systematics and biogeography of the great basin pocket
TRANSCRIPT
UNLV Retrospective Theses & Dissertations
1-1-1994
Systematics and biogeography of the Great Basin pocket mouse, Systematics and biogeography of the Great Basin pocket mouse,
Perognathus parvus Perognathus parvus
Carolyn Sue Ferrell University of Nevada, Las Vegas
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Systematics and Biogeography of the Great Basin Pocket Mouse,
Perognathus parvus
by
Carolyn Sue Ferrell
A thesis submitted in partial fulfillment of the requirements for the degree of
Master of Science
in
Biology
Department of Biological Sciences University of Nevada, Las Vegas
May 1995
DMI Number: 1374875
UMI Microform 1374875 Copyright 1995, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI300 North Zeeb Road Ann Arbor, MI 48103
The thesis of Carolyn S. Ferrell for the degree of Master of Science in Biology is approved.
Chairperson, Brett R. Riddle, Ph.D.
Examining Committee Member, Peter Starkweather, Ph.D.
/ /Examining Committee Member, Stanley Smith, Ph.D.
Graduate facu lty Representative, Rodney Metcalf, Ph.D.
Interim Dean of the Graduate College, Cheryl Bowles, Ed . D .
University of Nevada, Las Vegas May 1995
ABSTRACT
The Great Basin pocket mouse, Perognathusparvus, has a distribution that
extends across three geological regions: Columbia Plateau, Great Basin and Colorado
Plateau. Limited ability of P. parvus to disperse over large areas and across areas of
unsuitable habitat provide a foundation for studying biogeographic vicariance hypotheses
related to its distribution. Vicariance hypotheses involving late Tertiary geotectonic
events and dispersal hypotheses of Pleistocene glacial-interglacial climatic cycles that may
be responsible for phylogeographic structure in P. parvus were tested using mitochondrial
DNA (mtDNA) sequences and Restriction Fragment Length Polymorphisms (RFLPs).
High levels of sequence divergence support the hypothesis of late Tertiary isolation of
Columbia Plateau from Great Basin populations of P. parvus due to late Miocene uplift of
the Blue Mountains. Other species exhibit distributions similar to P. parvus and may show
the same type of phylogeographic disjunction between populations from the Columbia
Plateau and populations from the Great Basin.
Table of Contents
A B ST R A C T ............................................................................................................................... iii
LIST OF TABLES ........................................................................................................................ v
LIST OF FIGU RES.................................................................................................................... vi
ACKNOWLEDGEMENTS........................................................................................................ vii
CHAPTER 1 INTRODUCTION ............................................................................................... 1Perognathus p a rv u s ............................................................ 5Fossil Record of Heteromyids and Geologic H istory ...................................................9Molecular Data ................................................................................................................14
CHAPTER 2 MATERIALS AND METHODS ....................................................................17Specimen Collection........................................................................................................ 17DNA Extraction............................................................................................................... 17Polymerase Chain Reaction ...........................................................................................19DNA Sequences............................................................................................................... 20Restriction Fragment Length Polym orphism s............................................................ 21
CHAPTER 3 RESULTS .......................................................................................................... 26Sequence R esu lts .............................................................................................................26RFLP R esults....................................................................................................................31
CHAPTER 4 DISCUSSION ................................................................................................... 46Future R esearch............................................................................................................... 52Conclusions...................................................................................................................... 53
CHAPTER 5 Perognathus alticola and P. xanthonotus ..................................................... 55
APPENDIX I ...............................................................................................................................60
BIBLIOGRAPHY........................................................................................................................ 61
LIST OF TABLES
Table 1 Karyotype characteristics of P. p a r v u s .....................................................................10Table 2 Summary of nucleotide base substitutions................................................................27Table 3 Genetic distance matrix from mtDNA sequences ..................................................30Table 4 Composite haplotypes of specimens from the Great Basin/Colorado Plateau . . 32Table 5 Composite haplotypes of specimens from the Columbia P la te a u ........................ 35Table 6 Matrix of genetic distances and standard errors: Great Basin/Colo. Plat 37Table 7 Matrix of genedc distances and standard errors: Columbia Plateau ................. 41Table 8 Haplotype and nucleotide diversity values .............................................................44Table 9 Monte Carlo simulation X2 values and P-values ....................................................45Table 10 Karyotype characteristics of P. alticola and P. xan th o n o tu s .............................. 56
v
LIST OF FIGURES
Figure 1 Distribution of sagebrush-steppe and g rass land ........................................................6Figure 2 Distribution of Perognathus parvus .......................................................................... 7Figure 3 Phylogenetic tree from Riddle (1995) ................................................................... 28Figure 4 Estimated phylogeny of exemplars from Riddle (1995) ..................................... 29Figure 5 Distribution of haplotypes ........................................................................................ 36Figure 6 UPGMA dendrogram of Great Basin/Colorado Plateau haplotypes...................38Figure 7 UPGMA dendrogram of Columbia Plateau haplotypes ....................................... 40Figure 8 Distribution of haplotypes and composite populations ....................................... 43Figure 9 Distribution of Perognathus alticola and P. xanthonotus ..................................... 57
ACKNOWLEDGEMENTS
This study was conducted from January 1992 to January 1995.1 would like to
thank the members of my graduate committee, Dr. Brett Riddle, Dr. Peter Starkweather,
Dr. Stan Smith and Dr. Rodney Metcalf, for providing input on this thesis and for taking
the time to be on my committee. I would also like to thank Jeff Drumm, Bruce Ferrell
and Tyler Ferrell for helping me in the field, David Nickle and David Orange for
laboratory assistance as well as useful discussions about the research, and I would
especially like to thank Jeff Drumm for moral support throughout the progress of this
research. Thanks to CMBS and BBMilan for their hospitality and good food.
This research was funded by NSF grant BSR-9107520 awarded to Dr. Brett
Riddle and other grants awarded to Carolyn Ferrell by the Graduate Student Association
of the University of Nevada, Las Vegas; a Theodore Roosevelt Memorial Fund Grant
from the American Museum of Natural History; a Summer Research Enhancement Grant
and a Travel Grant both from the National Science Foundation, EPSCoR Women in
Science.
Approval for experimentation on vertebrate animals was obtained from the
Institutional Animal Care and Use Committee of the University of Nevada, Las Vegas
(Protocol # R701-1290-051).
Chapter 1
Introduction
Systematics is defined as "the scientific study of kinds of diversity of organisms
and of any and all relationships among them" (Simpson, 1961). A major task of
systematics is to classify organisms in a logical fashion and determine through
comparison what the unique properties of taxa are and also what properties taxa have in
common. Included in this task is the attempt to recover phylogenetic relationships
among organisms and to reconstruct a phylogeny based on these unique and common
properties. Phylogenetic systematics (cladistics-Hennig, 1966) uses branching points on
a phylogenetic tree to explain the evolutionary process of speciation (cladogenesis) while
ignoring anagenesis (Mayr and Ashlock, 1991). Cladistics has become the major
approach to recovering phylogenetic relationships among organisms (Brooks, 1981;
Brooks, 1990; Brooks and McLennan, 1991; Brooks and Wiley, 1985; Crisci and
Stuessy, 1980; Farris, 1983; Felsenstein, 1983; Miyamoto and Cracraft, 1991; Nelson
and Platnick, 1981; Wiley, 1981; Wiley et al., 1991).
Systematics has uses that are important not only in the recovery of phylogenies,
but in other fields of biology as well by providing a classification scheme for
populations, species and higher taxa used by all other scientists in biology. Without this
classification scheme, it would be impossible to explain interactions that-are studied in
1
ecology and behavior (Brooks and McLennan, 1991).
The nature of species has been a controversial subject for many years. Early
definitions of species used similarity of types as the criterion. Later, Buffon developed
the idea of sterility as a basis for species (Mayr and Ashlock, 1991). His idea then
became the principle on which the Biological Species Concept (BSC) was developed.
The BSC requires that species be made up of reproductive communities that are isolated
from one another. A major problem with this concept is that it does not allow for a test
for reproductive barriers in allopatric populations. A more fundamental problem is that
reproductive isolation is not necessarily concordant with phylogenetic relationships
(McKitrick and Zink, 1988).
Other concepts that have been developed have not used a biological criterion but
have been based on types. The Typological Species Concept (TSC) states that there are
several universal types. This concept does not reflect relationships among organisms,
and it does not take into account phenotypic plasticity or sexual dimorphism within a
species. The Nominalistic Species Concept (NSC) states that only individuals exist and
that species are abstractions created by people. The NSC precludes any type of
classification, and is therefore useless to biologists trying to determine patterns of
behavior or distribution (Mayr and Ashlock, 1991).
The species concepts that have become most useful to systematists are the
Evolutionary Species Concept (ESC) and the Phylogenetic Species Concept (PSC)
(Avise, 1994; Avise and Ball, 1990; Cracraft, 1983). The ESC states that a species is a
lineage that is evolving separately from others and has it's own unitary evolutionary role
3
and tendencies. This concept does not use reproductive criteria and so can include
allopatric populations. However, it does not take into account genetic variation among
populations. Such populations would be considered separate lineages and therefore
separate species. The PSC defines a species as a monophyletic group composed of "the
smallest diagnosable cluster of individual organisms within which there is a parental
pattern of ancestry and descent" (Cracraft, 1983). A monophyletic group is one in which
all members can be traced back to a single most recent common ancestor. This definition
seems to be the one most accepted by phylogenetic systematists. It too has problems, but
the requirement of monophyly is a fundamental component of cladistic principles. The
issue of monophyly involves the problem of determining what type of evidence is
required to diagnose a monophyletic group. Shared, derived characters, or
synapomoiphies, are the only type of characters that can be used to define a
monophyletic group. Shared ancestral characters can not be used in the construction of a
phylogeny because the common ancestor of the clade may not truly be the most recent
common ancestor.
Biogeography is the study of distributions of organisms both past and present
(Brown and Gibson, 1983). Biogeographers attempt to understand and describe patterns
of distribution, limits to distribution, and the roles of climate and topography in
determining the distribution of an organism (Brown and Gibson, 1983; Vrba, 1992).
Early biogeographers noticed several basic distributional patterns, including: the
observation that all taxa are endemic or restricted to a given area; that certain kinds of
organisms tend to occur together; and that similar kinds of organisms sometimes occur in
widely separated areas (Brown and Gibson, 1983). The underlying goal of modem
biogeography is to explain the causes of these patterns. Several scientists have made
contributions to the task of explaining distributions, most notably Charles Darwin.
Darwin (1859) made observations of biological variation between organisms from
separate islands in the Galapagos. He explained that this variation was due to natural
selection on certain traits that were advantageous to individual populations on different
islands. A. R. Wallace observed gaps in the distributions of animals in Southeast Asia.
As he got further south, the fauna changed and became more like the fauna in Australia
(Brown and Gibson, 1983). Both Darwin's and Wallace's observations involve a
dispersal explanation to account for the distributional patterns.
Explanations of biogeographic patterns became more robust in the 1960s and
1970s due to the acceptance of the theory of plate tectonics and continental drift (Brown
and Gibson, 1983). Acceptance of this theory made it possible for distributions to be
explained by vicariance events rather than dispersal. Vicariance events (i.e. movements
of tectonic plates, orogeny, river formation, desertification, glaciation) isolate once
continuous populations from each other, thus preventing gene flow between the two new
populations. Prolonged isolation will eventually lead to a unique evolutionary trajectory
for each of these populations and may result in speciation via an allopatric or peripatric
mode.
Phylogeography involves the study of the distribution of geographically localized
genealogical lineages and the processes governing those distributions (Avise, 1994).
Nuclear genotypes and mtDNA haplotypes are geographically localized in several
5
species. In general, differences in the ability of organisms to disperse and fragmentation
of suitable habitat exert strong influences on patterns of mtDNA phylogeographic
structure, and localization of mtDNA haplotypes often reflects limited gene flow among
populations (Avise, 1994). Intrinsic factors influencing distributional patterns of mtDNA
haplotypes also include rates of evolution. Rates of evolution can significantly affect the
amount of diversity and divergence in a taxon. MtDNA evolves at a rate faster than most
other single copy DNA (Vawter and Brown, 1986), but within the mtDNA genome,
different genes are evolving at different rates (Riddle, 1995). Therefore, sampling of
different gene regions or different genomes may reveal different patterns of diversity and
divergence within a single taxon.
Perognathus parvus
The genus Perognathus (family Heteromyidae, subfamily Perognathinae) consists
of arid-adapted rodents that are endemic to North America and whose range extends
throughout the desert shrub-steppe and grassland-steppe communities of the west (Figure
1) (Hall, 1981; Schmidley et al., 1993). Perognathus parvus is a geographically
widespread species that is distributed throughout the Great Basin, Columbia Plateau and
onto the Colorado Plateau (Figure 2).
The Perognathus parvus species group was originally thought to comprise the
species Perognathus parvus, P. alticola, and P. xanthonotus (Osgood, 1900; Williams,
1978) However, it is now postulated that P. xanthonotus is a subspecies of P. parvus
based on biochemical data (Sulentich, 1983; Williams, 1993).
The Great Basin pocket mouse was originally described by Peale in 1848 as
6
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re
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istrib
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h-st
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assla
nd
in w
este
rn
North
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7
Gre
at B
asin
, an
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to the
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do
Plat
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8
Cricetodipus parvus, based on a holotype from Oregon (Merriam, 1889). It has
subsequently had a complex nomenclatural history. Baird distinguished Perognathus
from Cricetodipus based on body size and whether or not there was fur on the soles of
the feet. Osgood (1900) assigned the name Perognathus parvus based on Peale's
description and the fact that the Wilkes expedition, of which Peale was a member, and
the location where Baird caught his specimen were determined to be the same.
Most research conducted on Perognathus parvus has consisted of ecological and
physiological studies (Cramer and Chapman, 1990; French, 1993; Kenagy and Banes,
1984; O'Farrell et al., 1975; Price and Brown, 1983; Small and Verts, 1983; Verts and
Carraway, 1986). Patton (1967) and Williams (1978) studied the karyological affinities of
P. parvus; but did not formally propose inter- or intraspecific relationships. No studies
have addressed the historical biogeography of this widespread species.
Patton (1967) first described the karyotype of P. parvus without suggesting any
phylogenetic hypotheses about it's evolutionary history relative to other members of the
genus Perognathus or intraspecific relationships among subspecies. Williams (1978)
examined the karyotypes of several subspecies of P. parvus as well as P. alticola and P.
xanthonotus, which were at the time considered closely related sister taxa and members
of the Parvus species group. He found that P. parvus columbianus from the Columbia
Basin had a karyotype that was extremely divergent from the karyotypes of P. parvus
subspecies that had been collected in the Great Basin (Table 1). The high fundamental
number (FN) and a submetacentric rather than an acrocentric Y chromosome in P. p_arvus
columbianus but not other subspecies of P. parvus represent derived traits, and indicate
9
Table 1. Karyotype characteristics of Perognathus parvus spp. from the Columbia Plateau and Great Basin/Colorado Plateau (Williams, 1978). Haplotype of P. p. columbianus is divergent from other subspecies of P. parvus based on high fundamental number and X and Y chromosome. 2N=diploid number, FN=fundamental number, BA=biarmed chromosome, A=uniarmed chromosome, ST=subtelocentric, SM=submetacentric, A=acrocentric.
Autosomal, pairs
Subspecies m EM BA A X Y.
P. parvus trumbullensis 54 70 8 18 ST A
P. parvus olivaceous 54 74 11 15 ST A
P. parvus clarus 54 76 12 14 ST A
P. parvus columbianus 54 106 0 26 SiM SM
10
independent evolution and divergence of P. parvus columbianus from other subspecies of
P. parvus (Hafner and Hafner, 1983). He also found that P. alticola and P. xanthonotus
had karyotypes that were identical to those found in P. parvus from the Great Basin.
Fossil Record of Heteromyids and Geologic History
Members of the family Heteromyidae first appear in the mid Oligocene fossil
record during the Orellan North American Land Mammal Age (NALMA) (Wahlert,
1993). Thus, the systematic and biogeographic history of members of the family should
reflect various aspects of geological, climatic, and vegetational history of western North
America during the past 30 million years.
The genus Perognathus also has a long history in North America. Fossil records
of Perognathus occur from the Miocene to the Pleistocene in western North America
(Wahlert, 1993). The first appearance in the fossil record is of a specimen from the early
Hemingfordian (mid Miocene) of the John Day formation of northern Oregon (James,
1963). However, it is thought that the subfamilies Heteromyinae and Perognathinae were
not yet clearly separated in the fossil record by the early Miocene (Wilson, 1960). A
specimen of P. maldei, now extinct, from the late Pliocene of Idaho is believed to most
closely resemble modem P. parvus, and it is thought to be ancestral to modem P. parvus,
which first appears in the fossil record during the Rancholabrean NALMA (mid
Pleistocene) of Idaho. There are slight differences between the fossils found in the John
Day formation of northern Oregon and the Idaho fossils, but they could be
phylogenetically related (Zakrzewski, 1969).
11
The region in which the oldest fossils of Perognathus occur is the Columbia
Plateau (James, 1963; Zakrzewski, 1969). The Columbia Plateau formed as a series of
basaltic volcanic plains during the mid to late Miocene, approximately the time
corresponding to the fossil record. As basalt accumulated in the Pasco Basin of central
Washington, tectonic deformation was taking place, resulting in a complex folding pattern
throughout the Columbia Plateau. In the late Miocene, the longest structure formed
during the height of volcanic activity was the Blue Mountains. This mountain chain
borders the Columbia Plateau along most of its southern boundary and separates it from
the Oregon Plateau. The High Lava Plains arose during the late Miocene and form a
geological transition between the Columbia Plateau and Great Basin. It separates an area
of declining volcanic activity on the Columbia Plateau from an area of tectonic extension
in the Great Basin (Christiansen and McKee, 1977). The Oregon Plateau is part of the
High Lava Plains and forms the northernmost region of the Basin and Range Province. It
is bordered on the north by the Blue Mountains and on the south by the Orevada rift
(Carlson and Hart, 1987). Fossil records of Perognathus from the Oregon Plateau are
reported from the Barstovian NALMA (mid Miocene). Basaltic volcanism in the Steens
Mountain area of southern Oregon was at its peak during the mid Miocene (Carlson and
Hart, 1987). Volcanism in the Steens Mountain area continued sporadically until
approximately 11 mya, and on the southeastern edge of the Oregon Plateau, volcanism has
continued until the Holocene. This southeastern edge forms the border between the
Oregon Plateau and the Great Basin. Late Miocene basalts from the Steens Mountain area
of southern Oregon are similar in composition to those found in the northern Great Basin,
12
and it is thought that they are related (Carlson and Hart, 1987).
Geologically the Great Basin is relatively young. The Great Basin had
considerable tectonic relief due to late Cretaceous and Tertiary orogeny, but the Basin and
Range topography did not exist until the late Miocene (Christiansen and McKee, 1977).
Volcanism appears to have begun along the northern axis of the Basin and Range
province. By 17 to 14 mya the Great Basin already had considerable tectonic relief.
Much of the surface of the region was covered by 14 to 12 mya. Numerous ashflow tuffs
of early Miocene age in Nevada indicate that there was little topographic relief comparable
to present-day Basin and Range topography (Christiansen and McKee, 1977). Although
much of the volcanic activity occurred prior to this time, the Great Basin has remained
tectonically active to present day. Areas of seismicity in this region are relegated primarily
to the eastern and southwestern edges, the Wasatch and Sierra Nevada ranges,
respectively, which form the east and west borders of the Great Basin.
Ancestral members of the lineage giving rise to extant Perognathus parvus were
distributed throughout most of the Columbia and Oregon Plateaus by tire mid Miocene
(Wahlert, 1993). Perognathus parvus fossils did not appear in the Great Basin fossil
record until the Rancholabrean NALMA (mid Pleistocene). An ancestor to P. parvus may
have evolved on the Columbia Plateau and subsequently migrated southward into the
Great Basin prior to the uplift of the Blue Mountains. One would predict that, based on
the geological and fossil records, the Blue Mountains uplift presented a significant barrier
to dispersal. This barrier would have isolated northern from southern populations as long
ago as the late Miocene. The loss of gene flow between populations from these areas
13
would have resulted in separate evolutionary trajectories for northern versus southern
populations of P. parvus. Williams' (1978) karyotype data support the hypothesis of a
separation of northern from southern populations.
P. parvus has long been postulated to be a single, widespread species. However,
several alternative vicariance hypotheses can be constructed relative to the response of P.
parvus to geological and climatic history. If P. parvus was split into two or more
geographically isolated populations by geological or climatic perturbations, I would
predict phylogeographic disjunction among populations and geographic variation
corresponding to geologic formations within the range of P. parvus.
Three alternative biogeographic models may be used to suggest possible
geographic mtDNA haplotype variation within and among populations of P. parvus on the
Columbia Plateau and in the Great Basin. First, late Tertiary geotectonic vicariance may be
responsible for isolating Columbia Plateau from Great Basin populations of P. parvus.
Second, late Quaternary glacial-interglacial climatic cycles causing expansion and
contraction of areas of suitable habitat may be responsible for isolating Columbia Plateau
and Great Basin populations. Finally, P. parvus populations on the Columbia Plateau may
have shifted their range southward during each Pleistocene glaciation in response to
decreased winter temperatures and the southward shift of shrub-steppe habitat followed by
northward expansion during interglacials. This model would predict no detectable "deep"
phylogenetic splits between Columbia Plateau and Great Basin populations.
In this study, I have addressed the following questions: 1) How is mtDNA
variation apportioned among populations of Perognathus parvusl 2) To what extent does
14
geographic variation in mtDNA distribution and divergence differentiate among the three
models presented above?
Molecular Data
The hypotheses stated above were tested using mitochondrial DNA (mtDNA)
sequences and Restriction Fragment Length Polymorphisms (RFLPs). The mitochondrial
genome contains non-recombining, maternally inherited DNA that evolves faster than
other single copy DNA (Vawter and Brown, 1986). Gene arrangement in mtDNA
appears generally stable, and nearly the entire genome is a coding region so that
hypervariable regions such as introns are not part of the data set (Avise, 1994). The
amount of variability in the mtDNA genome is largely due to its high rate of mutation,
thus it provides substantial numbers of characters for comparisons between closely related
species and populations. Many studies have empirically demonstrated geographic
variation within and among populations using mtDNA (Avise et al., 1987; Avise et al.,
1988; Avise, 1992; Moritz et al., 1987; Riddle et al., 1993).
Molecular data have several attributes that provide an advantage over
morphological data when reconstructing phylogenetic hypotheses. Morphological data
represent a small subset of genetic information that can be environmentally plastic and may
not reflect the true evolutionary relationships of organisms. Morphological characters in
some taxa may not be sufficient for inferring a robust phylogeny, and certain characters
can be misleading because they represent special environmentally induced adaptations that
may not have a genetic basis. Molecular data, on the other hand, have a distinct
15
advantage over morphological data in that the data are sampled directly from the genome
of the organism, thus ensuring that characters are hereditary (Avise, 1994; Hillis, 1987).
Genomes provide an enormous amount of information. The genome codes for proteins
and other cellular material, as well as retaining a record of evolutionary change in its
nucleotides. Molecular characters can provide an extensive, detailed data set, limited only
by the number of nucleotide pairs in the DNA, that permits the reconstruction of a well-
resolved phylogeny that can either refute or support a phylogeny that was constructed
using morphological characters (Hillis, 1987; Mayr and Ashlock, 1991). Morphological
and behavioral characters may often be convergent. For instance, long tails in kangaroo
rats (Dipodomys sp.) and jumping mice (Zapiis sp.) are believed to act as a rudder for
balance. Because the tail has a similar function in both of these mice, this character might
be incorrectly considered homologous. In reality these two taxa are only distantly related.
Because similar molecular characters are unlikely to be convergent, molecular data often
allow one to separate homologous characters from convergent ones by separating
morphologically similar organisms from one another based on genetic information rather
than shared morphological characteristics that may have evolved convergently.
Comparisons of distantly related taxa using morphological characters can be
confounded because of the lack of comparable characters between taxa. Molecular data
allow for the direct comparison of levels of genetic divergence among nearly any group of
organisms. Molecular data can also provide a means for estimating times of divergence
based on a molecular clock that is calibrated using fossil material or biogeographic
hypotheses, thus molecular characters can add an explicit temporal dimension by
16
calculating the distance between taxa and translating it into a time of divergence between
the taxa (Mayr and Ashlock, 1991; Moritz and Hillis, 1990).
The three models presented above can be related to phylogeographic hypotheses
that have derived from studies of mtDNA gene genealogies (Avise, 1994). Large
phylogenetic gaps between monophyletic groups usually arise from long-term barriers to
dispersal. A late Tertiary geotectonic vicariance would most likely result in a "deep" level
of divergence between northern and southern populations of P. parvus. Reciprocal
monophyly of northern and southern populations would have occured over time through
stochastic lineage sorting. The time of divergence calculated from a molecular clock
could be related to a geotectonic vicariance event such as the uplift of the Blue Mountains.
One would expect shallow levels of genetic divergence if barriers to dispersal had not been
present for long periods of time. Pleistocene glacial-interglacial expansion and contraction
of suitable habitat would result in shallower levels of divergence than would a long-term
isolation event from the late Tertiary. Phylogeographic population structure showing
shallow levels of divergence would be expected in species with life histories conducive to
dispersal or in populations that have few barriers to dispersal. If populations of P. parvus
migrated southward during periods of glacial maxima, low levels of divergence would be
found corresponding to a Pleistocene time of divergence. Reciprocal monophyly from a
Pleistocene divergence may not have occured in such a short time.
Chapter 2
Material and Methods
Specimen Collection
A total of 45 specimens were collected from throughout the range of Perognathus
parvus (Appendix 1). Specimens were caught in Sherman live traps using chicken feed
as bait. Specimens were euthanized using isohalane according to the University of
Nevada, Las Vegas Animal Care and Use protocol. Heart, liver, kidney, and brain tissue
were removed and stored in cryotubes in liquid nitrogen, or they were preserved at
ambient temperature in "Lysis Buffer" (Longmire and Baker, 1994). Tissues are stored
at the University of Nevada, Las Vegas , whereas skins, skeletons and skulls were
prepared as museum specimens and are housed at the Museum of Southwestern Biology,
University of New Mexico.
DNA Extraction
Total genomic DNA was extracted from heart or brain tissue using either of two
DNA isolation procedures. The first DNA isolation protocol, a slightly modified version
of Hillis et al. (1990), involved macerating approximately lOOmg of heart, liver, or brain
tissue and adding 500pl of STE buffer (0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5, 0.001 M
EDTA) with 25pl of (10 ug/pl) Proteinase K to a 1.5 ml centrifuge tube. Twenty-five
17
18
(25)pl of 20% SDS (sodium dodecyl sulfate) was then added to lyse cell membranes. The
mixture was incubated at 55°C for two hours with occasional mixing. After incubation, an
equal volume of phenol:chloroform:isoamyl alcohol (PCI) with a ratio of 25:24:1 was
added. The mixture was incubated at room temperature for 5 minutes with regular mixing
to prevent phases from separating. Tubes were then microcentrifuged for 5 minutes, and
the aqueous layer was removed and saved. This procedure was performed again and
followed by twice washing the removed aqueous layer with an equal volume of
chloroformrisoamyl alcohol (Cl, 24:1), incubating for 5 minutes at room temperature,
microcentrifuging for 3 minutes, and finally removing and saving the aqueous layer. The
extracted genomic DNA was then incubated overnight at -20°C in one ml of cold absolute
ethanol and 1/10 volume of 2M NaCl to precipitate the DNA. The next day the tubes
were centrifuged for five minutes, and the liquid was decanted. The remaining pellet in
the bottom of the tube was washed with 500 p. 1 of 80% ethanol by centrifuging for 5
minutes and decanting the ethanol. Once washed, the DNA pellet was vacuum
centrifuged for 20 minutes. When the pellet had dried, it was resuspended in 150 to
200 p. 1 of IX TE buffer (0.001 M Tris-HCl, pH 7.5, 0.0001 M EDTA) and stored at 4°C.
The second DNA isolation protocol of Longmire and Baker (1994) was much
easier to use in the field and was used for specimens captured during July and August of
1994. Approximately 0.4g of tissue (one entire heart or brain)was macerated in a petri
dish and placed in a 15ml polypropylene Coming tube with 5 ml of "Lysis Buffer." The
macerated tissue could then be stored indefinitely out of sunlight at ambient temperature.
19
"Lysis Buffer" consists of 0.5 M EDTA, 20% SDS, and Tris-HCl. For extraction, 250 |il
of (lOOpg/ml) Proteinase K was added to the tubes and rotated overnight at 37°C. After
overnight rotation, an equal volume of phenol buffered with TE was added and rotated for
another 30 minutes at 37°C. Tubes were then centrifuged at 2000 rpm for five minutes to
separate the phases. The aqueous layer was removed and placed in dialysis tubing.
Samples were dialyzed for 24 to 36 hours against three changes of IX TE buffer (0.001 M
Tris-HCl, pH 7.5, 0.0001 M EDTA). The genomic DNA remaining in the dialysis tubing
was placed in 15 ml tubes and stored at 4°C.
Polymerase Chain Reaction
The Polymerase Chain Reaction (PCR) was used to amplify target gene regions.
PCR uses oligonucleotide primers to amplify specific sequences of DNA in a cyclic
fashion. Template DNA is placed in a tube containing a 10X solution of reaction buffer
(buffers are different for each type of polymerase), lOmM of each oligonucleotide primer,
8 mM of each of four deoxynucleotides (dNTPs) (dATP, dCTP, dGTP, dTTP), and 4u/pl
of Vent® DNA polymerase (Hillis et al., 1990). Vent® DNA polymerase has a much lower
rate of error in incorporating wrong nucleotides during the primer extension phase of PCR
than Taq DNA polymerase does (Catalog reference). The mixture is then overlaid with
mineral oil to prevent evaporation. Template DNA is denatured at 95°C for one minute in
the first step. In the second step, the oligonucleotide primers anneal to the 5' ends of the
denatured DNA at 50 - 60°C for one minute. In the final step, extension of the primers
occurs by addition of the individual dNTPs to the 3' end of the primer at 72°C for 2
20
minutes. Each of these steps is repeated for 25 cycles, creating millions of copies of the
original double-stranded target sequence. Repeating this procedure using only one
oligonucleotide primer allows the generation of single-stranded DNA that is suitable for
DNA sequencing.
DNA Sequences
Most of the 784 base pair mt DNA Cytochrome oxidase HI (COIQ) gene was
amplified using PCR. Primers designed by Riddle (unpublished) and identified according
to the 3' base number in the published sequence of Miis (Bibb et al., 1981) are as follows:
L 8618 5' CAT GAT AAC ACA TAA TGA CCC ACC AA -3', H 9323 5'- ACT ACG
TCT ACG AAA TGT CAG TAT CA -3’. A section of this region containing 326 base
pairs was sequenced from three specimens, one from the lower Columbia Plateau and two
from the Great Basin, using the Sanger dideoxy sequencing reaction with Sequenase 2.0
DNA Sequencing kit (Allard et al., 1991; Sanger et al., 1977). Single-stranded DNA is
annealed to one primer (the sequencing primer). This mixture is separated into 4 sub
samples containing all four deoxynucleotides and a single dideoxynucleotide (ddNTP) that
lack a 3' hydroxyl (3'-OH) group. The DNA strand is made radioactive using 35‘S that has
been added to the ddNTPs. Primer extension is catalyzed by DNA polymerase, and
sequence extension occurs through the attachment of nucleotides to a free 3'-OH.
Wherever a ddNTP has been incorporated further strand extension is halted (Avise, 1994).
The reaction occurs such that DNA fragments of differing lengths are produced. Each of
the four reactions is electrophoresed through a polyacrylimide gel for approximately 5
21
hours and visualized on an autoradiograph. The DNA sequences obtained from the CO m
gene region were read into the MacVector program version 3.5 (International
Biotechnologies, Inc., New Haven, CT) for alignment and editing.
Transition substitutions in mtDNA accumulate at a stochastically-linear rate of
divergence initially but because of the high rate of substitution, multiple substitutions at a
site eventually produce a high level of phylogenetic noise. Even if transition substitutions
are saturated, transversion parsimony, which uses only transversion substitutions in
analysis, can still be employed because transversions accumulate in a linear fashion long
after the sequences have diverged beyond the saturation of transitions (Brown, 1985).
Riddle (1995) provided evidence that divergence among most geographically and
evolutionary distinct lineages of pocket mice exceeded the point of transition substitution
saturation. Therefore transversion sequence divergence was calculated using the Jukes-
Cantor formula (DNADIST in PHYLEP v.3—Felsenstein, 1993). Phylogenetic trees were
calculated using the branch and bound algorithm in PAUP version 3.0 (Swofford, 1991)
and mixed parsimony analysis, which employs generalized parsimony (transition and
transversion base substitutions weighted equally) for 1st and 2nd codon positions and
transversion parsimony (transition substitutions excluded) for 3rd positions, with 100
bootstrap iterations. Chaetodipiis hispidiis and C. intermedins were used as outgroups.
Restriction Fragment Length Polymorphisms
Restriction endonucleases cleave DNA at specific sites. The resulting fragment
pattern can then be used to infer restriction sites along the sequence of DNA being
22
analyzed (Avise, 1994). The fragments are separated by electrophoresis according to
molecular weight- Differences in fragment size may result from base substitutions within
cleavage sites, additions or deletions of DNA, or sequence rearrangements.
The NADH dehydrogenase 2 (ND2) gene region and a portion of the Cytochrome
oxidase I (COI) gene region containing 2150 base pairs was amplified using PCR with
primers designed by Dr. J. C. Patton (Riddle et al., 1993). Primer sequences are: L3880,
5'-TAA GCT ATC GGG CCC ATA CC-3'; and H6033, 5'-ACT TCA GGG TGC CCA
AAG AAT CA-3'. Ten microliters of the amplified product were subjected to restriction
endonuclease digestion according to manufacturer's protocols using the following 13
restriction enzymes: Bsp 12861. BslNI, BstUI, Haell. HaelU, H indi, HindlH. Hinfl,
Mbol. MspI, Rsal. Taql. and Xbal. Digested samples were electrophoresed through a
1.5% agarose gel for approximately 3 hours. Banding patterns were then visualized after
staining the gel with ethidium bromide and placing it on an ultra-violet transilluminator.
Polaroid photographs were taken of each gel.
After scoring fragments according to distance travelled on the gel, restriction sites
were inferred under assumptions of single-site substitution patterns (Dowling et al., 1990).
Data were analyzed using RESTSITE (Miller, 1991) and REAP (McElroy et al., 1991).
Distance matrices were calculated in RESTSITE using the Jukes-Cantor formula, and
were used to generate UPGMA dendrograms. Distance matrices plus standard errors of
distances were calculated using the DSE program in REAP. DSE estimates genetic
distance values using equations set out by Nei and Tajima (1981). Standard .errors are
computed according to Nei and Tajima (1983).
23
Haplotype diversity and nucleotide diversity and divergence were calculated in the
DA program of REAP. Haplotype diversity within populations (h) was calculated using
equation 7 of Nei and Tajima (1981):
h = n(l - Sxj2) / (n - 1),
where x; is the population frequency of the ith haplotype and n is the number of individuals
sampled. In this case X; is the frequency of haplotype (i) occurring in the ND2/COI region
of the mitochondrial genome. Standard error of haplotype diversity (VS11/2) was calculated
using equation 8.12 of Nei (1987):
VSI(h )= (2 / ( 2n - 1)) { 2 (2n - 2) [E X;3 - ( E x 3 )2] + 2 x 3 - ( 2 Xj2 )2},
where again n is the number of individuals sampled, and X; is the frequency of haplotype (i)
occurring in the ND2/COI region of the mitochondrial gene region. Estimated nucleotide
diversity within populations (n) was calculated using equation 10.19 of Nei (1987)
which corrects for small sample sizes:
n = ( n x / n x - 1 ) SgXiXjdg,
where nx is the number of sequences sampled, dy estimates the number of nucleotide
24
substitutions per restriction site between the ith and the jth haplotypes, and x{ and Xj are
the sample frequencies of the ith and jth haplotypes for the population being analyzed.
Nucleotide diversity among populations is calculated using Nei's equation:
^ x y = 2 , ^ y j d ^ ,
where d̂ - estimates the nucleotide substitutions between the ith haplotype from population
X and the jth haplotype from population Y. Nucleotide divergence between populations
(dA) is calculated using Nei's equation 10.21:
= X̂Y ' ( + dY ) / 2.
Geographic heterogeneity in haplotype distributions among populations was
estimated using the Monte program of REAP. This program uses a Monte Carlo
simulation to analyze the extent of geographic heterogeneity among populations (Roff and
Bentzen, 1989). The program generates, through a large number of randomizations of the
data set, the distribution of x2 expected if the null hypothesis of homogeneity were true for
the particular data set being studied. Because the frequency of some haplotypes is so low
that the expected distribution generated by this statistic may not conform to that of y2. the
Monte Carlo statistic is designated as X2. The probability, P, of obtaining a value of X2
equal to or larger than that obtain from the actual observations is given by the equation:
25
P = n / N,
where n is the number of randomizations that generate the large value of X2, and N is the
number of randomized sets. The standard error of P is:
s.e. = [P (1 - P ) / N]1/2.
Chapter 3
Results
Sequence Results
Analysis of sequence divergence in the C01H gene indicated that there are at least
two divergent mtDNA lineages within P. parvus. Average transversion sequence
divergence between specimens from Crook County, Oregon (Columbia Plateau) and two
specimens from the Great Basin (Cassia County, Idaho and Nye County, Nevada) was
estimated to be 7.3%. A summary of base substitutions among these three specimens is
given in Table 2.
Figure 3 depicts a phylogeny that was estimated using data from both the COm
gene and the mtDNA cyt b gene (Riddle, 1995). Bootstrap support for the P. parvus
node is 84% versus 54% (Figure 4) when exemplars from this data set included only
sequence from the COIII gene. Although the P. parvus clade from the smaller data set
had lower bootstrap support, P. parvus from the Columbia Plateau did not tend to cluster
with any other clade at a similar frequency as it did with the P. parvus clade. Bootstrap
support for Columbia Plateau P. parvus clustering with the P. apache clade or the P.
longimembris clade was 5% and 25%, respectively. A genetic distance matrix for these
sequences is shown in Table 3. Note that distances between Columbia Plateau and Great
26
27
Table 2. Pairwise comparison of nucleotide base substitutions from 326 base pairs of the mtDNA C O m gene obtained from 3 specimens of Perognathus parvus. Collection localities, Las Vegas Tissue (LVT) numbers and numbers under which sequences were published in GenBank are indicated. Substitutions are recorded as number of transitions/transversions per codon position.
Codon position
Locality LVT GenBank 1st 2nd 3rd
OR: Crook Co./ID: Cassia Co.
1921/794 U2 1624 4/1 3/1 37/20
OR: Crook Co./NV: Nye Co.
1921/1806 U2 1622 5/1 3/0 36/21
ED: Cassia Co./NV: Nye Co.
794/1806 U2 1621 1/0 0/0 0/2
28
CO COCO
COCO
CO CO CO
COCO
to00
C\C\
COC\C\ C\
a
evol
utio
n by
Ridd
le (1
995)
. Re
prod
uced
wi
th pe
rmis
sion
.
29
CO
CO
-c
§ CQ >> • 2?0 aUh §
Tabl
e 3.
Tran
sver
sion
dista
nce
matr
ix
of 32
6 ba
se
pairs
of
the
COIII
ge
ne
from
sequ
ence
s of
seve
ral
pock
et m
ouse
sp
ecie
s, ca
lcul
ated
us
ing
Juke
s-C
anto
r fo
rmul
a in
the
DN
AD
IST
algor
ithm
of
PHY
LIP.
Th
ese
spec
imen
s we
re
used
to
cons
truct
a ph
ylog
eny
incl
udin
g sp
ecim
ens
of Pe
rogn
athu
s pa
rvus
fro
m the
G
reat
Bas
in and
the
so
uthern
Co
lum
bia
Plat
eau.
G
RB
=Gre
at B
asin
, C
LP=C
olum
bia
Plat
eau,
HIS
P=H
ISPI
DU
S, A
PA=A
PAC
HE,
FA
SC=F
ASC
IATU
S, F
LAV
=FL
AV
US,
FLV
S=FL
AV
ESC
EN
S,
LON
G=L
ON
GIM
EMB
RIS
, PA
RV
1=PA
RV
US_
GR
B1,
PA
RV
2=PA
RV
US_
GR
B2,
PA
RV3=
PA
RV
US_
CLP
.
30
PARV
3
0.14
18
PARV
2
0.07
12
0.15
06
>06<
OOr***oo
i—tr-*o
t-Hinr-H
Hi o o o
O2OO n
O nOT“HCOC OO nO
vooi—H oCOy—4o o o o
FLVS
0.12
96
0.10
68
0.09
50
0.10
73
0.13
83
><frj
oCNr“-t
COV OC Oo
rtV Or-H i—H
CSooOO niny—4r—H
inV O
o o o O o o
FASC
oV OTft-H
lOO n
O
oocsN*1—4r--csCO
OCScsr—1r-t-Ho
N"r-»CNo O o o o o o
APA cn
o nmo
V Oc-*-CSO NcsCNO
inr—<
CNoor—(CNr-o\o
ooooO nO
V O 00 CO 1—<o O o o o o O o
-IISP
. voCOoCOxfr—'4
O nCNwoT—<r*Hr-(Ni—i
O NT—<COr”HV OoV O*—H
3mr*HCOV Omt—H
csxf1— I i—“4o o o o o o o o o
Spec
imen
HIS
PID
US
APA
CH
E
FASC
IATU
S1
FLA
VU
S
FLA
VES
CEN
S
LO
NG
IMEM
BR
IS1
PAR
VU
S_G
RB
1
PAR
VU
S_G
RB
2
PAR
VU
S_C
LP
INT
ERM
ED
IUS
31
Basin P. parvus (x = 7.3) is clearly less than average distances between either Columbia
Plateau or Great Basin P. parvus and the Flavus group and Longimembris group (x =
11.4, 10.3) or Fasciatus group (x = 10.6) representatives.
RFLP Results
Restriction Fragment Length Polymorphism (RFLP) analysis revealed eighteen
mtDNA haplotypes (7 from the Columbia Plateau and 11 from the Great Basin/Colorado
Plateau). The COIII sequence divergence among samples of P. parvus support a high
level of divergence between Columbia Plateau and Great Basin/Colorado Plateau
haplotypes (Table 2). Restriction site analysis methods employed in the study were unable
to discern specific site gains and losses between haplotypes from the Columbia Plateau
relative to haplotypes from the Great Basin/Colorado Plateau because double enzyme
digestions were not performed on restriction fragments. Additionally, sites that could be
scored as shared between regions could not be assumed to be uniquely derived due to high
probabilities of multiple losses and gains. Therefore, the Columbia Plateau and Great
Basin haplotypes were analyzed as separate restriction-site data sets (Tables 4 and 5).
Distribution of mtDNA RFLP haplotypes is shown in Figure 5.
A UPGMA dendrogram of haplotypes from the Great Basin/Colorado Plateau,
based on the genetic distance matrix in Table 6, is shown in Figure 6. The most common
haplotype found among Great Basin/Colorado Plateau samples was number 1 (Figure 5);
this haplotype was found in 15 specimens from five localities in central to southern
Oregon and from one specimen located in north-central Nevada (55% of the total).
Tabl
e 4.
Com
posit
e ha
plot
ypes
of
spec
imen
s fro
m the
G
reat
Bas
in/C
olor
ado
Plate
au
used
in
restr
ictio
n sit
e an
alys
is.
Enzy
me
clas
s (E
nz.c
l), e
nzym
e, a
nd
Las
Vega
s Ti
ssue
(L
VT)
are
indi
cate
d.
Com
posit
e ha
plot
ype
(CO
MP)
num
bers
are
id
entif
ied
in the
las
t co
lum
n.32
COMP
t“ 4 cs cn St NO NO <o WO r - H 1— 4 1— 4 N O r~ OO
n© < < < < < < < < <C < < < < <
TT3
c PQ < < < < < < < < < < < <
Tf PQ po pq pq < < < < PQ PQ PQ PQ PQ PQ
Tf pq < < < < < < < PQ PQ PQ PQ PQ PQ
Tf < < < < < < < < < < < < < <
1 < U h < < < < < PQ PQ PQ < Q O
n©a
< < < < < < < < < < < < < <
5.33
i< < < < < < < < < < < < < <
Tf»— ( t— 1
< < < pq < < < < < < < < < <
5.33
l< < < PQ < < < < < < < < < <
>-4
< < < < < < < < < < < < < <
4.67
>— <
1< < < < PQ PQ PQ PQ < < < < < <
5.33
> -4
OO< sT—^ < < < < < ■ < < < < < < < < <
. s iC
f c d
H
>mO N
r ~ -
u ooC Or —4
N Oo0 0
C ~ -oO Oi— i
C OT—H
o o
• N ft— 4C Or-H
> o1—HC Oi—H
N O
O OT— t
C OcsO N 1“ •
w ocsO N1— 4
r -csO N 1— <
O N C S O N 1— t
1— 4mO ni— 4
csC OO NT-H
Tabl
e 4
Con
tinue
d.
Com
posit
e ha
plot
ypes
of
spec
imen
s fro
m the
G
reat
Bas
in/C
olor
ado
Plate
au
used
in
restr
ictio
n sit
e an
alys
is.
Enzy
me
class
(E
nz.c
l), e
nzym
e, a
nd
Las
Veg
as T
issue
(L
VT)
are
in
dica
ted.
Co
mpo
site
hapl
otyp
e (C
OM
P) n
umbe
rs
are
iden
tifie
d in
the
las
t co
lum
n.33
COMP
OO VO On »—H
v©X
hal
< < < < < < < < < < < < <
T f < < < < < < < < < < < < <
T f
Rsa
l
pq m f f l m CQ pq pq pq pq pq pq pq pq
T f pq < pq pq pq pq pq pq pq pq pq
T f < < < < < < < < < < < < <
T f
Hin
fl
u < < PQ pq pq W pq pq pq pq pq pq
VD
Hin
din
< < < < < < < < < < < < <
5.33
i< < < < < < < < < < < < <
T f
Hae
in < < < < < < < < < < < < <
5.33
1< < < < < < < < < < < < <
T f
1< < < < < < < < < < < < <
4.67 Z < < < < < < < < < < < < <
5.33
Bsp
l286
I
< < < < < < < < < < < < <
CW L
VT
1933
1934
1935
1937
1938
1939
1940
1941
1942
1943
1944
1945
'19
46
Tabl
e 4
Con
tinue
d.
Com
posit
e ha
plot
ypes
of
spec
imen
s fro
m the
G
reat
Bas
in/C
olor
ado
Plate
au
used
in
restr
ictio
n sit
e an
alys
is.
Enzy
me
class
(E
nz.c
l), e
nzym
e, a
nd
Las
Veg
as T
issue
(L
VT)
are
in
dica
ted.
Co
mpo
site
hapl
otyp
e (C
OM
P)
num
bers
are
iden
tifie
d in
the
las
t co
lum
n.34
| CO
MP
T— •
VO < < < < < <
T f
3< < < < < <
T f
clm m m m m m
T T m « m m m <
T f < < < < < <
T f
1m m m
no1
< < < < < <
5.33
Hi
nc
II
< < < < < <
rr a < < < < < <
5.33
1< < < < < <
T f | < < < < < <
l >V D
T f
z1
< < < < < <
5.33
t—<SOO Of S < < < < < <
Enz
.cl
H
>r -T fO Nr-H
o oT fOn
onT fO NT— (
T—ii nO s1- H
c smOn
t -> nO N
Tabl
e 5.
Com
posit
e ha
plot
ypes
of
spec
imen
s fro
m the
Co
lum
bia
Plate
au
used
in
restr
ictio
n site
an
alys
is.
Enzy
me
class
(E
nz.c
l),
enzy
me,
and
La
s V
egas
Tiss
ue
(LVT
) are
in
dica
ted.
Co
mpo
site
hapl
otyp
e (C
OM
P) n
umbe
rs
are
iden
tifie
d in
the
last
colu
mn.
.35
COM
P
CSr-H
COr-H
T fr~A
wor-H
wot—H
csr-H
VOr-H
VOT—Hc~~r-H
r~r -H
OOy*H oor-H
OOr-H
vo
XhnI z Z z Z z z o o Z z o o O
T f1 o O o o o o z z o o z z z
T f1 o Cu o o o o P i P i o a z z z
T f
Mbo
l
z Z o z z z P a Pa z z o o o
T f z Z z z z z z Z z z z z z
T fi
cl, CL, o o o Oh z Z P i pi a a a
v©
Hinr
lHI
Z Z z z z z z Z z z z z z
5.33
Hinc
II o o o o o o z z o o z z z
T f
Hne
lll z o z z z z Pa z z O' a a
5.33
l-H
z z z z z z CL, Pa z z o o o
T fP1
z z z z z z z Z z z o o o
4.57
>-HZ z z z z z z CL, CL, z z o o o
5.33
VOoori o o o o o o o O o o z z z
cw L
VT
#
1915
1916
1917
1918
1919
1920
1921
1950
1953
1954 in vn CTs 1956
1958
36
ca
CQc3o ~
- 32 « cd<u cs ■3 CL — <DCL 002 "8.O Ns -ar3̂ CO
Ua 2o S3
C GO
o S.S2 oS CL C rt <3 X i Cl 32 2 "S rtS ECXO O O TDL .
,5°U_O
COC<Ds
rtt-.Oou"s
COC3CQo
& cs " 2 o O<y cQ, C3 2P 3 ° 8rt i 2 J= CL <£ rtZ £ Q SS - c o'o Uao4—»3
X)•n *—*
V i3
pao3CUoT3rt
*> r93 ^ CO TD» cUh rt
Tabl
e 6.
Matr
ix
of ge
netic
di
stanc
es
and
stand
ard
erro
rs
(Nei
and
Tajim
a, 1
981)
from
so
uthe
rn
popu
latio
ns
of P.
par
vus.
Gen
etic
di
stan
ces
amon
g ele
ven
com
posit
e mt
DNA
hapl
otyp
es
are
show
n be
low
the
maj
or d
iago
nal,
and
stand
ard
erro
rs are
sh
own
abov
e th
e m
ajor
dia
gona
l.
37
O n O O V O C S r - c - in C O r -
O O T"H T“ H o T f cs CS m 0 0 cs1—1 o t“H T“H t- H t- H r - H t- H t - H o r H
r H p p o o O o o O o O
o o o o O o o o o o
O n O n T t cs T f r - in m CSO O V O Tt O O cs cs m m oo O T—H T—H t- H r - H t- H t- H t- H T—HO p o o o o o o o oo o o o o o o o o o
r- oo t- H Tf CO in oo C O O n
cs r- CO r-- V N oo in t- H m T ft- h o o o o T“H t- H t- H Owr o o p o r H
. o p O O O
o o o o o o o o o O
r- m O n C O O C O wo 0 0 C O C O
cs T f C O cs V O oo in O N in inT -H t—h T—H t- H t- H o T -H o t- H t- H
o o p o o p o o O O
o o O o o o o o o o
Q \ O n cs T f r- C O C O cs csOO r- V O T f oo cs in in o oO t—H t - H y—f t- H r - H r - H t- H r H T“ H
o O o o o o o o o oo o o o o o C D o o o
C T \ 0 0 r- V O cs cs O N O N cs o )
o o t- H t—H o T f o T f T f o oV/“ i o r — 4 t- H t- H t- H t- H O o t- H T—H
p o O o o o o o o p
o o o o o o o o C D o
c n O n V O o O n oo V O O n oV O C O C S t- H o O T f T f O oi 4 r - H r -H t- H cs C O cs t- H C O cs
m ) p O O o o o o O o oo O O o o o o o o o
O n o T f in in r - T-H T f r - m
C S o C O T f O n O n T f T f
t—H t- H t- H cs t- H cs t—H O C S T—HX T o o o p o p o O p o
o o o o o o o O o o
r - - t- H r*** 0 0 O N cs o T f cs O n
t J* t- H o in in r - T—H O r - mt- H cs cs T—H cs cs t- H cs ■ i
no o o o p o o O o p
o o o o o o o O o o
v o C O O n C O o o C O r - r - C O o o
t o in C O O n O n o T f T f o O n
T—H 1—H t—H t- H O cs t- H O cs Or s
p o o O O p o O o O
o o o o O o C D o o O
C O O n O n O n t- H t - H C S cs t- H T—H
in t- H O n m m in o o m ini—H cs t- H cs o o T“H T—H o o
r HO p o o p p p p p p
o o o o o o o o o o
<D
%s i - H c s C O t T l o V O 00 O N 10 r H
r H
- c d
ffi
38
Figu
re
6. UP
GMA
dend
rogr
am
of G
reat
Bas
in/C
olor
ado
Plate
au
mtDN
A ha
plot
ypes
ge
nera
ted
from
gene
tic
dist
ani
matr
ix
calc
ulat
ed
usin
g eq
uatio
ns
of Ne
i (1
987)
in
REST
SITE
(M
iller
, 19
91).
Haplotype number 10 was the next most closely related to haplotype 1 and was found
together with haplotype 1 in southeastern Oregon. Haplotype 7 clustered with haplotypes
1 and 10 but was found in a population that was located approximately 120 miles from the
nearest haplotype 1. Haplotype 7 was found in conjunction with one other haplotype,
number 8,with which it did not cluster and to which it was not found to be most closely
related. Haplotypes 6 and 9, from east-central and northeastern Nevada, respectively,
clustered together with approximately 0.25% sequence divergence between them.
Haplotypes 2 and 11 also clustered with 6 and 9 and were from the Nevada Test Site and
central Nevada, respectively; haplotype 8, from the same locadon as haplotype 7,
clustered outside this group. The most divergent haplotype was number 5. This was
found in specimens collected from south-central Utah on the Colorado Plateau. It was
found to be approximately 2.6% divergent from the nearest haplotypes, numbers 3 and 4,
which were both found on the Nevada Test Site.
Eight composite haplotypes were found from six localities from the Columbia
Plateau specimens. The UPGMA dendrogram (Figure 7), constructed from the distance
matrix in Table 7, showed a distinct demarcation between specimens collected in Oregon
and those collected in Washington. Haplotype 18, which clustered with 16, was found in
eastern Oregon. Both of the localities from which these specimens were collected were
along the southernmost border of the Columbia Plateau. This cluster of haplotypes was
found to be approximately 8.1% divergent from other haplotypes on the Columbia
Plateau. The five haplotypes found in Washington all clustered relatively closely with one
another. Haplotypes 12 and 13 were both from the same locality, and they clustered most
40
CMT —oo
COCMO
incoo
coo
COinoo
oN.O
COo
cd6<doccd «—*C/3’5o<ucoCQ
0)oaCO
f ge x ’-1
< sz 2 Q wc Wca cn B H d cn
^ alo .s s c*■s Soo , >w‘' 0 *53s zcd d— & ° 2 g
- o o c -zsO cd "O 3
< STS ep O -SCL 2}
r -d>
-oOJcd
j jLl o
Tabl
e 7.
Matr
ix
of ge
netic
di
stanc
es a
nd
stand
ard
erro
rs
(Nei
and
Tajim
a, 1
981)
fro
m no
rther
n po
pula
tions
of
P. p
arvu
s. G
enet
ic
dist
ance
s am
ong
seve
n co
mpo
site
mtDN
A ha
plot
ypes
are
sh
own
below
the
m
ajor
dia
gona
l, and
sta
ndar
d er
rors
are
sh
own
abov
e th
e m
ajor
dia
gona
l.
41
18
0.0
26
7 r -cnoo 0
.02
77
0.0
25
5 OOOOr-Hoo ' 0
.02
31
Tf* m c s 0 0 c-~ r -O r o OS •'tj- o o i n
t" CN cn i—t r-H o oT—4 O o o o o o
o o o o o o
o r - CO o s i nr—< r*H o CO
v o cn cn CN CN VO i n^4 o o O o o p
o o o o o o
CO 0 0 o o s r -o TT OS xf- VO VOT——1 r-H o r - fH CSo p o o o oo o o o o o
r-* (N CN c-- CN VOi n C \ oo T t VO o o
r r l-H o OO CN oo o p o p r—Ho o o O O o
CN o s r-- o i no VO VO VO o ocn r—-< CN T—< OO CO oo O o o O 1—<o o o o o o
VO VO i n o o CN i nCO o o VO r - o
<N o o r-* CN oi—4 o o p o o 1—<
o o o o o d
<D&
<N cn TT i n VO t> 0 0r—i i—4 1-4 rH i-4 f—( r-H rHCL,cd
42
closely with haplotypes 14 and 15 which were also from the same locality, which was
located approximately 60 miles from where 12 and 13 were found. Haplotype 17
clustered outside this group and was found to be about 2.3% divergent from it.
A total of six composite populations were compiled for use in a Monte Carlo
simulation; three populations within the Columbia Plateau and three populations within the
Great Basin/Colorado Plateau. Composite populations and distribution of haplotypes are
as shown in Figure 8. Columbia Plateau and Great Basin/Colorado Plateau populations
were analyzed separately, and then the composite populations in each region were
combined and analyzed as two populations. X 2 values were calculated from the original
data matrices and then compared to the X2 calculated after repeated random sampling of
the data matrix. Haplotype diversity (h) and nucleotide diversity in the Columbia Plateau
were much higher than in the Great Basin/Colorado Plateau (Table 8). Monte Carlo
simulation results demonstrated significant geographic heterogeneity in both regions
(Table 9).
43
o\md.O'!
ci O.o o
O.cx
V)XT'
<u5tooCo
a
*w 8. .i c*
i H
S3 d> o k s^ « g CQ •5 73^ Ccoc: £> ^ *3 C4 a,
33CTOCO C <D,§ x$ ocx Joo 3E .2?5 ^ 2 * *— DCO CJ
& * Sr u^ <o.2 J=
£ a<zQ
S ° .2 ~
3X)•c3uo
00 cQ 2oo 3
3 73 CQ O£ 3
Table 8 . Haplotype and nucleotide diversity values among composite mtDNA haplotypes in the Great Basin/Colorado Plateau and on the Columbia Plateau calculated in the DA program of REAP using equations from Nei (1987) and Nei and Tajima (1983).
G eographic location Haplotype Diversity (h)
Nucleotide diversity (7t)
G reat Basin/Colorado Plateau
0.3797 ± 0.08289 0.005087 ± 0.0000112
Colum bia Plateau 0.7556 ± 0.00605 0.02743 ± 0.0002384
45
Table 9. Monte Carlo simulation X2 values and P-values among composite mtDNA haplotypes from the Great Basin/Colorado Plateau and the Columbia Plateau and between the Great Basin/Colorado Plateau and Columbia Plateau. X2 values were calculated in the Monte program of REAP using the equations of Roff and Bentzen (1989).
M onte C arlo sim ulation X2 values P-values
Among Great Basin/Colorado Plateau populations
66.00 0.0001
Among Columbia Plateau populations
22.75 0.003
Between Great Basin/Colorado Plateau and Columbia Plateau populations
46.00 0.0001
Chapter 4
Discussion
Biogeographic hypotheses based upon Pleistocene glacial and interglacial cycles
have been used to explain distributions of rodents in western North America (Schmidley et
a l , 1994;) as well as diversity patterns of flora (Spaulding, 1990; Thompson, 1990).
Analyses reveal that there is considerable variation both within and among populations of
Perognathus parvus from the Columbia Plateau and the Great Basin. Prior studies hinted
at the variation between populations from the Columbia Plateau versus the Great Basin
(Williams, 1978), but none exhibited this amount or pattern of variation. Variation within
P. parvus occurs in a well-defined hierarchy. Riddle (1995) demonstrated hierarchical
structuring among other species of Perognathus, Chaetodipus, and Onychomys. DNA
sequence divergence levels between Columbia Plateau and Great Basin/Colorado Plateau
haplotypes are veiy high, while genetic distance values from RFLP analyses show much
smaller levels of divergence among haplotypes within either region. Additional
phylogeographic structure may be present among northern and southern Columbia Plateau
populations.
Molecular clocks based on generation time, body size and metabolic rate allowed
for the calibration of a time of divergence between populations of P. parvus (Li et al.,
46
47
Martin and Palumbi, 1993). The Mus - Rattus split about 10 mya provides a standard
against which rates of divergence can be calibrated in rodents of similar body size (Jaeger
et al., 1986). Riddle (1995) estimated a transversion substitution rate of about 1% per
million years in the COIU gene of Mus and Rattus. The transversion sequence divergence
of about 7.2% between northern and southern haplotypes of P. parvus therefore suggests
a late Miocene time of divergence. The demarcation between Columbia Plateau and
Great Basin populations of P. parvus occurs in central Oregon at the Blue Mountains.
The estimated time of divergence of approximately 7.2 million years ago roughly
corresponds to the uplift of the Blue Mountains. The high level of bootstrap support for
the P. parvus clade in Riddle (1995) supports the monophyletic nature of the P. parvus
clade, and the level of transversion sequence divergence between Columbia Plateau and
Great Basin sequences supports the hypothesis that P. parvus is actually made up of at
least two distinct lineages. Genetic distances between Columbia Plateau and Great Basin
sequences are greater than the distances between P. apache and P.fasciatus (5.9%), P.
flavus and P.flavescens (4.9%), and between P. apache and P . flavescens (2.3%), which
are all considered to be separate species (Nickle, 1994). This level of DNA sequence
divergence is exceptionally high for an intraspecies comparison and indicates that what has
traditionally been known as one species may actually represent two distinct lineages that
diverged from one another following a large-scale vicariance event, thus warranting
unique specific status for each. Further support for recognizing two distinct species
comes from the high degree of karyotypic divergence that Williams (1978) found between
northern and southern populations of P. parvus (Table 1).
48
The deep level of sequence divergence between the Columbia Plateau and Great
Basin populations of P. parvus and the estimated time of divergence at approximately 7.2
ma provide evidence for support of the hypothesis of a late Tertiary geotectonic vicariance
event The time of the Blue Mountains orogeny (late Miocene) roughly corresponds to
the time of isolation between Columbia Plateau and Great Basin populations of P. parvus.
Late Pleistocene vicariance as the result of habitat expansion and contraction would be
reflected in lower levels of sequence divergence between northern and southern
populations, as would Pleistocene dispersal due to climatic changes during glacial maxima.
Therefore, Pleistocene vicariance and dispersal hypotheses can be rejected in favor of a
late Tertiary vicariance explanation for the distribution of northern and southern mtDNA
haplotypes.
Nucleotide diversity among haplotypes from the Columbia Plateau is an order of
magnitude higher than diversity found among the Great Basin haplotypes. Genetic
distance values among haplotypes on the Columbia Plateau are also significantly higher
than in the Great Basin. The high levels of diversity and divergence on the Columbia
Plateau are largely attributed to comparisons among Washington and Oregon haplotypes.
Haplotypes 16 and 18 from Oregon are more closely related to each other than they are to
haplotypes from Washington; they also exhibit the largest amount of genetic divergence
from the other haplotypes on the Columbia Plateau. Haplotypes 12 and 15 from one
location are separated from haplotypes 13, 14, and 17, as well as another 12 and 15 by the
Columbia River. Gene flow probably occurred between these populations before the
Columbia River formed a possible barrier to dispersal. If the Columbia River does form a
49
barrier, it has not existed long enough to cause large amounts of divergence to occur
between populations in Washington. Additional data are required to assess geographic
distribution of haplotypes 16 and 18, which are most divergent from other Columbia
Plateau haplotypes. Although sequences from Washington haplotypes would allow for
estimation of a more detailed phylogeny of the P. parvus clade, RFLP data indicate that
additional phylogeographic structure is included in P. parvus.
Fossil pollen records from the Columbia Plateau indicate that xeric shrub-steppe
habitat did not exist in that area as a dominant component until about 14 to 12 ma
(Leopold and Denton, 1987). Prior to this time the area contained mesic deciduous forest
vegetation. The change from a mesic to xeric vegetation is believed to be due to the high
level of active volcanism occurring during this time period. Volcanic activity can cause
what appears to be climatic change, causing a moist habitat type to shift into a xeric one
(Cross and Taggart, 1982). Although xeric grassland and shrub-steppe habitat was not
the dominant vegetation type in the area, it was present at lower elevations. Uplift of the
Cascades during the mid Miocene created a rain shadow that decreased the amount of
precipitation east of the mountain range and facilitated the expansion of xeric habitat
(Leopold and Denton, 1987). P. parvus on the Columbia Plateau during the Miocene may
have expanded it's range with the expansion of xeric shrub-steppe vegetation and decline
of woodland habitat An hypothetical scenario that would explain the amount of diversity
in the Columbia Plateau would be that an ancestral Perognathus parvus was present on
the Columbia Plateau during middle Miocene. Beginning in the Miocene, the Columbia
Plateau was dominated by mesic woodland with patches of drier shrub-steppe type habitat
50
at lower elevations (Leopold and Denton, 1987). Advancement of woodland habitat
would have forced widespread P. parvus populations into increasingly smaller patches of
suitable habitat. Diversity that remained in those populations was a remnant of the earlier,
more widespread populations.
Another possible scenario to explain the amount of diversity on the Columbia
Plateau is that there was considerable basaltic volcanism occurring in the area during the
Miocene. Vertical tectonic rotation associated with volcanism caused a sharp folding
pattern to the basaltic plains (Swanson et al., 1989). This folding could have caused
isolation of populations and subsequent genetic differentiation from one another.
Readvancement of shrub-steppe habitat approximately 3 mya (Leopold and Denton, 1987)
would have allowed populations to expand, and genetic diversity within and among
populations would be left over from a time when populations were isolated from one
another and evolving separately. Pleistocene climatic reconstructions show evidence for
severe decreased winter temperatures on the Columbia Plateau. P. parvus most likely
persisted on the Columbia Plateau during the height of Pleistocene glaciation instead of
shifting its range southward off of the Columbia Plateau due to the fact that P. parvus is
able to hibernate, slowing its metabolic rate so that it can withstand periods of unsuitable
weather (French, 1993).
The amount of divergence occurring among haplotypes in the Great
Basin/Colorado Plateau is on average much lower than that on the Columbia Plateau.
This suggests that long-term effective population sizes of P. parvus on the Columbia
Plateau have been higher than in the Great Basin. The low levels of divergence among
51
Great Basin/Colorado Plateau haplotypes, and the wide distribution of haplotype 1
indicate few barriers to dispersal and gene flow in the northern part of the Great Basin, or
may indicate recent expansion of the range of P. parvus into the northern Great Basin
from southern refugia. Pleistocene glacial-interglacial cycles and expansion and
contraction of areas of suitable habitat in the Great Basin may be responsible for the
genetic divergence found among haplotypes from the Great Basin. During the Wisconsin
glaciation, shrub-steppe plant communities existed in the intermontane basins of the Great
Basin (Thompson and Mead, 1982). The distribution of this habitat may have provided
corridors for dispersal of organisms inhabiting i t Subsequent climatic warming forced
shrub-steppe communities upslope to mid elevations where they now exist. Populations of
P. parvus, which had a once more continuous distribution in the intermontane basins,
became isolated from one another when suitable habitat became fragmented.
The correspondence of geological, fossil, and molecular data appears to be
chronological, with the oldest basalt and fossils, and the greatest amount of genetic
diversity occurring in the north on the Columbia Plateau. As one moves southward into
the Great Basin, the relative age of basalt is younger, fossil records appear later, and the
genetic diversity among haplotypes is lower.
There are other taxa that exhibit similar distribution patterns to P. parvus.
Spermophilus beldingi is distributed throughout the Great Basin much like Perognathus
parvus, while S. columbianus is distributed on the Columbia Plateau north of the Blue
Mountains (Hall, 1981). This is very similar to northern and southern populations of P.
parvus. The Blue Mountains orogeny likely had extreme influence on the distributions of
52
animal and plant taxa and the genetic variation present in those taxa that underwent a
vicariance event of such magnitude. There is evidence for the presence of sciurid rodents
in North America subsequent to the Barstovian NALMA (mid Miocene) (Savage and
Russell, 1983). If they were present on the Columbia Plateau as well as in the Great
Basin, populations would have been subjected to the same vicariance events as P. parvus.
I propose the hypothesis that S. beldingi and S. columbianus, and possibly other species
of small rodents exhibit a pattern of genetic variation very similar to that between northern
and southern populations of P. parvus.
On another temporal scale, there are rather high levels of haplotype diversity in
both northern and southern populations of P. parvus. Pleistocene glacial perturbations
probably contributed to geographic variation among populations of P. parvus. Fossil
pollen records show that the overall composition of vegetation in the Great Basin has
changed very little since the Pleistocene, but there have been shifts in the distribution of
the vegetation since the latest glacial episode (Thompson, 1990). Glacial-interglacial
cycles are known to have caused expansion and contraction of biotic environments that in
turn may be related to maintenance of haplotype diversity among populations of P.
parvus.
Future Research
There are some concerns about interpreting molecular variation that comes from
only one source (i.e. a single gene region or genome). It would be prudent to use nuclear
DNA to test conclusions in this study. Assuming that the northern and southern
53
populations have been separated for a long period of time, differences should also
accumulate in the nuclear genome (Avise et al., 1987). Use of allozymes, sequencing of
nuclear DNA, DNA-DNA hybridization, microsatellites, and microcomplement fixation
techniques would provide independent data sets.
The area of overlap between northern and southern populations of P. parvus in
central Oregon contains populations that have both northern and southern haplotypes
present. Because this study involved only the use of mtDNA, which is maternally
inherited, it is impossible to assess whether or not hybridization may be occurring between
northern and southern populations or if this area merely represents a zone of overlap in the
distributions of the two reproductively-isolated populations of P. parvus. In order to test
the hypothesis of hybridization, it would be necessary to use analyses of nuclear DNA (i.e.
allozymes, micro satellite DNA) to determine if there had been any gene flow between
populations. Analyses of nuclear DNA would allow for the determination of
heterozygosity in individuals as well as identification of origin of parental genotypes
present in offspring.
Conclusions
The results of this study have clearly shown that although Perognathus parvus is
monophyletic, it consists of at least two distinct lineages: one on the Columbia Plateau,
north of the Blue Mountains, one in the Great Basin, south of the Blue Mountains, and
possibly another in central Oregon along the southern edge of the Columbia Plateau.
Divergence values between the Columbia Plateau and Great Basin lineages suggest that
northern and southern populations were isolated from one another during the mid to late
Miocene corresponding to the uplift of the Blue Mountains in northern Oregon.
Haplotype diversity on the Columbia Plateau is higher than in the Great Basin and
suggests that effective population size has remained much higher over time.
Chapter 5
Perognathus alticola and P. xanthonotus
Perognathus alticola and P. xanthonotus were originally hypothesized to be
members of the Perognathus parvus Species Group (Osgood, 1900). Williams' (1978)
study of karyotypes supported this hypothesis. Sulentich (1983) reviewed the systematics
of the Perognathus parvus Species Group in southern California and based on biochemical
data, he determined that P. xanthonotus was a subspecies of P. parvus and that P. alticola
alticola and P. alticola inexpectatus should be separated at the specific level. Williams
(1993) then accepted Sulentich's proposal that P. xanthonotus is a subspecies of P.
parvus, but he did not accept the separation of P. alticola subspecies, thus disagreeing
with his own karyotype data showing that both P. alticola and P. xanthonotus had
identical karyotypes to P. parvus from the Great Basin (Table 10).
The status of these two other members of the Perognathus parvus Species Group
has not been satisfactorily resolved. These two species reside in the northern Mojave
Desert of southern California in isolated areas of habitat characterized by Joshua trees
(Yucca brevicauda), sagebrush (Artemisia sp.), juniper (Juniperus sp.) and pinyon pine
(Pinus sp) (Figure 9). Woodrat middens in the Mojave Desert suggest a cooler
temperature regime for the area as evidenced by shadscale-juniper association during the
55
l
56
Table 10. Karyotype characteristics of species included in the Perognathus parvus species group showing similarity between P. p. olivaceous and P. alticola, and similarity between . p. clarus and P. xanthonotus. 2N=diploid number, FN=fundamental number,BA=biarmed chromosome, A=uniarmed chromosome, ST=subtelocentric, SM=submetacentric, A=acrocentric. Data are from Williams (1978).
Autosomal pairs
SPECIES m EN BA A X X
Perognathus parvus olivaceous 54 74 11 15 ST A
Perognathus parvus clarus 54 76 12 14 ST A
Perognathus alticola 54 74 11 15 ST A
Perognathus xanthonotus 54 76 12 14 ST A
57
.a a 'C ^r-!
Q So
rteIc3U<u*£"3o<Z)CO)•N*
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OOO>*.
co3
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£
58
middle Wisconsin. Extirpation of shadscale around 19,000 ybp was followed by the
immigration of pinyon pine and shrubs (Spaulding, 1990), and desertscrub became the
dominant vegetation type during the late Pleistocene and early Holocene. These areas are
likely to be post-Pleistocene refugia, and P. xanthonotus and P. alticola may represent
isolated populations of previously more southern distribution of P. parvus. These areas
also support other taxa that may be Pleistocene relicts (Riddle, pers. comm.). Based on
the distribution, historical ecological evidence and Williams' (1978) karyotype data, I
propose the hypothesis that both P. alticola inexpectatus and P. xanthonotus are
subspecies of P. parvus. P. alticola alticola may truly represent a unique taxon based on
its distribution in relation to the other two species and the possibility that the Sierra
Nevada acts as a barrier to dispersal. P. alticola inexpectatus and P. xanthonotus reside
on the eastern side of the Sierra Nevada, while P. alticola alticola resides on the western
side. Uplift of the Sierra Nevada during the early Miocene would have separated these
populations long ago if they were present at that time.
It is not known whether or not P. alticola or P. xanthonotus are extant. Neither
have been caught in several years (not for lack of effort), and P. alticola is a protected
species in California. For this reason it will be difficult to test the hypotheses presented
above using whole specimens. Recent techniques in isolating and analyzing DNA from
museum skins and bones of extinct species have opened new areas of research (Higuchi et
al., 1984, 1987; Hoss and Paabo, 1993; Paabo, 1985; Paabo, 1989; Paabo et al., 1989).
Isolation of DNA from museum specimens of P. alticola and P. xanthonotus, and use of
primers designed for amplification of small diagnostic sequences of DNA would allow one
59
to compare sequences of these two species with those of P. parviis and determine levels of
divergence among them. Taxonomic status of P. alticola and P. xanthonotus could then
be assessed and biogeographic hypotheses evaluated.
60
APPENDIX I. List of specimens and localities used to construct phylogenetic relationships within Perognathus parvus and calculate genetic distances within and among mtDNA haplotypes in composite populations.
IDAHO: Cassia Co., (LVT 794, LVT 795)
NEVADA: Esmeralda Co., 11 mi. N, 7 mi. W Dyer, T1S, R34E, S l/2 , NE 1/4 sec. 19; (LVT 1931, LVT 1932, LVT 1933, LVT 1934). Nye Co., 36 mi. N, 24 mi. W Mercury, Nevada Test Site, (LVT 1805, LVT 1806); 30 mi. N, 17 mi. W Mercury, Nevada Test Site, (LVT 1807); 1 mi. W Manhattan, (LVT 1957). White Pine Co., (LVT 1929).
OREGON: Baker Co., 1 mi. S, 5 mi. E Baker, T9S, R40E, (LVT 1955, LVT 1956, LVT 1958). Crook Co., 10 mi. N, 5 mi. E Redmond; T B S , R14E sec. 34, (LVT 1921, LVT 1949, LVT 1950, LVT 1951, LVT 1952). Harney Co., 5 mi. S, 4 mi. W Hines; T24S, R30E sec. 8, (LVT 1923, LVT 1925, LVT 1927). Lake Co., 2 mi. S, 8 mi. E Hampton; T22S, R21E, (LVT1943, LVT 1944, LVT 1945, LVT 1946, LVT 1947, LVT 1948). Malheur Co., 1 mi. N, 1 mi. E Basque Station, (LVT 1937, LVT 1938, LVT 1939, LVT 1940, LVT 1941, LVT 1942).
WASHINGTON: Adams Co., 4 mi. S, 6 mi. E Ritzville; T19N, R36E sec. 3, (LVT 1919, LVT 1920). Douglas Co., 16 mi. N, 5 mi. E Wenatchee; T25N, R21W sec. 25, (LVT 1915, LVT 1916). Okanogan Co., 4 mi. S, 3 mi. W Oroville, Wannacut Lake; T39N, R27E, (LVT 1953, LVT 1954); 1 mi. S, 1.5 mi. W Okanogan; T33N, R26E sec. 19,(LVT 1917, LVT 1918).
Bibliography
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