., 1999). The levels of average sequence divergence of simulus versus boylii from the Mexican Plateau (Samples 5– 8, 25, and 26) were among the lowest
values (2.7%) obtained for among-species comparisons
(Table 3), whereas sequence divergence between madrensis and boylii or stephani and boylii was 3.8 and
4.2%, respectively. Compared to other studies of rodents, values for among-species sequence divergence
found for oryzomyines ranged from 5 to 17% and variation within species was as high as 3.0% (Myers et al.,
1995). Smith and Patton (1991) reported among-species divergence values between Akodon and Microxus
of 3 to 15% and conspecific sequence divergence values
of 0.25 to 5%. Sullivan et al. (1997) depicted values for
the aztecus species group of less than 3.5% for withinspecies comparisons, whereas among-species comparison values ranged from 4.5 to 9%. A possible explanation for the low divergence values reported in this
study would be to assume that boylii and simulus were
isolated only recently or that the genetic rate of evolution for simulus is slower than that for other branches
in the boylii group. Genetic divergence of other species
examined in this and other studies, and the fact that
the karyotype of simulus is identical to that of northern
boylii (Schmidly and Schroeter, 1974; Carleton et al.,
1982), raises questions about the specific distinction
between simulus and boylii. However numerous studies support the validity of simulus and boylii as distinct
species. For example, simulus was elevated to specific
status based on its sympatric occurrence in Nayarit
with populations of spicilegus (Carleton, 1977; Carle-
MOLECULAR PHYLOGENETICS OF Peromyscus boylii
ton et al., 1982) and its distinct skull (Carleton et al.,
1982; Schmidly and Bradley, 1995) and phallus (Carleton et al., 1982), which approaches the level of distinction observed between boylii and madrensis. Allozymically, simulus was reported to be distinct from
rowleyi, beatae, and levipes (Sullivan et al., 1991; Bradley et al., 1996). Additionally, simulus is geographically
isolated from taxa with which it has been morphologically and karyotypically affiliated: rowleyi by a distance of approximately 2000 m of mountainous terrain
and madrensis, which is distributed on the Tres Marı́as
Islands. In light of this evidence, the hypothesis that
simulus is a valid species is well supported.
A close phylogenetic relationship of stephani and
madrensis to boylii was depicted by the cytochrome b
sequence data. The specific differentiation of stephani
is supported by allozymic data, which depicted a genetic distance between stephani and boylii similar to
that observed between attwateri and boylii (Avise et
al., 1974a,b). The average genetic distances of the cytochrome b sequences for madrensis and stephani to
rowleyi (4.2 and 3.8%, respectively; Table 3) were
within the range of among-species sequence divergences for this study (2.7 to 9.8%). P. stephani and
madrensis are island derivatives of a mainland boylii
and apparently, based on karyotypic and morphological evidence (Lawlor, 1971; Carleton et al., 1982), were
isolated recently from a parental boylii stock. Therefore, the recognition of stephani and madrensis as distinct species in the boylii species group is well supported.
P. beatae and levipes form a sister taxon relationship
depicted by a clade distinct from the clade containing
simulus, boylii, stephani, and madrensis. Based on
support from cytochrome b and D-loop sequence data
(Castro-Campillo et al., 1999; Bradley et al., 2000) and
to some extent from allozymic (Rennert and Kilpatrick,
1987; Sullivan et al., 1991; Bradley et al., 1996) and
morphological (Bradley and Schmidly, 1987; CastroCampillo et al., 1999) data, it appears that levipes and
beatae shared a common ancestor following a divergence from boylii. Despite the allozymic (Rennert and
Kilpatrick, 1987), karyotypic (Houseal et al., 1987),
and morphological (Bradley and Schmidly, 1987;
Schmidly et al., 1988) evidence that supports the recognition of levipes and beatae as distinct species, Carleton (1989) retained beatae as a subspecies of boylii.
The sequence divergence between beatae and boylii
(8.6%) obtained for this study was similar to that between boylii and levipes (8.8%) and was larger than for
other comparisons among species of the boylii group.
Based on these results, the recognition of beatae and
levipes as distinct species is warranted.
Several studies have focused on the systematics and
phylogenetic relationships of “boylii taxa” in the western region of Mexico in the states of Durango, Jalisco,
Nayarit, and Michoacán (Houseal et al., 1987;
375
Schmidly et al., 1988; Bradley et al., 1989; Carleton et
al., 1982). These studies suggest that boylii forms with
atypical fundamental numbers found in that region
represent at least three undescribed taxa, although the
monophyletic affiliation of these atypical forms to the
boylii group has not been tested. In this study, the
phylogenetic analysis of these undescribed forms resulted in three separate clades affiliated with levipes.
The first clade contained undescribed forms found in
central and southwestern Durango, characterized by a
FN of 54 –56 (Samples 17–20). These forms appear to
be a distinct species based on three lines of evidence.
First, karyotypically, these specimens were characterized with a FN of 54 –56 (Lee et al., 1972; Schmidly and
Schroeter, 1974; Kilpatrick and Zimmerman, 1975;
Houseal et al., 1987). This karyotype differs substantially from those of simulus (FN ⫽ 52) and rowleyi
(FN ⫽ 52). Carleton (1977, 1989) reported that, in this
region, many of the boylii specimens classified by Osgood (1909) as spicilegus were ultimately not assignable to a given species (particularly boylii and levipes),
based on morphology or geographic range. Second, allozymic (Kilpatrick, 1984; Kilpatrick and Zimmerman,
1975) and phallic (Bradley et al., 1989) data supported
the differentiation of populations from Durango with a
FN of 54 –56 from other boylii taxa. Third, levels of
DNA sequence divergences among the undescribed
taxon from Durango (Samples 17–20) and other species
of the boylii group within the same clade (levipes/P. sp.
clade) were similar to other among-species comparisons in the boylii group (Table 3). In addition, the close
phylogenetic relationship of this undescribed taxon to
levipes implies its classification within the boylii species group. No name is available for this taxon.
The second clade was formed by a specimen from
Ocota, Nayarit (Sample 22). This sample was placed in
chromosomal Group IV, FN ⫽ 65– 68, by Houseal et al.
(1987). The genetic divergence between this form and
other species within the same clade (levipes/undescribed taxa clade) was similar to the values reported
for the undescribed taxon from Durango (Table 3).
Because this undescribed form is allopatric with other
members of the boylii species group, the occurrence of
reproductive isolation in sympatry cannot be used to
measure its specific distinctiveness, as has been the
case for other members of the group (Carleton, 1977;
Carleton et al., 1982; Schmidly et al., 1988). The morphological (Carleton, 1982; Bradley et al., 1996) and
karyotypic (Houseal et al., 1987) differentiation of the
populations from Nayarit with a FN of 65 to 68 from
other boylii taxa further supports that this taxon represents a second undescribed species. No name is available for this taxon.
The third clade was formed by the undescribed taxon
from Dos Aguas, Michoacán (Sample 21) and the sample of levipes from Amealco, Querétaro (Sample 11).
The interpretation of this association poses some diffi-
376
TIEMANN-BOEGE ET AL.
culty. Karyotypically, the specimen from Dos Aguas
(FN ⫽ 65– 68; Sample 21) is different from the levipes
from Amealco, Querétaro (FN ⫽ 58 – 60; Sample 11),
with these populations being placed into two distinct
chromosomal groups (Group IV and Group III, respectively; Houseal et al. 1987). Additionally, it is morphologically different (Bradley et al., 1989) from the other
FN ⫽ 65– 68 form from Nayarit (Sample 22). The same
study identified two distinct forms of levipes based on
presence or absence of spines on the phallus, although
the division of levipes into spined and spineless taxa
has not been recognized formally (Bradley et al., 1989).
Spineless populations were collected from the same
locality in Querétaro as levipes (Sample 11) of this
study. The question is why has the spineless levipes
been associated phylogenetically with the undescribed
taxon from Michoacán and what taxonomic rank
should these taxa be given? The genetic distance calculated between the undescribed form (Sample 21) and
levipes (Sample 11) was 2.2%. The sequence divergence
values between the undescribed form (Sample 21) and
the closest taxon (levipes 23/ambiguus) was 2.9% and
between levipes (Samples 23 and 24) was 3.2%. Based
on these divergence values, it is not likely that the
undescribed form (Sample 21) and levipes (Sample 11)
are differentiated sufficiently from other levipes taxa to
be considered a distinct species. The alternative is to
recognize the undescribed taxon (Sample 21) and levipes (Sample 11) as two additional subspecies of levipes,
but this classification would ignore the karyotypic differentiation of the undescribed high-FN form and contradicts the Houseal et al. (1987) distinction of chromosomal Groups III and IV. The solution to this dilemma
requires the analysis of more specimens from the
Transvolcanic Belt and the western coast of Mexico.
Evaluation of Phylogenetic Relationships among
Subspecies
The close relationship of ambiguus to levipes is supported by allozymic (Rennert and Kilpatrick, 1987),
morphologic (Bradley and Schmidly, 1987; Schmidly et
al., 1988; Castro-Campillo et al., 1999), and DNA sequence (Castro-Campillo et al., 1999) data and is congruent with the results obtained in this study. CastroCampillo et al. (1999) recognized ambiguus as an
eastern subspecies of levipes. Similarly, all subspecies
of boylii formed a monophyletic unit, except for sacarensis, which was affiliated with beatae. Karyotypically, sacarensis is more similar to beatae than to boylii
(Bradley and Ensink, 1987), and based on comparisons
of DNA sequences it was proposed as a subspecies of
beatae (Bradley et al., 2000). The remaining subspecies
of boylii (boylii, glasselli, rowleyi, and utahensis)
formed a monophyletic unit within which boylii and
utahensis and one sample of rowleyi (Sample 9) formed
a smaller unresolved clade. This clade possessed lower
levels of genetic differentiation than observed between
TABLE 4
Proposed Taxonomy of the Peromyscus boylii Species Group and Related Taxa Based on the Results
Obtained from DNA Sequences of the Cytochrome b
Gene
Proposed classification
P. boylii species group
P. boylii (boylii, glasselli, rowleyi, and utahensis)
P. beatae (beatae and sacarensis)
P. levipes (ambiguus and levipes)
P. madrensis
P. simulus
P. stephani
P. sp. nv. (Durango, FN ⫽ 52–54)
P. sp. nv. (Nayarit, FN ⫽ 65–68)
P. sp. nv. (Michoacán, FN ⫽ 65–68)
P. aztecus species group
P. aztecus (aztecus, evides, and oaxacensis)
P. hylocetes
P. spicilegus
P. winkelmanni
P. truei species group
P. difficilis assemblage
P. attwateri
P. difficilis
P. nasutus
P. truei assemblage
P. gratus
P. truei
incertae sedis
P. pectoralis
P. polius
P. sagax
Note. Subspecies are listed in parentheses. General localities and
karyotypic information are provided to assist in distinguishing
among the three P. sp. nv, for which no names are currently available.
other subspecies comparisons (e.g., levipes and ambiguus; rowleyi and glasselli). This genetic homogeneity among subspecies in the southwestern United
States can be explained in two ways: (1) gene flow
occurs among boylii, rowleyi, and utahensis from California, Nevada, and Utah or (2) samples for boylii and
utahensis for this study occurred near the boundary of
rowleyi and it is possible that all of these specimens
represent rowleyi. If the latter statement is true then
the northern rowleyi are genetically different from
southern populations of rowleyi. Populations of rowleyi
from the southwestern United States occur in montane
pine and oak forests, whereas populations found on the
Mexican Plateau are common in grasslands and cactus–acacia scrub habitats (Schmidly et al., 1988). This
ecological isolation may explain the distinctiveness of a
northern and a southern form of rowleyi.
Taxonomic Implications
A summary of the results obtained in this study
(Table 4) includes the formation of a monophyletic
MOLECULAR PHYLOGENETICS OF Peromyscus boylii
clade referred to as the boylii species group and comprises the following taxa: simulus, boylii, beatae, levipes, madrensis, stephani, and three undescribed taxa
from western Mexico. The elevation to specific status of
two of the three undescribed forms is recommended in
this study. Only four of the five subspecies of boylii
should remain under the boylii epithet (utahensis, boylii, glasselli, and rowleyi); sacarensis should be placed
within beatae. The placement of ambiguus as a subspecies of levipes is corroborated in this study. P. attwateri
is associated with difficilis and should be removed from
the boylii species group and placed in the truei species
group. Similarly, pectoralis, polius, and sagax should
be removed from the boylii species group. Although
some analyses depicted a relationship between these
taxa and the truei group, we recommend that they be
treated as incertae sedis until data are available for all
members of the truei group. At this point it is unclear
whether the truei species group as classically defined is
a valid species group due to its polyphyletic nature. A
proposed taxonomical treatment of the truei group
would be to recognize attwateri, difficilis, and nasutus
as one assemblage and truei and gratus as a second
assemblage within the truei species group. The aztecus,
boylii, and truei species groups were distinct from each
other but the relationships among these were unresolved.
ACKNOWLEDGMENTS
We thank R. J. Baker, C. Jones, C. W. Edwards, D. S. Carroll, and
M. L. Clary for previous comments on the manuscript. Special
thanks go to R. J. Baker (Museum, Texas Tech University), Zadock
Thompson Natural History Collections, University of Vermont, M. D.
Engstrom (Royal Ontario Museum), J. W. Bickham (Texas Cooperative Wildlife Collection, Texas A&M University), J. L. Patton (University of California, Berkeley), M. A. Bogan (Museum of Southwestern Biology, University of New Mexico), R. D. Fisher (National
Museum of Natural History, Smithsonian Institution), and P. Myers
(Museum of Zoology, University of Michigan) for tissue loans. S. A.
Smith, K. M. Davis, T. W. Houseal, J. Ensink, D. Hale, P. D. Rennert,
D. Werbitsky, J. A. Markert, J. M. Sullivan, P. Nunez, and M. Allard
assisted in collection of specimens. This research was supported by
grants from the National Science Foundation (DEB 81-17447 to
D.J.S. and DEB 81-18996 and VT-EPSCoR to C.W.K.), the National
Institutes of Health (DHHS A141435-01 to R.D.B.), and the Department of Biological Sciences and Graduate School of Texas Tech
University (minigrant and summer research award to I.T-B.).
REFERENCES
Avise, J. C., Smith, M. H., and Selander, R. K. (1974a). Biochemical
polymorphism and systematics in the genus Peromyscus. VI. The
boylii species group. J. Mammal. 55: 751–763.
Avise, J. C., Smith, M. H., Selander, R. K., Lawlor, T. E., and
Ramsey, P. R. (1974b). Biochemical polymorphism and systematics in the genus Peromyscus. V. Insular and mainland species of
the subgenus Haplomylomys. Syst. Zool. 23: 226 –238.
Avise, J. C., Smith, M. A., and Selander, R. K. (1979). Biochemical
polymorphism and systematics in the genus Peromyscus VII. Geo-
377
graphic differentiation in members of the truei and maniculatus
species groups. J. Mammal. 60: 177–192.
Bibb, M. J., Van Etten, R. A., Wright, C. T., Walberg M. W., and
Clayton, D. A. (1981). Sequence and gene organization of mouse
mitochondrial DNA. Cell 26: 167–180.
Bradley, R. D., and Ensink, J. (1987). Karyotypes of five cricetid
rodents from Honduras. Texas J. Sci. 39: 171–175.
Bradley, R. D., and Schmidly, D. J. (1987). The glans penes and
bacula in Latin American taxa of the Peromyscus boylii group. J.
Mammal. 68: 595– 612.
Bradley, R. D., Schmidly, D. J., and Kilpatrick, C. W. (1996). The
relationships of Peromyscus sagax to the P. boylii and P. truei
species groups in Mexico based on morphometric, karyotypic, and
allozymic data. In “Contributions in Mammalogy: A Memorial
Volume in Honor of J. Knox Jones, Jr.” (H. H. Genoways and R. J.
Baker, Eds.), pp. 95–106, The Museum of Texas Tech University,
Lubbock.
Bradley, R. D., Schmidly, D. J., and Owen, R. D. (1989). Variation in
the glans penes and bacula among Latin American populations of
the Peromyscus boylii species complex. J. Mammal. 70: 712–725.
Bradley, R. D., Tiemann-Boege, I., Kilpatrick, C. W., and Schmidly,
D. J. (2000). Taxonomic status of Peromyscus boylii sacarensis:
Inferences from DNA sequences of the mitochondrial cytochrome b
gene. J. Mammal., in press.
Bremer, K. (1994). Branch support and tree stability. Cladistics 10:
295–304.
Carleton, M. D. (1977). Interrelations of populations of Peromyscus
boylii species group (Rodentia: Muridae) in western Mexico. Occas.
Papers Mus. Zool. Univ. Michigan 675: 1– 47.
Carleton, M. D. (1989). Systematics and evolution. In “Advances in
the Study of Peromyscus (Rodentia)” (G. L. Kirkland, Jr. and J. N.
Layne, Eds.), pp. 7–141. Texas Tech Univ. Press, Lubbock.
Carleton, M. D., Wilson, D. E., Gardner, A. L., and Bogan, M. A.
(1982). Distribution and systematics of Peromyscus (Mammalia:
Rodentia) from Nayarit, Mexico. Smithsonian Contrib. Zool. 352:
1– 46.
Castro-Campillo, A., Roberts, H. R., Ramirez-Pulido, J., Schmidly,
D. J., and Bradley, R. D. (1999). The systematic status of Peromyscus boylii ambiguus based on morphological and molecular data.
J. Mammal. 80: 1214 –1231.
DeWalt, T. S., Zimmerman, E. G., and Planz, J. V. (1993a). Mitochondrial-DNA phylogeny of species of the boylii and truei groups
of the genus Peromyscus. J. Mammal. 74: 352–362.
DeWalt, T. S., Sudman, P. D., Hafner, M. S., and Davis, S. K.
(1993b). Phylogenetic relationships of pocket gophers (Cratogeomys and Pappogeomys) based on mitochondrial DNA cytochrome b
sequences. Mol. Phylogenet. Evol. 2: 193–204.
Eriksson, T. (1997). Autodecay. Version 3.03. Botaniska Institutionen, Stockholm Univ. Stockholm.
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791.
Gadaleta, G., Pepe, G., De Candia, G., Quagliariello, C., Sbisà, E.,
and Saccone, C. (1989). The complete nucleotide sequence of the
Rattus norvegicus mitochondrial genome: Cryptic signals revealed
by comparative analysis between vertebrates. J. Mol. Evol. 28:
497–516.
Hafner, M. S., Gannon, W. L., Salazar-Bravo, J., and Alvarez-Castañeda, S. T. (1997). “Mammal Collections in the Western Hemisphere: A Survey and Directory of Existing Collections,” Allen
Press, Lawrence, KS.
Hall, E. R. (1981). “The Mammals of North America,” Vol. II, Wiley,
New York.
Hasegawa, M., Kishino H., and Yano T. (1985). Dating of the hu-
378
TIEMANN-BOEGE ET AL.
man–ape splitting by a molecular clock of mitochondrial DNA. J.
Mol. Evol. 21: 160 –174.
Hoffmeister, D. F. (1951). A taxonomic and evolutionary study of the
piñon mouse, Peromyscus truei. Illinois Biol. Monogr. 21: 1–104.
Hooper, E. T. (1968). Classification. In “Biology of Peromyscus (Rodentia)” (J. A. King, Ed.), pp. 27–74. Special Publication 2, Am.
Soc. Mammal.
Houseal, T. W., Greenbaum, I. F., Schmidly, D. J., Smith, S. A., and
Davis, K. M. (1987). Karyotypic variation in Peromyscus boylii
from Mexico. J. Mammal. 68: 281–296.
Irwin, D. M., Kocher, T. D., and Wilson, A. C. (1991). Evolution of the
cytochrome b gene in mammals. J. Mol. Evol. 32: 37–55.
Janecek, L. L. (1990). Genic variation in the Peromyscus truei group
(Rodentia: Cricetidae). J. Mammal. 71: 301–308.
Kilpatrick, C. W. (1984). Molecular evolution of the Texas mouse,
Peromyscus attwateri. In “Festschrift for Walter W. Dalquest in
Honor of his Sixty-Sixth Birthday” (H. Horner, Ed.), pp. 87–96,
Midwestern State Univ., Wichita Falls, TX.
Kilpatrick, C. W., and Zimmerman, E. G. (1975). Genetic variation
and systematics of four species of mice of the Peromyscus boylii
species group. Syst. Zool. 24: 143–162.
Kishino, H., and Hasegawa, M. (1989). Evaluation of the maximum
likelihood estimate of the evolutionary tree topologies from DNA
sequence data, and the branching order in Hominoidea. J. Mol.
Evol. 29: 170 –179.
Lara, M. C., Patton, J. L., and da Silva, M. N. (1996). The simultaneous diversification of South American echimyid rodents (Hystricognathi) based on complete cytochrome b sequences. Mol. Phylogenet. Evol. 5: 403– 413.
Lawlor, T. E. (1971). Evolution of Peromyscus on northern islands in
the Gulf of California, Mexico. Trans. San Diego Soc. Nat. Hist. 16:
91–124.
Lee, M. R., Schmidly, D. J., and Huheey, C. C. (1972). Chromosomal
variation in certain populations of Peromyscus boylii and its systematic implications. J. Mammal. 53: 697–707.
Lessa, E. P., and Cook, J. A. (1998). The molecular phylogenetics of
tuco-tucos (genus Ctenomys, Rodentia: Octodontidae) suggests an
early burst of speciation. Mol. Phylogenet. Evol. 9: 88 –99.
Maddison, W. P., and Madisson, D. R. (1992). MacClade 3.04. Sinauer, Sunderland, MA.
Myers, P., Lundrigan, B., and Tucker, P. K. (1995). Molecular phylogenetics of oryzomyine rodents: The genus Oligoryzomys. Mol.
Phylogenet. Evol. 4: 372–382.
Osgood, W. H. (1909). Revision of the mice of the American genus
Peromyscus. N. Am. Fauna 28: 1–285.
Pääbo, S., Gifford, J. A., and Wilson, A. C. (1988). Mitochondrial
DNA sequences from a 7000-year old brain. Nucleic Acids Res. 16:
9775–9787.
Rennert, P. D., and Kilpatrick, C. W. (1986). Biochemical systematics
of Peromyscus boylii. I. Populations from east-central Mexico with
low fundamental numbers. J. Mammal. 67: 481– 488.
Rennert, P. D., and Kilpatrick, C. W. (1987). Biochemical systematics
of Peromyscus boylii. II. Chromosomally variable populations from
eastern and southern Mexico. J. Mammal. 68: 799 – 811.
Robbins, L. W., and Baker, R. J. (1981). An assessment of the nature
of rearrangements in eighteen species of Peromyscus (Rodentia:
Cricetidae) Cytogenet. Cell Genet. 31: 194 –202.
Rogers D. S., Greenbaum, I. F., Gunn, S. J., and Engstrom, M. D.
(1984). Cytosystematic value of chromosomal inversion data in the
genus Peromyscus. J. Mammal. 65: 457– 465.
Saiki, R. K., Gelfand, D. H., Stoeffl, S., Scharf, S. J., Higuchi, R.,
Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed
enzymatic amplification of DNA with thermostable DNA polymerase. Science 239: 487– 491.
Schmidly, D. J., and Bradley, R. D. (1995). Morphological variation
in the Sinaloan Mouse Peromyscus simulus. Rev. Mex. Mast. 1:
44 –58.
Schmidly, D. J., and Schroeter, G. L. (1974). Karyotypic variation of
Peromyscus boylii (Rodentia, Cricetidae) from Mexico and corresponding taxonomic implications. Syst. Zool. 23: 333–342.
Schmidly, D. J., Bradley, R. D., and Cato, P. S. (1988). Morphometric
differentiation and taxonomy of three chromosomally characterized groups of Peromyscus boylii from east-central Mexico. J.
Mammal. 69: 462– 480.
Schmidly, D. J., Lee, M. R., Modi, W. S., and Zimmerman, E. G.
(1985). Systematics and notes on the biology of Peromyscus hooperi. Occas. Papers Mus. Texas Tech. Univ. 97: 1– 40.
Smith, M. F., and Patton, J. L. (1991). Variation in mitochondrial
cytochrome b sequence in natural populations of South American
akodontine rodents (Muridae: Sigmodontinae). Mol. Biol. Evol. 8:
85–103.
Smith, S. A. (1990). Cytosystematic evidence against monophyly of
the Peromyscus boylii species group (Rodentia: Cricetidae). J.
Mammal. 71: 654 – 667.
Smith, S. A., Greenbaum, I. F., Schmidly, D. J., Davis, K. M., and
Houseal, T. W. (1989). Additional notes on karyotypic variation in
the Peromyscus boylii species group. J. Mammal. 72: 681– 696.
Stangl, F. B, Jr., and Baker, R. J. (1984). Evolutionary relationships
in Peromyscus: Congruence in chromosomal, genic, and classical
data sets. J. Mammal. 65: 643– 654.
Sullivan, J. M., and Kilpatrick, C. W. (1991). Biochemical systematics of the Peromyscus aztecus assemblage. J. Mammal. 72: 681–
696.
Sullivan, J. M., Kilpatrick, C. W., and Rennert, P. D. (1991). Biochemical systematics of the Peromyscus boylii species group. J.
Mammal. 72: 669 – 680.
Sullivan, J. M, Markert, J. A., and Kilpatrick, C. W. (1997). Phylogeography and molecular systematics of the Peromyscus aztecus
species group (Rodentia: Muridae) inferred using parsimony and
likelihood. Syst. Biol. 46: 426 – 440.
Swofford, D. L. (1999). PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4.0b2.0a. Sinauer, Sunderland, MA.
Tamura, K., and Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in
humans and chimpanzees. Mol. Biol. Evol. 10: 512–526.
Watrous, L. E., and Wheeler, Q. D. (1980). The outgroup comparison
method of character analysis. Syst. Zool. 30: 1–11.
Yang, H., Golenberg, E. M., and Shoshani, J. (1997). Proboscidean
DNA from museum and fossil specimens: An assessment of ancient
DNA extraction and amplification techniques. Biochem. Genet. 35:
165–179.
Yang, Z., Goldman, N., and Friday, A. (1994). Comparison of models
for nucleotide substitution used in maximum-likelihood phylogenetic estimation. Mol. Biol. Evol. 11: 316 –324.
Zimmerman, E. G., Hart, B. J., and Kilpatrick, C. W. (1975). Biochemical genetics of the boylii and truei groups of the genus Peromyscus (Rodentia). Comp. Biochem. Physiol. 52: 541–545.