Content uploaded by Gerardo Ceballos
Author content
All content in this area was uploaded by Gerardo Ceballos on Mar 25, 2014
Content may be subject to copyright.
Genetic divergence of Microtus pennsylvanicus chihuahuensis and
conservation implications of marginal population extinctions
RURIK LIST,* OLIVER R. W. PERGAMS,JESU
´SPACHECO,JUAN CRUZADO,AND GERARDO CEBALLOS
Instituto de Ecologı
´a Universidad Nacional Auto´noma de Mexico, Apartado Postal 70-275, Ciudad Universitaria, 04510
Me´xico Distrito Federal, Mexico (RL, JP, JC, GC)
Department of Biological Sciences, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7060,
USA (ORWP)
* Correspondent: rlist@miranda.ecologia.unam.mx
Microtus pennsylvanicus is represented in Mexico only by the Chihuahuan meadow vole (M. p. chihuahuensis),
known from only 1 disjunct population in a small and isolated marsh in the arid lands of northern Chihuahua.
Livetrapping conducted between 2000 and 2004 provided no specimens of M. p. chihuahuensis, nor was any
sign of this vole observed. By the end of this study the marsh providing water had been drained, thereby
destroying the vole’s habitat. Surveys of other marshes in northern Chihuahua also failed to produce evidence of
the species. We therefore conclude that M. p. chihuahuensis has been extirpated from its only known locality.
Using ‘‘ancient’’ DNA from museum specimens we evaluated genetic divergence between museum specimens
of M. p. chihuahuensis and 46 extant Microtus species and subspecies. Our results support the subspecific status
of M. p. chihuahuensis. The loss of this subspecies is an example of population extinction, a very severe form of
biodiversity loss. Until recently such losses have been mostly neglected. DOI: 10.1644/09-MAMM-A-168.1.
Key words: ancient DNA, biodiversity loss, Chihuahua, marginal populations, meadow vole, Microtus, mitochondrial
DNA, population extinctions
E2010 American Society of Mammalogists
The importance of marginal populations for the conserva-
tion of species has been widely debated. Marginal populations
are separated spatially from central or core populations and
tend to be found in suboptimal habitats. These populations
have a greater risk of extinction than core populations (Lienert
et al. 2002; Tomback et al. 2005). However, investment of
resources for conservation of continuous marginal populations
is criticized in favor of conservation efforts in core distribution
areas (Bunnell et al. 2004). This contradicts available evidence
from some species in which contraction and fragmentation of
their geographic ranges has affected both marginal and core
populations at global scales. Furthermore, focusing on core
populations does not take into account the loss of populations,
which at local scales is as important as species loss (Ceballos
and Ehrlich 2002). Yet, populations with peripheral distribu-
tion are more tolerant of disturbance and tend to be
substantially different from core populations in traits such as
genetics, behavior, life history, or size (Gaines et al. 1997;
Garavello et al. 1998; Snyder and Peterson 1999). Sometimes
these differences accrue rapidly, over centuries or even
decades rather than geologic time (Ashley et al. 2003; Hendry
and Kinnison 1999; Pergams and Kareiva 2009). This means
that even relatively recently diverged populations may deserve
conservation attention for their intrinsic value (Ehrlich 1988;
Joyal et al. 2000). More important, marginal populations often
differ genetically from other populations of the same species,
even after a relatively short time of separation, playing a role
in allopatric speciation (Fritz et al. 2006; Huang et al. 2005;
Waters et al. 2000). This is particularly relevant for
conservation because loss of marginal populations implies
the loss of genetic diversity, the loss of that portion of the gene
pool that is not present in other parts of the range of a species
(Eckstein et al. 2006; Johannesson and Andre´ 2006). Some
studies have reported range contraction (Channell and
Lomolino 2000). Overall, however, we ignore the magnitude
of genetic diversity we lose when marginal populations
disappear as a consequence of geographic range contraction
due to anthropogenic causes. We use the Chihuahuan meadow
vole (M. pennsylvanicus chihuahuensis) as a case study of a
marginal population to assess genetic divergence and
determine current conservation status at the southern portion
of the species’ range.
www.mammalogy.org
Journal of Mammalogy, 91(5):1093–1101, 2010
1093
The meadow vole (Microtus pennsylvanicus) inhabits moist
habitats (Reich 1981). It is represented in Mexico by 1
subspecies, the Chihuahuan meadow vole (M. p. chihuahuen-
sis), known from only 1 disjunct population. This population is
found in a small and isolated marsh in the arid lands of
northern Chihuahua, approximately 700 km south of the core
range of the species and 400 km south-southeast from the
closest relict population (both in the United States—Anderson
and Hubbard 1971). Like most mammals it was designated a
subspecies on the basis of morphology (Bradley and Cockrum
1968). It represents the most geographically restricted species
of mammal in Mexico and is considered endangered
(Secretarı
´a del Medio Ambiente y Recursos Naturales 2002),
but its current status has been considered unknown (Ceballos
2007; Ceballos et al. 1998). Hence, the objectives of our
research were to determine its current status, if M. p.
chihuahuensis maintained its morphologically assigned sub-
specific status using metrics of genetic divergence and
distinction from its most closely related taxa, and a
conservation strategy if the subspecies still existed.
MATERIALS AND METHODS
Study area.—The study area is located in the northwest
portion of the Chihuahuan Desert, at 30u039320N, 107u359290W
and an altitude of 1,461 m (Galeana marsh; Fig. 1). The climate
is arid with hot summers and cold winters. Mean annual
temperature is 16.9uC (Garcı
´a 1973), with extremes ranging
between 212uC in winter and 48uC in summer. Annual
precipitation averages 294 mm, most of it occurring in July
and August and, to a lesser extent, during the winter
(Rzedowski 1981). The area is a small (34-ha), isolated marsh
surrounded by grasslands and desert scrub dominated by
Prosopis sp. The vegetation of the marsh consists of sedges
and rushes, partly immersed in water in the lowest parts of the
marsh (Bradley and Cockrum 1968). In 2000 the marsh was fed
from 2 springs that surfaced near the base of a hill and were
separated from each other by 300 m. The longest spring formed
a natural pond 45 m long and 32 wide and ran into a stream for
2,340 m, the marsh being 30–260 m wide along the stream. The
shortest stream was channeled at the spring into 3 consecutive
artificial pools built by local people. After leaving the pools the
water continued along a stream for 320 m, blending with the
marsh, which ranged from 87 to 200 m wide. The depth of the
ponds surpassed 80 cm in some places. In 2004 the only water
available was in 2 small ponds approximately 50 and 120 cm in
diameter and separated by 15 m, with an average water depth of
3.5 cm and greatest depth of 15 cm. In 2005 the marsh, streams,
and springs had completely disappeared. Three additional
marshes exist in the region. One was transformed into a
recreational area (Casas Grandes marsh; Fig. 1), but trapping
for voles was conducted in the other 2 (Ojitos and Ojo Caliente
marshes; Fig. 1) despite the absence of vole records.
Small mammal sampling.—We conducted our survey to
capture meadow voles during 5 sampling periods in May
2000, November 2002, February 2003, November 2003, and
September 2004. We used 120 Sherman traps (7.5 393
23 cm; H. B. Sherman Traps, Inc., Tallahassee, Florida) set in
linear transects within the flooding area. Trapping effort
consisted of a system of linear transects arranged in parallel
lines separated by 20 m. Trap interval was 10 m, and traps
were baited with a mixture of rolled oats and peanut butter. All
of the marsh was sampled. Traps were set from 3 to 5
consecutive days. All individuals collected or observed in the
area were identified in situ and then released. No mark–
recapture techniques were used, because the objective of the
study was solely to locate the Chihuahuan vole. The total
trapping effort consisted of 1,920 trap days. We complement-
ed our trap data with direct observations, conducting random
walks across the marsh and searching for runways. Animal
trapping and handling was conducted according to guidelines
approved by the American Society of Mammalogists (Animal
Care and Use Committee 1998).
Genetic analyses.—Four museum skins of M. p. chihua-
huensis—numbers 7231, 7134, 7138, and 7139—borrowed
from the University of Nevada at Las Vegas were sequenced
for this study. Museum tags show that all were collected by R.
Mauer and G. Austin 3 miles south of Galeana, Chihuahua,
Mexico, in 1964. Mitochondrial DNA (mtDNA) cytochrome-b
gene (Cytb) sequences from all Microtus species present
on GenBank on 17 August 2004 were downloaded. A total
of 46 Microtus species with complete (1,143 base pairs
[bp]) Cytb sequences was deposited in GenBank, plus Cytb
sequences of 5 Myodes species used as outgroups. Fortuitous-
ly, the GenBank M. pennsylvanicus sequence (accession
number AF119279) came from the subspecies geographically
closest to M. p. chihuahuensis,M. p. modestus, caught in San
Juan County, New Mexico (Conroy and Cook 2000) about
400 km north of our study site (Hoffmann and Koeppl 1985).
FIG.1.—Map of northwestern Chihuahua, Mexico, showing the
region’s marshes: 1) Galeana, 2) Casas Grandes, 3) Ojitos, and 4) Ojo
Caliente. The reported distribution of Microtus pennsylvanicus
chihuahuensis was limited to the Galeana marsh.
1094 JOURNAL OF MAMMALOGY Vol. 91, No. 5
For further comparison an individual of subspecies M. p.
pennsylvanicus was sequenced. This specimen was collected
in 2003 near State College, Pennsylvania, the approximate
center of the ranges of both the subspecies and the entire
species and about 3,000 km northeast of the location of M. p.
chihuahuensis.
Museum skins were sampled as follows. An approximately
1.5 312-mm strip was removed from ventral seam of the skin.
Because we found that proteins produced from hair during the
Chelex extraction interfered with the polymerase chain
reaction (PCR) process (Pergams et al. 2003; Pergams and
Lacy 2007), we shaved the strips of skin with single-edged
razors. Each strip was then minced into approximately 1.5-mm
squares. Museum DNA extraction was performed using a
Chelex 100 protocol (Walsh et al. 1991) modified by the
addition of proteinase K as in Steinberg (1999). However, we
performed trials to determine what proportion of proteinase K
was most effective and found that this proportion was 10 mlof
20 mg/ml proteinase K solution added to 490 mlof5%Chelex
solution (Pergams et al. 2003; Pergams and Lacy 2007). We
also found that freezing the resulting DNA-laden supernatant
to 20uC in a Boekel Polar Block model 260012 benchtop
cooler/heater (Boekel Scientific, Feasterville, Pennsylvania),
thawing the supernatant fully, and repeating the process 1 or 2
more times helped to separate out residual proteins and other
substances that seemed to inhibit PCR (Pergams et al. 2003).
We subjected the supernatant that remained after the
freeze–thaw process to phenol–chloroform extraction (Man-
iatis et al. 1982). The DNA pellet was resuspended in 20 mlof
1 mM TE buffer. Amplification was performed by PCR using
2 sets of 2 nested primers. All primers were designed using the
program Primer3 (Rozen and Skaletsky 2000) from GenBank
M. pennsylvanicus sequence AF119279. Primers MPC1 (59-
TCT TCG CCT TCC ACT TCA TT-39and MPC2 (59-CCT
GCG ATT GGC ATA AAG AT-39) were designed to amplify
577 bp. PCR was performed for 20 cycles with each 2-part
cycle 93uC for 1 min and 60uC for 20 min. We then performed
a nested PCR, moving in 25–100 bp from the ends of the
previous segment and using the previous PCR product as a
template. Primers MPC5 (59-TCC CAC CGG TCT AAA CTC
AG-39) and MPC8 (59-GGT TGA CCA CCG ATT CAT GT-
39) amplified 405 bp. Nested PCR is useful with very low
numbers of target templates (van Pelt-Verkuil et al. 2008).
PCR was performed for 22 cycles with each cycle 93uC for
1 min and 65uC for 20 min. Important were the use of KlenTaq
LA DNA polymerase and PCR buffer (DNA Polymerase
Technology, Inc., St. Louis, Missouri), because 59-exonucle-
ase–deficient Taq polymerase provides improved fidelity and
thermostability (Barnes 1992, 1995; Cheng et al. 1994;
Korolev et al. 1995), the long extension cycles (Barnes
1994), and the use of betaine (Barnes 1994). Bovine serum
albumin was added to prevent proteins from further inhibiting
PCR (Pa¨a¨ bo et al. 1988; Thomas et al. 1990). PCR products in
agarose check gels were stained with ethidium bromide and
viewed under an ultraviolet transilluminator. Single bands
resulted for most runs for most samples, but not when bands of
the appropriate width (based on size standards) were cut from
the gels with a single-edge razor blade. These gel slices were
processed using Montage Gel Extraction Kits (Millipore, Inc.,
Billerica, Massachusetts). PCR product was cleaned with
QIAquick PCR Purification Kits (Qiagen, Inc., Valencia,
California) and quantified using a GeneQuant RNA/DNA
Calculator spectrophotometer (Amersham Pharmacia Biotech,
GE Healthcare UK Ltd., Buckinghamshire, United Kingdom).
Sequencing was performed on an ABI PRISM 3100 Genetic
Analyzer (Life Technologies Corp., Carlsbad, California) at
the University of Illinois at Chicago DNA Sequencing
Facility. Sequences were aligned with the program ClustalW
(Higgins et al. 1994). The program DnaSP (Rozas et al. 2003)
was used to format sequence files and to examine synonymous
and nonsynonymous substitutions. Microtus species geneti-
cally closest to M. pennsylvanicus (based on the entire Cytb
sequence) were determined by constructing phylogenetic trees
using all Microtus species in GenBank. Unweighted pair-
group method with arithmetic mean, neighbor-joining, mini-
mum-evolution, and maximum-parsimony methods (using 2-
parameter distances—Kimura 1980) were constructed using
Mega2 (Kumar et al. 2001). These genetically closest species
were then compared to the 3 M. pennsylvanicus subspecies,
again using 4 tree-building methods. However, simple p-
distances instead of Kimura 2-parameter distances were used
because of the reduced amount of divergence.
RESULTS
Disappearance of Microtus pennsylvanicus chihuahuen-
sis.—In June of 1988 we visited the type locality and saw 2 M.
pennsylvanicus. At that time the marsh had been partly
transformed into a recreation area with 2 swimming pools. In
2000 the swimming pools were still filled by water from the
spring that fed the marsh, but the surrounding vegetation, where
the specimens of M. p. chihuahuensis had been collected in 1968
and observed in 1988, was heavily overgrazed. That year we
observed what seemed to be runways of this species, but no
specimens were captured in .120 trap days (day being a 24-h
period). In addition, despite intensive trapping efforts, no
specimens were captured in 2002, 2003, or 2004. In 2003 the
condition of the marsh had improved and the flooded vegetation
had recovered, because cattle had been excluded from the
swimming pool and the surrounding areas. In 2004, however,
several center-pivot irrigation systems had been installed in
croplands located .2 km away from the marsh. As a result the
stream had completely disappeared, and the marsh was reduced to
a few small and scattered ponds. The recreational area was closed
and decaying. In 2005 the former marsh was entirely gone;
exposed soil and dry grasses had replaced it. No trapping was
conducted, because appropriate habitat for the meadow vole was
absent. We saw no sign of the species (Fig. 2). Our effort in the
other marshes was more limited: 400 trap days in Ojo Caliente in
September 2000 and 800 trap days in Ojitos in 2000 and 2003.
Taxonomic status.—Bradley and Cockrum (1968) described
M. p. chihuahuensis on the basis of morphology. We tested
October 2010 LIST ET AL.—MEADOW VOLE AND POPULATION EXTINCTIONS 1095
whether genetic differences corroborated a subspecies desig-
nation. A neighbor-joining tree was constructed from 46
Microtus species in GenBank, plus 5 Myodes species used as
outgroups. M. p. modestus and M. montanus form a clade, and
with M. canicaudus and M. townsendii form a larger clade
(Fig. 3). Similar to what Conroy and Cook (2000) reported,
identical topologies for these 4 species were obtained using
unweighted pair-group method with arithmetic mean, mini-
mum-evolution, and maximum-parsimony methods. These 4
genetically most similar species then were compared to M. p.
chihuahuensis and M. p. pennsylvanicus.
For all museum specimens 379 bp were sequenced. All 4
museum skins yielded the same haplotype (GenBank acces-
sion number GU177626). A comparison with the sequence for
M. p. modestus shows only 3 substitutions along this length (p
50.00782), all of which are 3rd-position and synonymous.
The genetic distances between M. p. modestus or M. p.
chihuahuensis to M. p. pennsylvanicus are about 3.5 times as
great (range of pairwise p50.026–0.029), but the
substitutions are still all synonymous. The distances from M.
p. chihuahuensis to M. montanus,M. canicaudus, and M.
townsendii are about 10 times as great (range of pairwise p5
0.071–0.090). A neighbor-joining tree using only these taxa
further illustrates these relationships (Fig. 4). Again, identical
topologies for these 6 taxa were obtained using unweighted
pair-group method with arithmetic mean, minimum-evolution,
and maximum-parsimony methods. Also, we note that there
were 1 or 2 nonsynonymous substitutions between M. p.
chihuahuensis and M. montanus and M. townsendii, although
none were found between M. p. chihuahuensis and M.
canicaudus.
DISCUSSION
The genetic divergences we found are consistent with
divergences noted in coding mitochondrial genes among other,
known murid rodent subspecies. Using mtDNA restriction
fragment length polymorphisms, Plante et al. (1989) found
divergences of 0.007–0.045 in M. p. pennsylvanicus,M. p.
drummondi, and M. p. aphorodemus from Canada. However,
restriction fragment length polymorphism divergence, even
when corrected, is not fully comparable to nucleotide sequence
divergence. Fink et al. (2004) found a divergence range of
0.007–0.248 in 1,044 bp of Cytb among populations of Mictotus
arvalis spanning almost all of Europe, which must have
included a number of subspecies (26 total subspecies are found
in Europe—Niethammer and Krapp 1982). A slightly reduced
subset of these specimens resulted in a slightly narrower
divergence range of 0.010–0.019 (Haynes et al. 2003). Within
the species Peromyscus maniculatus, 6 known California
Channel Island and adjoining mainland subspecies exhibited a
mean pof 0.00714 using 603 bp of cytochrome c oxidase
subunit II (Pergams and Ashley 2000; Pergams et al. 2000), a
value essentially identical to the 0.00782 divergence we
calculated between M. p. chihuahuensis and M. p. modestus.
Were M. p. chihuhuensis to be considered a separate
species, divergence probably should have been greater. For
comparison we have 2 recent and comprehensive works on
interspecific Cytb divergence between a large number of
Microtus species, Conroy and Cook (2000), cited earlier, and
Jaarola et al. (2004). Among the 46 Microtus species used in
Conroy and Cook (2000), divergence in 1,143 bp of Cytb
ranged from 0.015 (M. abbreviatus and M. miurus) to 0.18 (M.
FIG.2.—Habitat transformation of the Chihuahuan meadow vole
marsh in Galeana, Chihuahua, Mexico, from A) 2000 to B) 2003 to
C) 2006.
1096 JOURNAL OF MAMMALOGY Vol. 91, No. 5
FIG.3.—Neighbor-joining tree using Kimura 2-parameter distances (scale at bottom) and 1,143 base pairs of the cytochrome-bgene from 46
Microtus species (with genus abbreviated as M.) plus 5 Myodes species as outgroups. Microtus pennsylvanicus (modestus) and M. montanus
form clade 1, and with M. canicaudus and M. townsendii form a larger clade 2. These 4 genetically most-similar species were compared to M. p.
chihuahuensis and M. p. pennsylvanicus in this study, and identical topologies for these 4 species were obtained using unweighted pair-group
method with arithmetic mean, minimum-evolution, and maximum-parsimony methods. Numbers after species are GenBank accession numbers.
October 2010 LIST ET AL.—MEADOW VOLE AND POPULATION EXTINCTIONS 1097
oregoni and M. gregalis). Jaarola et al. (2004) found a similar
divergence range of 0.042–0.180 in 1,140 bp of Cytb among
49 Microtus species. Considering the outcomes of these
studies, we conclude that M. p. chihuahuensis is genetically
distinct enough to be considered a subspecies, thereby
corroborating its morphological assignment. Although genet-
ically closely related to the geographically closest subspecies,
M. p. modestus,M. p. chihuahuensis nevertheless is geneti-
cally irreplaceable.
Between 2002 and 2004 we found 7 species of small
mammals in the Galeana marsh, compared to 5 species found
in 1968. Only the deer mouse (Peromyscus maniculatus) was
trapped within the marsh in both studies (Table 1). We did not
catch any Chihuahuan voles, nor did we see any sign of them.
Including the marsh and adjacent dry grassland, we trapped 7
species that Bradley and Cockrum (1968) did not trap in either
of these habitats (Table 1). One was an introduced species, the
black rat (Rattus rattus). In the dry grassland we trapped 1
species associated with surrounding scrub, Merriam’s kanga-
roo rat (Dipodomys merriami). In addition to the Chihuahuan
vole, we did not trap 3 species (Reithrodontomys montanus,
Reithrodontomys megalotis, and Sigmodon hispidus) previ-
ously reported in the marsh and dry grassland. We also
collected 3 species of fish from the springs and streams of
Galeana: Tex-Mex gambusia (Gambusia speciosa), black
bullhead (Ameiurus melas), and whitefin pupfish (Cyprinodon
albivelis), an undescribed species at that time (Minckley et al.
2002). The red shiner (Cyprinella lutrensis), which was
collected in the springs by Edwards et al. (2003), was not
found during our study. During fieldwork we also observed
mud turtles (Kinosternon flavescens) and many bullfrogs
(Rana catesbeiana), the latter an introduced species. During
the 2005 visit, after the area had dried out, the only signs of
the former aquatic life were buried skulls of A. melas.
The lack of success in trapping Chihuahuan meadow voles
in the Galeana marsh and the other marshes of the region
indicates the possibility that the subspecies was not present.
The disappearance of the Galeana marsh practically confirms
the extirpation of this population. Although an unknown
population could exist elsewhere in northwestern Mexico, this
possibility is remote, and for practical purposes this subspecies
is most likely extinct. The size of the marsh in the early part of
our study was similar to that reported by Bradley and
Cockrum (1968) 3 decades earlier. The replacement of rodent
FIG.4.—Neighbor-joining tree using p-distances (scale at bottom) and 379 base pairs of the cytochrome-bgene from Microtus pennsylvanicus
chihuahuensis,M. p. modestus,M. p. pennsylvanicus,M. montanus,M. canicaudus, and M. townsendii. Numbers after taxa are GenBank
accession numbers.
TABLE 1.—Changes in the small mammal community composition in the Ojo de Galeana marsh, Chihuahua, Mexico, from 1968 to 2004.
Rodentia species
Bradley and Cockrum (1968) This study (2000–2004)
Marsh Adjacent dry grass Marsh Adjacent dry grass
Heteromyidae
Dipodomys merriami X
Chaetodipus hispidus X
Chaetodipus intermedius X
Chaetodipus penicillatus X
Muridae
Microtus pennsylvanicus X
Neotoma albigula X
Onychomys torridus X
Peromyscus leucopus XXX
Peromyscus maniculatus XXXX
Reithrodontomys megalotis XX
Reithrodontomys montanus XX
Ratus rattus X
Sigmodon fulviventer XX
Sigmodon hispidus XX
No. species 5675
1098 JOURNAL OF MAMMALOGY Vol. 91, No. 5
species (compared to our earliest surveys) indicates that rodent
composition had changed to one more characteristic of
scrubland habitats. Thus, the decline of the Chihuahuan
meadow vole likely began before the drilling of wells for
center-pivot irrigation. The loss of a system of desert springs
always represents an important loss, but in this case the
wetland also was the only known lowland locality of the
whitefin pupfish (C. albivelis), and the only known locality
outside the Rı
´o Papigochic drainage (Minckley et al. 2002).
The establishment of wells and irrigation channels for the
growing town and agricultural area probably caused the loss of
the wetland. The diurnal nature of the Chihuahuan meadow
vole made it more sensitive to the development of a
recreational area in the springs and the increased human
activity and presence of pets. A subspecies that was isolated in
a very small but stable site for thousands of years was, not
surprisingly, sensitive to change. This supports the idea that
populations at the margins of species ranges are more
susceptible to extinction than core populations (Lienert et al.
2002; Tomback et al. 2005).
We have trapped the few other existing isolated marshes
between Galeana and the United States, but we have been
unable to trap or find evidence of other isolated populations of
M. p. chihuahensis in northern Chihuahua. Therefore, we
conclude that the Chihuahuan meadow vole has become
extinct in Mexico. In this case 1 or 2 deep wells caused the
disappearance of the desert spring that maintained the mesic
habitat of M. p. chihuahuensis in an otherwise arid region.
Similar accounts in the literature include that of Peromyscus
guardia from Estanque Island off Baja California, which
became extinct in only a few years as the result of the
introduction of a single cat (Va´ zquez et al. 2004). The
extirpation of M. pennsylvanicus from the only known locality
in Mexico increases to .50 the number of vertebrate species
that have become either extirpated or extinct in the country in
the last century (Ceballos and Oliva 2005).
The disappearance of M. p. chihuahuensis represents the
extinction of a subspecies and contraction of the geographic
range of the species with the loss of the extreme southern
component of its distribution. Because isolation is one of the
main sources of allopatric speciation (Bush 1975), the loss of
marginal or relict populations such as the Chihuahuan
meadow vole reduces not only the species richness of a
country or region but also the evolutionary potential of the
earth’s biota. Although the allocation of resources to the
conservation of marginal populations is controversial (Bunnell
et al. 2004), in the case of M. p. chihuahuensis the absence of
conservation efforts resulted in the extinction of an endemic
subspecies.
The loss of M. p. chihuahuensis is an example of population
extinction, a very severe form of biodiversity loss mostly
neglected until recently (Ceballos and Ehrlich 2002; Hughes et
al. 1997). According to Ceballos and Ehrlich (2002), millions
of populations have become extinct in recent decades due to
human activities. The loss of those populations reduces
morphological, genetic, and ecological diversity. Although
relatively little information exists about the magnitude of the
negative impacts of such population losses, it is well
established that population losses at a local or regional level
must be treated as total extinctions. Their disappearance
modifies the structure and function of communities and
ecosystems and the delivery of ecosystems services (Corlet
2007; Luk et al. 2003; Mayfield et al. 2005; S¸ ekerciog˘lu et al.
2004), regardless of the persistence of the same species
elsewhere.
The disappearance of a population often reduces the
geographic range of a species and makes it more vulnerable
to extinction both by human and natural causes. Many species
are distributed across political boundaries, either state or
national (e.g., Bunnell et al. 2004; Manzano-Fischer et al.
2006). This pattern of persistence of marginal populations is
repeated in other parts of the world (Burbidge and McKenzie
1989; Channell 1998; Channell and Lomolino 2000). Main-
taining marginal populations reduces the risk of global
extinction due to policy differences among countries, political
instability, economic trends, and other factors that are less
likely to manifest themselves similarly in 2 adjacent countries
(Ceballos and Ehrlich 2002). The Chihuahuan meadow vole is
a good example of the high vulnerability to extinction of
subspecies with both restricted geographic ranges and
marginal populations. It represents the species with the most
restricted geographic range of all mammals in Mexico. Its
extremely rapid extinction indicates the vulnerability to
extinction of range-restricted subspecies caused by anthropo-
genic causes.
RESUMEN
Microtus pennsylvanicus esta´ representado en Me´xico so´lo
por el metorito de Galeana (M. p. chihuahuensis), conocido
u´ nicamente de 1 poblacio´ n disyunta en un pantano pequen˜o y
aislado en las zonas a´ridas del norte de Chihuahua. Los
muestreos se realizaron entre 2000 y 2004, sin lograr la
captura de ningu´ n espe´cimen de M. p. chihuahuensis,nise
observo´ evidencia alguna de su presencia. Al final de este
estudio el pantano se habı
´a secado, desapareciendo comple-
tamente el ha´bitat del metorito. Muestreos en otros pantanos
en el noroeste de Chihuahua tampoco aportaron prueba de la
presencia de esta especie. Por lo que concluimos que M. p.
chihuahuensis ha sido extirpada de la u´nica localidad conocida
en Me´ xico. Se utilizo´ ADN de ejemplares de museo para
evaluar la divergencia gene´ tica entre M. p. chihuahuensis y
otras 46 especies y subespecies existentes de Microtus.
Nuestros resultados apoyan el estatus subespecı
´fico de M. p.
chihuahuensis.Lape´rdida de esta subespecie es un ejemplo de
la extincio´ n de una poblacio´ n y una forma muy severa de la
pe´ rdida de la biodiversidad. Hasta hace poco estas pe´rdidas
habı
´an sido menospreciadas.
ACKNOWLEDGMENTS
This research was supported by the J. M. Kaplan Fund and The
Nature Conservancy through P. Kareiva. We thank B. Vieyra, E.
October 2010 LIST ET AL.—MEADOW VOLE AND POPULATION EXTINCTIONS 1099
Ponce-Guevara, G. Santos, and R. Sierra for assistance with the
fieldwork. The comments from P. Ortega and 2 anonymous reviewers
helped to improve the manuscript.
LITERATURE CITED
ANDERSON, S., AND J. P. HUBBARD. 1971. Notes on geographic
variation of Microtus pennsylvanicus (Mammalia, Rodentia) in
New Mexico and Chihuahua. American Museum Novitates
2460:1–8.
ANIMAL CARE AND USE COMMITTEE. 1998. Guidelines for the capture,
handling, and care of mammals as approved by the American
Society of Mammalogists. Journal of Mammalogy 79:1416–1431.
ASHLEY, M. V., M. F. WILSSON,O.R.W.PERGAMS, D. J. O’DOWD,
S. M. GENDE,AND J. S. BROWN. 2003. Evolutionarily enlightened
management. Biological Conservation 111:115–123.
BARNES, W. M. 1992. The fidelity of Taq polymerase catalyzing PCR
is improved by an N-terminal deletion. Gene 112:25–29.
BARNES, W. M. 1994. Methods and reagents—tips and tricks for long
and accurate PCR reply. Trends in Biochemical Sciences 19:342.
BARNES, W. M. 1995. Thermostable DNA polymerase with enhanced
thermostability and enhanced length and efficiency of primer
extension. 1995 July 25. United States patent 5,436,149.
BRADLEY, W. G., AND E. L. COCKRUM. 1968. A new subspecies of the
meadow vole (Microtus pennsylvanicus) from northwestern
Chihuahua, Mexico. American Museum Novitates 2325:1–7.
BUNNELL, F. L., R. W. CAMPBELL,AND K. A. SQUIRES. 2004.
Conservation priorities for peripheral species, the example of
British Columbia. Canadian Journal of Forest Research 34:2240–
2247.
BURBIDGE, A. A., AND N. L. MCKENZIE. 1989. Patterns in the modern
decline of western Australia’s vertebrate fauna, causes and
conservation implications. Biological Conservation 50:143–198.
BUSH, G. L. 1975. Modes of animal speciation. Annual Review of
Ecology and Systematics 6:339–364.
CEBALLOS, G. 2007. Conservation priorities for mammals in
megadiverse Mexico: the efficiency of reserve networks. Ecolog-
ical Applications 17:569–578.
CEBALLOS, G., AND P. R. EHRLICH. 2002. Mammal population losses
and the extinction crisis. Science 296:904–907.
CEBALLOS, G., AND G. OLIVA. 2005. Los mamı
´feros silvestres de
Me´xico. CONABIO–Fondo de Cultura Econo´mica, Me´xico,
Distrito Federal, Me´xico.
CEBALLOS, G., P. RODRI
´GUEZ,AND R. MEDELLI
´N. 1998. Assessing
conservation priorities in megadiverse Mexico: mammalian
diversity, endemicity, and endangerment. Ecological Applications
8:8–17.
CHANNELL, R. 1998. A geography of extinction, patterns in the
contraction of geographic ranges. M.S. thesis, University of
Oklahoma, Norman.
CHANNELL, R., AND M. V. LOMOLINO. 2000. Dynamic biogeography
and conservation of endangered species. Nature 403:84–86.
CHENG S., C. FOCKLE,W.M.BARNES,AND R. HIGUCHI. 1994. Effective
amplification of long targets from cloned inserts and human
genomic DNA. Proceedings of the National Academy of Sciences
91:5695–5699.
CONROY, C. J., AND J. A. COOK. 2000. Molecular systematics of a
Holarctic rodent (Microtus: Muridae). Journal of Mammalogy
81:344–359.
CORLET, R. L. 2007. The impact of hunting on the mammalian fauna
of tropical Asian forests. Biotropica 39:292–303.
ECKSTEIN, R. L., R. A. O’NEILL,J.DANIHELKA,A.OTTE,AND W.
KO
¨LER. 2006. Genetic structure among and within peripheral and
central populations of three endangered foodplain violets. Molec-
ular Ecology 15:2367–2379.
EDWARDS, R. J., G. P. GARRETT,AND E. MARSH-MATTHEWS. 2003.
Conservation and status of the fish communities inhabiting the Rı
´o
Conchos basin and middle Rio Grande, Me´xico and U.S.A.
Reviews in Fish Biology and Fisheries 12:119–132.
EHRLICH, P. R. 1988. The loss of diversity: causes and consequences.
Pp. 21–27 in Biodiversity (E. O. Wilson and F. M. Peter, eds.).
National Academy Press, Washington, D.C.
FINK, S., L. EXCOFFIER AND G. HECKEL. 2004. Mitochondrial gene
diversity in the common vole Microtus arvalis shaped by historical
divergence and local adaptations. Molecular Ecology 13:3501–
3514.
FRITZ, U., M. BARATA,S.D.BUSACK,G.FRITASCH,AND R. CASTILHO.
2006. Impact of mountain chains, sea straits and peripheral
populations on genetic and taxonomic structure of a freshwater
turtle, Mauremys leprosa (Reptilia, Testudines, Geoemydidae).
Zoologica-Scripta 35:97–108.
GAINES, M. S., J. E. DIFFENDORFER,R.H.TAMARIN,AND T. S. WHITTAM.
1997. The effects of habitat fragmentation on the genetic structure
of small mammal populations. Journal of Heredity 88:294–304.
GARAVELLO, J. C., O. ROCHA,E.G.ESPINDOLA,A.C.RIETZLE,AND A.
C. LEAL. 1998. Diversity of fauna in the interdunal lakes of
‘‘Lencois Maranhenses’’, II, the ichthyofauna. Anais da Academia
Brasileira de Ciencias 70:797–803.
GARCI
´A, E. 1973. Modificaciones al sistema de clasificacio´n clima´tica
de Ko¨epen. Instituto de Geografı
´a, Universidad Nacional Auto´n-
oma de Me´xico, Me´xico, Distrito Federal, Me´xico.
HAYNES, S., M. JAAROLA,AND J. B. SEARLE. 2003. Phylogeography of
the common vole (Microtus arvalis) with particular emphasis on
the colonization of the Orkney archipelago. Molecular Ecology
12:951–956.
HENDRY, A. P., AND M. T. KINNISON. 1999. Perspective: the pace of
modern life, measuring rates of contemporary microevolution.
Evolution 53:1637–1653.
HIGGINS, D., J. THOMPSON,T.GIBSON,J.D.THOMPSON,D.G.HIGGINS,
AND T. J. GIBSON. 1994. ClustalW: improving the sensitivity of
progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Research 22:4673–4680.
HOFFMANN, R. S., AND J. W. KOEPPL. 1985. Zoogeography. Pp. 84–115
in Biology of New World Microtus (R. H. Tamarin, ed.). Special
Publication 8, The American Society of Mammalogists.
HUANG, Z., N. LIU,AND T. ZHOU. 2005. A comparative study of genetic
diversity of peripheral and central populations of chukar partridge
from northwestern China. Biochemical Genetics 43:613–621.
HUGHES, J. B., G. C. DAILY,AND P. R. EHRLICH. 1997. Population
diversity: its extent and extinction. Science 278:689–692.
JAAROLA, M., ET AL. 2004. Molecular phylogeny of the speciose vole
genus Microtus (Arvicolinae, Rodentia) inferred from mitochon-
drial DNA sequences. Molecular Phylogenetics and Evolution
33:647–663.
JOHANNESSON, K., AND C. ANDRE
´. 2006. Life on the margin: genetic
isolation and diversity loss in a peripheral marine ecosystem, the
Baltic Sea. Molecular Ecology 15:2013–2029.
JOYAL, L. A., M. MCCOLLOUGH,AND L. HUNTER-MALCOLM. 2000.
Population structure and reproductive ecology of Blanding’s turtle
(Emydoidea blandingii) in Maine, near the northeastern edge of its
range. Chelonian Conservation and Biology 3:580–588.
1100 JOURNAL OF MAMMALOGY Vol. 91, No. 5
KIMURA, M. 1980. A simple method for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide
sequences. Journal of Molecular Evolution 16:111–120.
KOROLEV, S., M. NAYAL,W.M.BARNES,E.DICERA,AND G. WAKSMAN.
1995. Crystal structure of the large fragment of Thermus aquaticus
DNA polymerase I at 2.5-angstrom resolution—structural basis for
thermostability. Proceedings of the National Academy of Sciences
92:9264–9268.
KUMAR, S., K. TAMURA,I.B.JAKOBSEN,AND M. NEI, M. 2001.
MEGA2, molecular evolutionary genetics analysis software.
Arizona State University, Tempe.
LIENERT, J., M. FISCHER,AND M. DIEMER. 2002. Local extinctions of
the wetland specialist Swertia perennis L. (Gentianaceae) in
Switzerland: a revisitation study based on herbarium records.
Biological Conservation 103:65–76.
LUK, G. W., G. C. DAILY,AND P. R. EHRLICH. 2003. Population
diversity and ecosystem services. Trends in Ecology & Evolution
18:331–336.
MANIATIS, T., E. F. FRITSCH,AND J. SAMBROOK. 1982. Molecular
cloning: a laboratory manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York.
MANZANO-FISCHER, P., R. LIST,G.CEBALLOS,AND J. L. E. CARTRON.
2006. Avian diversity in a priority area for the conservation of
North American grasslands: the Janos–Casas Grandes Prairie Dog
Complex in northwestern Mexico. Biodiversity and Conservation
15:3801–3825.
MAYFIELD, M. M., M. F. BONI,G.C.DAILY,AND D. ACKERLY. 2005.
Species and functional diversity of native and human-dominated
plant communities. Ecology 86:2365–2372.
MINCKLEY, W. L., R. R. MILLER,AND S. M. NORRIS. 2002. Three new
pupfish species, Cyprinodon (Teleostei, Cyprinodontidae) from
Chihuahua, Mexico and Arizona, USA. Copeia 2002:687–705.
NIETHAMMER, J., AND F. KRAPP (EDS.). 1982. Microtus arvalis (Pallas
1779)—Feldmaus. Pp. 282–318 in Handbuch der Sa¨ugetiere
Europas Band 2/I Rodentia II. Akademische Verlagsgessellschaft
Wiesbaden, Wiesbaden, Germany.
PA
¨A
¨BO, S., J. A. GIFFORD,AND A. C. WILSON. 1988. Mitochondrial
DNA sequences from a 7000-year old brain. Nucleic Acids
Research 16:9775–9787.
PERGAMS, O. R. W., AND M. V. ASHLEY. 2000. California Island deer
mice, genetics, morphometrics, and evolution. Pp. 278–288 in
Proceedings of the Fifth California Islands Symposium 29 March to
1 April 1999 (D. R. Browne,K. L. Mitchell, and H. W. Chaney, eds.).
Santa Barbara Museum of Natural History, Santa Barbara, California.
Sponsored by the United States Minerals Management Service,
Pacific Outer Continental Shelf Region. OCS Study 99-0038.
PERGAMS, O. R. W., W. M. BARNES,AND D. NYBERG. 2003. Rapid
change of mouse mitochondrial DNA. Nature 423:397.
PERGAMS, O. R. W., AND P. KAREIVA. 2009. Support services: a focus
on genetic diversity. Pp. 642–651 in The Princeton guide to
ecology (S. A. Levin et al., eds.). Princeton University Press,
Princeton, New Jersey.
PERGAMS, O. R. W., AND R. C. LACY. 2007. Rapid morphological and
genetic change in Chicago-area Peromyscus. Molecular Ecology
17:450–463.
PERGAMS, O. R. W., R. C. LACY,AND M. V. ASH LEY.2000.
Conservation and management of Anacapa Island deer mice.
Conservation Biology 14:819–832.
PLANTE, Y., P. T. BOAG,AND B. N. WHITE. 1989. Macrogeographic
variation in mitochondrial DNA of meadow voles (Microtus
pennsylvanicus). Canadian Journal of Zoology 67:158–167.
REICH,L.1981.Microtus pennsylvanicus. Mammalian Species
159:1–6.
ROZAS, J., J. C. SA
´NCHEZ-DEL BARRIO,X.MESSEGUER,AND R. ROZAS
LIRAS. 2003. DnaSP, DNA polymorphism analyses by the
coalescent and other methods. Bioinformatics 19:2496–2497.
ROZEN, S., AND H. J. SKALETSKY. 2000. Primer3 on the WWW for
general users and for biologist programmers. Pp. 365–386 in
Bioinformatics methods and protocols: methods in molecular
biology (S. Krawetz and S. Misener, eds.). Humana Press, Totowa,
New Jersey.
RZEDOWSKI, J. 1981. Vegetacio´n de Me´xico. Limusa, Mexico City,
Mexico.
S¸ EKERCIOG
˘LU, C. H., G. C. DAILY,AND P. R. EHRLICH. 2004. Ecosystem
consequences of bird declines. Proceedings of the National
Academy of Sciences 101:18042–18047.
SECRETARI
´A DEL MEDIO AMBIENTE Y RECURSOS NATURALES. 2002.
NORMA Oficial Mexicana NOM-059-ECOL-2001, proteccio´n
ambiental—especies nativas de Me´xico de flora y fauna silves-
tres—categorı
´as de riesgo y especificaciones para su inclusio´n,
exclusio´n o cambio—lista de especies en riesgo. Diario Ofical de la
Federacio´n 582:1–80.
SNYDER, D. J., AND M. S. PETERSON. 1999. Life history of a peripheral
population of bluespotted sunfish Enneacanthus gloriosus (Hol-
brook), with comments on geographic variation. American
Midland Naturalist 141:345–357.
STEINBERG, E. K. 1999. Characterization of polymorphic microsatel-
lites from current and historic populations of North American
pocket gophers (Geomydidae, Thomomys). Molecular Ecology
8:1075–1092.
THOMAS, E. H., S. PA
¨A
¨BO,F.X.VILLABLANCA,AND A. C. WILSON. 1990.
Spatial and temporal continuity of kangaroo rat populations shown
by sequencing mitochondrial DNA from museum specimens.
Journal of Molecular Evolution 31:101–112.
TOMBACK, D. F., A. W. SCHOETTLE,K.E.CHEVALIER,AND C. A. JONES.
2005. Life on the edge for limber pine: seed dispersal within a
peripheral population. Ecoscience 12:519–529.
VAN PELT-VERKUIL, E., A. VAN BELKUM,AND J. P. HAYS. 2008.
Principles and technical aspects of PCR amplification. Springer,
New York.
VA
´ZQUEZ, E., G. CEBALLOS,AND J. CRUZADO. 2004. Extirpation of an
insular subspecies by a single introduced cat: the case of the
endemic deer mouse Peromyscus guardia on Estanque Island,
Mexico. Oryx 38:347–350.
WALSH P. S., D. A. METZGER,AND R. HIGUCHI. 1991. ChelexH100 as a
medium for simple extraction of DNA for PCR-based typing from
forensic material. BioTechniques 10:506–513.
WATERS, J., J. M. EPIFANIO,T.GUNTER,AND B. L. BROWN. 2000.
Homing behaviour facilitates subtle genetic differentiation among
river populations of Alosa sapidissima: microsatellites and
mtDNA. Journal of Fish Biology 56:622–636.
Submitted 13 May 2009. Accepted 8 April 2010.
Associate Editor was David L. Reed.
October 2010 LIST ET AL.—MEADOW VOLE AND POPULATION EXTINCTIONS 1101
