m i d AiO.ZLA' MOLECULAR PHYLOGENY AND EVOLUTION OF ...

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AiO.ZLA'

MOLECULAR PHYLOGENY AND EVOLUTION OF THE

AMERICAN WOODRATS, GENUS NEOTOMA

(MURIDAE).

DISSERTATION

Presented to the Graduate Council of the

University of North Texas in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

John Valentine Planz

Denton, Texas

August, 1992

Planz, John V., Molecular Phvlogenv and Evolution of the American

Woodrats. Genus Neotoma fMuridaeX Doctor of Philosophy (Biology), August,

1992, 164pp., 5 tables, 21 figures, references, 146 titles.

The evolutionary relationships of woodrats (Neotoma) were elulcidated

through phylogenetic analyses of mitochondrial DNA restriction site and allozyme

data. DNA samples from eleven nominal species from the genus Neotoma and

two outgroup taxa, Ototylomys phyttotis and Xenomys nelsoni, were cleaved using a

suite of 17 Type II restriction endonucleases. Mitochondrial DNA restriction

profiles were visualized following electrophoresis of restriction digests via methods

of Southern transfer and hybridization with 3 2 P- and digoxigenin-labeled mtDNA

probes. Restriction mapping resulted in the identification of 37 unique mtDNA

haplotypes among the woodrat taxa examined. Proteins representing 24

presumptive structural gene loci were examined through starch gel electrophoresis.

Binary-coded allozyme data and allozyme frequency data were analyzed using

PAUP and FREQPARS, respectively. Phylogenetic analyses of the mtDNA

restriction site data incorporated three different character type assumptions:

unordered binary characters, Dollo characters, and differentially weighted

unordered characters employing the STEPMATRIX option of PAUP. Proposed

phylogenies for Neotoma are based on majority-rule consensus trees produced

using bootstrap procedures. Phylogenetic analyses of the woodrat data sets

revealed a distinct dichotomy among populations of white-throated woodrats

(N. albigula) suggesting the presence of cryptic species within that taxon. MtDNA

and allozyme data support the specific status of N. devia as distinct from N. lepida,

and additionally reveal the presence of a third cryptic species referable to N.

intermedia among the desert woodrats. Phylogenetic analyses of the genetic data

also suggest subgeneric status for the desert woodrats, which is in agreement with

evidence from morphology. The genetic data revealed a sister group relationship

between N. stephensi and samples of N. mexicana, suggesting the placement of N.

stephensi into the N. mexicana species-group. Neotoma fuscipes and N. cinerea

formed a monophyletic lineage basal to the remaining members of the subgenus

Neotoma which supports the assignment of N. fuscipes to the subgenus Teonoma

with N. cinerea. Although stringent, Dollo parsimony methods produced the best

supported phylogenies among the species of Neotoma. The STEPMATRIX

approach was unable to resolve species relationships within species-groups but

clearly delineated the higher taxonomic levels between species-groups and

subgenera.

ACKNOWLEDGEMENTS

I would like to thank my committee members, Drs. Thomas L. Beitinger, Robert C. Benjamin, Gerard A. O'Donovan, and Duane A. Schlitter for their patience, assistance, and cooperation with the various phases of this project. I also wish to extend special thanks to Dr. Benjamin for sharing his knowledge of molecular biology methodology and his willingness to provide laboratory supplies when they were in short supply. Most of all, I wish to extend my thanks to Dr. Earl G. Zimmerman, whose patience and support while serving as my major professor allowed me to expand this project to the level it has achieved. His moral support and encouragement in the lab and field, especially during difficult times, were crucial to the success of this project.

The completion of this project would not have been possible without the assistance of many colleagues over the past five years. Drs. Sarah George, William Kilpatrick, Terry Yates, Robert Baker, and Robert Dowler provided additional tissue samples of Neotoma and other taxa used in this study. G. Lance Brooks, Darrin R. Akins, Cheryl Lewis, William Gannon, Cheryl Watts, Jesus Maldonado, Ely Garzae, and Drs. R. Edward DeWalt, J. Bruce Moring, Chris McAllister, David Hafner and David Huckaby assisted in the field collection specimens. Sincere thanks are extended to Theresa S. DeWalt, whose companionship and hard work made collection trips and long nights in the lab both successful and enjoyable.

Darrin Akins also provided technical advice and obtained the cloned mitochondrial genome of Mus domesticus from Evan Hermel of Southwestern Medical Center, which was used as a probe in this study. Thanks are extended to Candice Workman, Lesley Duesman, and Latasha Williams for assistance in the laboratory.

Special thanks are also extended to Cliff Tyner (Long X Ranch), G. Fry, and C. Russell for allowing access to their lands, and the Game and Fish Departments of Texas, Arizona, New Mexico, Colorado, California, Utah, West Virginia, Oklahoma, and Kansas for granting permits for the collection of specimens.

Portions of this work were supported by grants from the Theodore Roosevelt Memorial Fund of the American Museum of Natural History, the North American Mammal Research Fund of The Carnegie Museum of Natural History, and financial contributions from my parents, Hans L. and Anna Planz, to whom this work is dedicated.

in

TABLE OF CONTENTS

Page

LIST OF TABLES v

LIST OF FIGURES vi

CHAPTER

I. Introduction 1

II. Methods 24

III. Results 38

IV. Discussion 65

V. Conclusions 90

APPENDIX I 93

APPENDIX II 101

APPENDIX III 104

APPENDIX IV 117

APPENDIX V 120

APPENDIX VI 127

APPENDIX VII 134

LITERATURE CITED 145

IV

LIST OF TABLES

Table Page

1. Distribution of estimates of mitochondrial DNA sequence divergence (6) across various taxa 6

2. Taxonomic arrangement of the Genus Neotoma and related genera 10

3. Goodness-of-fit statistics for consensus trees produced by Wagner, Dollo, and Generalized Parsimony methods for the Neotoma lepida and Neotoma floridana species groups 44

4. Goodness-of-fit statistics for consensus trees produced by Wagner, Dollo, and Generalized Parsimony methods for the genus Neotoma 53

5. Genetic distance matrix consisting of Rogers' distance and estimated level of nucleotide sequence divergence (5) for Neotoma and two outgroup species. . 59

LIST OF FIGURES

Figure Page

1. Geographic distribution of Neotoma floridana, N. magister, N. micropus, N. stephensi, and N. angustapalata 11

2. Geographic distribution of Neotoma albigula and Hodomys alleni 12

3. Geographic distribution of Neotoma fuscipes, N. mexicana, and N. chtysomelas 13

4. Geographic distribution of members of the Neotoma lepida species group 14

5. Geographic distribution of Neotoma cinerea 15

6. Geographic distribution of Neotoma goldmani, N. phenax, Xenomys nelsoni, and Ototylomys phyllotis 16

7. Geographic distribution of collection localities of Neotoma, Ototylomys phyllotis, and Xenomys nelsoni used in this study 25

8. Bootstrapped consensus trees produced by employing Wagner (above) and Dollo (below) parsimony methods for the Neotoma floridana species group. . 40

9. Bootstrapped consensus tree produced by the Generalized Parsimony criteria described in the text for the Neotoma floridana species group 41

10. Bootstrapped consensus tree produced by employing Wagner (above) and Dollo (below) parsimony methods for the Neotoma lepida species group 46

11. Bootstrapped consensus tree produced by employing the Generalized Parsimony criteria described in the text for the Neotoma lepida species group.. 47

12. Bootstrapped consensus tree produced by employing Wagner (above) and Dollo (below) parsimony methods for the genus Neotoma 50 13. Bootstrapped consensus tree produced by employing the Generalized Parsimony criteria described in the text for the genus Neotoma 51

14. Bootstrapped consensus tree produced from mtDNA restriction sites and binary coded allozyme data subjected to Wagner Parsimony criteria 56

VI

15. Bootstrapped Wagner consensus tree (above) produced from binary coded allele data, FREQPARS cladogram produced from allele frequency data (below) 57

16. Phenogram produced from UPGMA cluster analysis of allozyme data for 14 Neotoma taxa and two outgroup species 60

17. Phenogram produced from UPGMA cluster analysis of 14 Neotoma taxa and two outgroup species using the estimated level of nucleotide sequence divergence 62

18. Phenogram produced under least squares criteria for 14 Neotoma taxa and two outgroup species using the estimated level of nucleotide sequence divergence 63

19. Proposed ancestral drainage pattern of the Rio Grande River during the late Pliocene through early Pleistocene 71

20. Proposed variations in the drainage pattern of the Rio Grande River in the vicinity of the Franklin Mountains during the Pleistocene 73

21. Geographic location of critical fossil sites for the Neotoma lepida species group referred to in text 85

vn

CHAPTER I

INTRODUCTION

Background.- An ultimate goal in the study of organismal diversity is an

understanding of the speciation process. Traditionally, the vast majority of

organisms to which the designation of "species" has been applied stem from

comparisons of morphological similarity. The organization of these taxa into

schemes of higher taxonomy, i.e. species-groups, subgenera, and genera, has

therefore relied on characteristics that may be subject to convergence, which may

be environmentally or stocastically mediated, and did not take into account the

genetic heritability of the traits in question (Templeton, 1981). With the advance

of current techniques and analytical procedures, the evolutionary change

accompanying the speciation process can be studied using several unique and

often independent characters. A number of genetic markers have been employed

in a variety of studies which attempt to correlate genetic changes with population

demographics and speciation events (Avise, 1976; Avise et al., 1983; Patton and

Sherwood, 1983; Jansen et al., 1990; Mindell and Honeycutt, 1990). Thus, at this

time, the genetic consequences of the speciation process can be studied from

levels of chromosomal morphology, alterations in structural nuclear genes,

mitochondrial DNA polymorphism, and DNA sequence analysis. Each of these

1

2

procedures has traditionally been employed independently. By applying these

approaches in concert to a model group whose species are at various levels of

differentiation, concordance among the various stages of evolution and genetic

differentiation can be measured.

One of the most thoroughly employed techniques is the analysis of

structural gene variability by allozyme electrophoresis (Selander et al., 1971).

Studies of nuclear gene products have been used to visualize the conversion of

intra- to interspecific variability accompanying the speciation process among taxa

of insects, teleost fish, reptiles, mammals, and birds (Ayala et al., 1975; Avise and

Smith, 1974; Zimmerman et al., 1978; Zimmerman and Nejtek, 1977; Smith and

Zimmerman, 1976). From these studies, it has been found that genetic distance

generally increases as higher levels of differentiation are reached, however,

it is evident that the degrees of divergence are not consistent among animal taxa

nor within specific groups. For instance, among rodent taxa, interspecific levels of

genetic distance range from 0.036 among semispecies of ground squirrels,

(Spermophilus, Cothran et al., 1977) to 0.64 for voles of the genus Clethrionomys

(Tegelstrom, 1987; 1988). A variety of factors, including effectiveness of

geographic and reproductive barriers, population demographics, and mode by

which speciation events occur, all play unique roles in determining the degree of

genetic distinctiveness among related species.

Recently, genetic markers of cytoplasmic origin, such as mitochondrial

DNA (mtDNA) and chloroplast DNA (cpDNA), have found rapid acceptance in

3

both intra- and interpopulation studies (Avise et al., 1987; Jansen et al., 1990).

The mitochondrial genome of mammals consists of a covalently closed, circular

DNA molecule, 16.3 to 19.2 kbp in length, coding for 13 proteins, two rRNAs, and

22 tRNAs (Brown, 1985). The gene content of mtDNA varies little among

multicellular animals (Rastl and Dawid, 1979). MtDNA evolves primarily by

nucleotide substitution in parts of the control region (D-loop) and at codon

positions that do not cause amino acid replacement (Brown, 1983). Substitutions

do not appear to be random, since there is a strong bias for transitions over

transversions among closely related taxa (Brown et al., 1982). A useful feature of

mtDNA is its rapid rate of molecular evolution compared to that of the nuclear

genome. Brown et al. (1982) estimated the mtDNA genes of hominoid primates

evolve five to ten times faster than single copy nuclear genes. However the rates

determined for primates cannot be used as indicative of other mammals due to a

graded decrease in evolutionary rate in the descent of the simian primates (Bailey

et al., 1991). Increased rates of mtDNA evolution are also reported in

rodents and frogs (Moritz et al., 1987). However, lower rates (two times for

Drosophila) and an equal rate (for sea urchins) have been reported (Solignac et

al., 1986; Smith, 1988). MtDNA is maternally inherited, and as a consequence it

offers great benefits as a genetic marker. While nuclear DNA can introgress

through male or female mediated hybridization between species or populations,

mtDNA is transmitted only by the female (Avise and Lansman, 1983).

The value of mtDNA as a phylogenetic tool can further be exemplified by

4

its sensitivity to two prominent factors in speciation: population subdivision and

population growth (Avise et al., 1988). In subdivided or rapidly expanding

populations, localized retention of variable mtDNA genotypes will aid in

establishing polymorphism among the resultant daughter species/populations. The

degree of mtDNA polymorphism within a species is reflected by the degree of

gene flow resulting from dispersal between subdivided populations.

To date, work has been done relating the structuring of the mtDNA

genome of populations, closely related species, and hybrid populations (Table 1).

The inclusion of mtDNA into a suite of characters for a phylogenetic analysis

contributes an independent genetic system that, while linked to the nuclear

genome due to split production of gene products integral to the electron transport

chain and ATP synthesis, evolves at its own unique rate and lacks the complicated

features of introns, repetitive DNA, and recombination found in the nuclear

genome. Although chromosomal and allozymic methods target the nuclear

genome, they are uncoupled with regard to the level of evolution and information

they can provide. The strictly maternal mode of inheritance of the mitochondrial

genome also contributes greatly to the sorting processes needed for establishing

lineages (Neigel and Avise, 1986).

Characteristics of the mitochondrial genome lead to the proposal of several

possible outcomes for a speciation event, dependent upon the geographic origin of

samples of the gene pool involved in the speciation event and the degree of

isolation provided by a vicariance barrier. If random populations within a parent

5

species are isolated by a speciation event, the population of mtDNA within the

daughter species would exhibit paraphyletic status for an extended number of

generations. However, if there is some degree of geographic sorting and

differentiation of individuals at the extremes of the parental range, monophyletic

status is achieved in relatively fewer generations (Neigel and Avise, 1986).

Testing of theoretical concepts such as these that have been obtained by computer

simulations relies on studies which employ several independent genetic markers

across a broad range of taxonomic levels. Studies, such as those on Drosophila

(Solignac et al.,1986; Latorre et al., 1988), Lepomis (Avise and Saunder, 1984),

Onychomys (Riddle, 1990), and Peromyscus species groups (De Walt et al,

submitted; Avise et al., 1983, Zimmerman et al., 1978) have addressed the level of

single generation daughter species formation. Additional studies on levels of

genetic differentiation between named species-groups and higher levels of

organization which reflect possible evolutionary affinities would increase

understanding of the long term changes in the overall genome accompanying

speciation during the development of a species assemblage, in addition to

formulating a phylogenetic test of existing taxonomic arrangements.

A common point of difficulty encountered in addressing phylogenetic issues,

with regard to taxonomy, revolves around species concepts and the definition of

species and other nomenclatoral categories. The most popularly held of the

modern species concepts is the biological species concept. Biological species, as

defined by Mayr (1969), are "groups of interbreeding natural populations that are

Table 1. Distribution of estimates of mitochondrial DNA sequence

divergence (S of Nei and Li, 1979) across various taxa.

Taxa S Source

Intraspecific

Geomys pinetis 0.034 Avise et al., 1979

Rattus 0.05-0.09 Baverstock et al., 1983

Peromyscus sp. 0.002-0.059 Avise et al., 1983

Odocoileus virginiana 0.014 Carr et al., 1986

Clethrionomys glareolus 0.008-0.01 Tegelstrom, 1988

C. rutilus 0.127 Tegelstrom, 1988

Lepomis macrochirus 0.075 Avise and Saunder, 1984

L. cyanellus 0.014 Avise and Saunder, 1984

Artibius jamaicensis 0.004 Pumo et al., 1988

A. j. jamaicensis -

A. j. schwartzi 0.092-0.105 Pumo et al., 1988

Ursus americanus 0.012 Zimmerman, unpubl.

Interspecific

Mus sp. 0.05-0.14 Ferris et al., 1983

Rattus sp. 0.16 Baverstock et al., 1983

Lepomis sp. 0.206* Avise and Saunder, 1984

Clethrionomys sp. 0.139 Tegelstrom, 1988

Peromyscus sp. 0.02-0.16 Avise, 1986

Apodemus sp. 0.10 Tegelstrom, 1988

Spermophilus sp. 0.028-0.075 MacNeil and Strobeck, 1987

Drosophila sp. 0.02-0.097 Solignac et al., 1986

* This value represents a median value due to the biasing effect values of >. 1.0 have on an arithmetic mean.

reproductively isolated from other such groups". This concept relies heavily on

demic gene flow as a cohesive factor for maintainance of specific boundaries, but

is inflexible with regard to cases of secondary contact and chance hybridization,

which would be judged as evidence for incomplete speciation. Although very

useful, the biological species concept lacks a structural framework with which

closely related taxa can be examined with regard to their evolutionary

relationships.

During this study the evolutionary species concept will be the foundation

for assessing specific rank and designation. The evolutionary species concept

(Simpson, 1961) can be regarded as an appropriate bridge principle (Hempel,

1966) in the study of evolutionary process with regard to pattern of descent.

Wiley (1978) defines an evolutionary species as "a single lineage of ancestor-

descendant populations which maintains its identity from other such lineages and

which has its own evolutionary tendencies and historical fate." A corollary of this

concept states that a phylogenetic tree is actually composed of evolutionary

8

species, and that all terminal taxa and linkages between terminal taxa are species

(Wiley, 1981). This concept, however, also dictates that species must be

reproductively isolated from each other, such that they maintain separate

identities, tendencies, and fates. This requirement again brings up the difficulties

when secondary contact and hybridization are encountered. Simpson (1961),

however, clarifies the context in which hybridization should be taken with regard

to the evolutionary species concept: "the important question is not whether two

species hybridize, but whether two species do or do not lose their distinct

ecological and evolutionary roles. If, despite some hybridization, they do not

merge, then they remain separate species in the evolutionary perspective."

The term cryptic species will be used throughout this manuscript in

replacement of the terminology "sibling species" introduced by Mayr (1942).

Cryptic species are morphologically similar or identical species that represent

reproductively isolated gene pools that may or may not be closely related

evolutionarily. The term "sibling" has a connotation referring to closely related

taxa, possibly to be confused with "sister species" or "sister groups" which refers to

species that hypothetically share a genealogical common ancestor.

Neotoma as a model of speciation.- American woodrats of the Genus

Neotoma provide an excellent model for the study of genetic differentiation

accompanying the speciation process. The genus has been divided into five

subgenera, Neotoma, Hodomys, Homodontomys, Teonoma, and Teonopus,

comprising twenty-one recognized taxa (Goldman, 1910; Hall, 1981; Ryckman et

9

al., 1981) (Table 2). Within the subgenus Neotoma, there are several "species-

groups" and nominal taxa which are comprised of species and populations at

various levels of differentiation, from reproductively isolated forms to those which

are largely allopatric but hybridize in narrow zones of contact (Birney, 1976;

Huheey, 1972). The other four subgenera represent monotypic forms.

Woodrats are considered to be members of the cosmopolitan rodent family

Muridae, belonging to the New World subfamily Sigmodontinae (Carlton, 1980).

The earliest fossil record of Neotoma is from the middle Hemphillian (Late

Miocene), approximately 6.6 million years ago (Dalquest, 1983). Neotoma ranges

over much of North and Central America, with the center of its distribution lying

in northern Mexico (Carleton, 1980) (Figure 1-6). Although the group is thought

to have had a South American origin, two taxa have ranges extending north into

New England and the Yukon Territory {Neotoma magister and Neotoma cinerea,

respectively). The southernmost limit of the genus is in Nicaragua, represented by

N. chrysomelas (Hall, 1981). The majority of species are associated with more

xeric desert/grassland and montane biomes. Woodrats tend to be dietary

generalists when associated with plant communities of high species diversity

(Vaughan, 1990). When associated with plant communities of low species

diversity, they tend to be dietary specialists, as is the case when several woodrat

species are found in sympatry (Dial and Czaplewski, 1990).

Woodrats are medium sized, long-tailed rodents attaining adult weights of

approximately 100-400g. The molariform teeth are prismatic and flat-crowned,

10

Table 2. Taxonomic arrangement of the Genus Neotoma and related

genera (Carleton, 1980; Goldman, 1910; Hall, 1981; Ryckman et al., 1981).

(• denotes taxa included in this study).

subgenus Neotoma

-floridana species-group — - mexicana species-group ~

Neotoma floridana • Neotoma mexicana

Neotoma magister Neotoma chrysomelas

Neotoma micropus -- unassigned species -

Neotoma albigula Neotoma goldmani

Neotoma varia • Neotoma stephensi

Neotoma nelsoni • Neotoma fuscipes

Neotoma palatina subgenus Teonoma

Neotoma angustapalata • Neotoma cinerea

lepida species-group -- subgenus Teonopus

Neotoma lepida • Neotoma phenax

Neotoma devia

Neotoma anthonyi Hodomys alleni

Neotoma bryanti • Xenomys nelsoni

Neotoma bunkeri Ototylomys phyllotis

Neotoma martinensis

11

£•] N. magister

| | N. ffoddana

M mtcropus

• N. steptensf

N. anQustfpaiata kUomettra 0 200 400

-40

20

Figure 1. Geographic distribution of Neotoma floridana, N. magister, N.

micropus, N. stephensi, and N. angustapalata.

12

N. aid/gum

H. alien f

kilometers

0 200 400

-30

- 2 0

Figure 2. Geographic distribution of Neotoma albigula, and Hodomys

(Neotoma) alleni.

13

*

N. fusc/pes

N. mexicana

N. chrysomefas

kilometers

0 200 400

Figure 3. Geographic distribution of Neotoma fuscipes, N. mexicana, and N.

chrysomelas.

14

f

a

b c

d

— 40

N. devia

N. iep/da

N. /. intermedia

N. anthonyi

N. martinensis

N. bryanti

N. bunkeri

— 30

kffometers

200 400

Figure 4. Geographic distribution of members of the Neotoma lepida species

group.

15

N. c/nerea

J-l kifometers

0 200 400

Figure 5. Geographic distribution of Neotoma cinerea.

16

H Xenomys nelson!

£3 Ototyfomys phyHotis

Q Neotoma phemix

j<3 Neotoma goktmanf kifomifrs 0 200 400

Figure 6. Geographic distribution of Neotoma goldmani, N. phenax, Xenomys

nelsoni, and Ototylomys phyllotis.

17

and the caecum is well inflated, features that would be expected in rodents

associated with a fibrous diet of low nutritional value (Fitch and Rainey, 1956;

Vorhies and Taylor, 1940). Pelage is typically soft and ranges in coloration from

pale buff among desert dwellers to dark russet among inhabitants of more mesic

or semitropical habitats (Hall, 1981; Vaughan, 1990). Woodrats are long lived in

comparison to other small rodents, with lifespans of three to five years being

reported (Feldman, 1935; Finley, 1958; Fitch and Rainey, 1956; Linsdale and

Tevis, 1951). The group, as a whole, is noted for its construction of conspicuous

dens ranging in size from a small accumulation of sticks and debris among rocks

or cacti to large collections of material several cubic meters in volume. These

dens serve as buffers against temperature extremes and safe refuge from

predators (Finley, 1958; Vaughan, 1990). Dens may be accumulations of material

deposited over several generations of a species or of alternating species occupying

the den over time (Dial and Czaplewski, 1990; Finley, 1958, 1990). These

middens have been examinrd extensively by paleoecologists due to the well

preserved fossil plant and animal materials obtainable from them (Spaulding,

1990, 1983; Thompson and Hattori, 1983, 1990; Van Devender, 1973, 1990a,

1990b).

The systematics of the genus Neotoma has received some attention throughout

the 1900's, originating with Goldman's (1910) revision of the genus. Several

putative taxa have since been synonomyzed once more precise information on

species ranges became available. Most of the systematics of the group is based on

18

external phenotypic and cranial characteristics, but several attempts have been

made to add new characters which would better segregate the sometimes

confusing taxa. Most notably used were the male accessory organs, the baculum,

and glans penis (Burt and Barkalow, 1942; Hooper, 1960). Due to the nature of

these structures and the link that has been made in their importance as

mechanisms of reproductive isolation, the current systematic arrangement of the

genus is based on these characters (Carleton, 1980).

Within the subgenus Neotoma, there has been increased interest recently in

establishing proper evolutionary histories for the members of the N. floridana and

N. lepida species groups. Members of the former group have been addressed with

craniometric and allozymic studies (Anderson, 1969; Birney, 1973; Zimmerman

and Nejtek, 1977). Three species in this group, N. floridana, N. micropus, and N.

albigula, are reported to represent the classic definition of semispecies proposed

by Mayr (1963). They appear to have been isolated in the past and have recently

established secondary contact in parts of their ranges. Hybridization between N.

micropus and N. albigula has been reported at locations at the northern limits of

both species' ranges in southeastern Colorado and western Oklahoma (Birney,

1976; Huheey, 1972). However, over most of their ranges where these two species

are sympatric, they appear to maintain their genetic integrity by habitat

separation.

Neotoma floridana has been reported to hybridize with N. micropus at a single

locality in northern Oklahoma where the two species are sympatric (Birney, 1973;

19

Spencer, 1968). Birney (1976) describes this as a case of stasipatric distribution

(Key, 1968), where impaired fecundity of hybrids rather than ecological

competition is the major factor preventing broad species overlap. However, at a

sympatric locality in south-central Texas, Dalbey (1980) found no evidence of

hybridization between these two species.

Within the N. floridana group, there has recently been considerable debate as

to the status of the northeastern subspecies, N. f . magister. Goldman (1910)

recognized this taxon as a species belonging to the "pennsylvanica" group of the

subgenus Neotoma. His separation of this group from the N. floridana group has

often been interpreted to mean that they are distantly related. Based on bacular

and craniometric studies, N. magister was relegated to subspecific status under N.

floridana (Burt and Barkalow, 1942; Schwartz and Odum, 1957). However,

attempts to mate this subspecies with other subspecies in N. floridana were

unsuccessful, leading Birney (1976) to suspect that N. f . magister and N. floridana

are cryptic species that maintain separate gene pools. Recently, Hayes (1990)

studied allozymes, morphology, and mtDNA of N. floridana from eastern North

America and found significant differences between N. f . magister and other

members of N. floridana, supporting the validity of N. magister as a valid species.

The taxonomy of woodrats in the Neotoma lepida group has also been

addressed recently. Chromosomal studies of N. lepida revealed a 2N = 52

karyotype for the species, with marked variability in the autosomal arm number in

populations east and west of the Colorado River in Arizona and California

20

(Mascarello and Hsu, 1976). Variations in allozymes, differentially stained

chromosomes, penile morphology, and cranial characters are concordant,

indicating the two chromosomal races represent cryptic species. In comparisons of

the bacula and glans penes, N. I intermedia, a western coastal subspecies,

possesses significantly different characters compared to those of inland desert

woodrats (Mascarello, 1978; D. Huckaby, pers. comm.). Although allozymic

markers verified the distinctness of this form, Mascarello (1978) did not address

the issue in his study. The evidence provided by this karyotypic and allozymic

study, however, has been questioned based on a morphometric analysis of these

taxa in Arizona (Hoffmeister, 1986).

Various levels of speciation represented by the Genus Neotoma have yet to be

studied. As demonstrated above, the evolutionary history of even the most well

studied forms remains questionable. The relationships of other well differentiated

species such as N. stephensi and N. mexicana to other members of the subgenus

Neotoma have yet to be clarified, as are the relationships of several endemic

island forms to their presumed mainland counterparts. On a larger scale, the

validity of and relationships among the five subgenera within the genus Neotoma,

along with their position within the family Muridae, has received only minor

attention (Carleton, 1980).

OBJECTIVES

The objective of this study is to determine the degree of nuclear and mtDNA

21

differentiation among members of the genus Neotoma and use the resultant data

base in a phylogenetic analysis of the group. From this data base, the degree of

genetic change accompanying the speciation process can be estimated across

several levels of organization, i.e., between subspecies, semispecies, cryptic species,

members of species-groups, and between species-groups and subgenera within the

genus. By considering as many of the species as possible within an assemblage

such as Neotoma, the genetic changes accompanying speciation can be correlated

with cladogenesis of the several levels of organization presented by their

taxonomy. These data will be employed in a phylogenetic analysis to determine

the evolutionary relationships of the members of this group and with closely allied

members of the family Muridae, which will serve as outgroups. Four major null

hypotheses will be tested during this study (see Materials and Methods for

specifics).

Hypothesis 1: Differentiation of the mitochondrial genome is not concordant with

that found in the nuclear genome. This hypothesis will be rejected if levels of

differentiation of the nuclear genome differ significantly from restriction site

differentiation of the mtDNAs of the various species. This will be accomplished

by converting mitochondrial restriction site and allozymic data into matrices of

genetic distance measurements which can be analyzed statistically.

Hypothesis 2: No relationship exists between the degree of differentiation of the

mitochondrial genome and levels of speciation. Hypothesis 2 will be rejected if

levels of mtDNA sequence divergence increase significantly in an orderly fashion

22

as pair-wise comparisons are made ranging from closely-related to more distantly

related taxa. Should this hypothesis be rejected and a pattern of increasing

sequence divergence be found as higher taxonomic levels of organization are

crossed, a careful examination of the phylogenies which result from cladistic and

phenetic analyses of the data and a study of the fossil record will be employed to

develop a time frame for the evolutionary separations of the various groups and

species. A reasonable estimate of the unique rate of molecular evolution with

regard to the mitochondrial genome can be developed from which estimated times

of divergence of the mitochondrial genomes can be compared against the fossil

record and biogeographical events which may have been responsible for lineage

splitting.

Hypothesis 3: Rates of differentiation of the nuclear and mitochondrial genomes

do not differ. Hypothesis 3 will be rejected if significant deviations between the

rates of differentiation of the nuclear and mitochondrial genomes are detected

among the various taxa. These results can be compared to data already published

on hominoid primates (Brown et al., 1982; Smouse and Li, 1987), other

vertebrates, and invertebrates (reviewed in Moritz et al., 1987).

Hypothesis 4: Rates of mtDNA differentiation do not differ among the taxa in

this assemblage. Hypothesis 4 will be rejected if significant differences in log-

likelihood values based on time-depth estimates are found among comparisons of

the clades described by the phylogenetic analysis. Heterogeneity in the rates of

mtDNA evolution between the various taxa of Neotoma could obscure proper

23

lineage relationships. Felsenstein (1985) demonstrated that if events of parallel

evolution occur in greater number than unique or nonreversible changes,

parsimony analyses can suggest incorrect topologies as the number of characters is

increased.

In addition, the monophyletic status of the members of the genus Neotoma

will be tested by outgroup analysis with members of closely related taxa. The

phylogenetic relationships among the members of the genus Neotoma derived

from this analysis will be tested against phylogenies presented from strictly

morphological studies (Carleton, 1980; Hoffmeister, 1986).

CHAPTER II

METHODS

Collection of specimens.-Woodrats (n=257), representing 11 of the 21 named

species of Neotoma (see Table 2), were collected throughout their ranges (Figure

7) using Sherman™ and Tomahawk™ livetraps following the guidelines for

acceptable field methods approved by the American Society of Mammalogists

(Committee, 1987). Additionally, tissue samples of 51 individuals representing

eight species of Neotoma, three samples of Xenomys nelsoni, and five samples of

Ototylomys phyttotis were obtained through the assistance of several collectors.

Specific locality, sample size, and specimen information are provided in Appendix

I.

Animals were prepared as standard museum skin and skull, skin and

skeleton, full skeletons or fluid preserved specimens and will be deposited in the

mammal collection of the Carnegie Museum of Natural History, Pittsburgh, PA

(CM). Additional specimens are deposited at the following institutions: Museum

of Southwestern Biology, University of New Mexico (MSB), The Museum, Texas

Tech University (TTU), Los Angeles County Museum (LACM), Angelo State

Natural History Collection (ASNHC), and University of Vermont (UV). Heart,

liver, kidney, and muscle tissues were removed and placed in liquid nitrogen until

24

25

N. aibigula a N. cinema • N. devia A N. florktana

N. fusdpes o N. • N. magister o N. mexfcana a N m/cropus • N. phenax © N. Stephens! • Ototyfomys phyl/otfs <$ Xenomys nelson!

kilometers

0 400 800

Figure 7. Geographic distribution of collection localities of Neotoma,

Ototyfomys phyllotis, and Xenomys nelsoni used in this study.

26

they could be stored in an ultracold freezer (-80° C) prior to DNA isolation.

Karyotypes of selected individuals and populations were prepared following a

modification of the technique of Lee and Elder (1980) (Appendix II). Accessory

collections, such as parasites, preserved glans penes, karyotypes, and tissue

samples were retained for later use or sent to corroborating specialists.

Mitochondrial DNA was extracted according to four different procedures

throughout the course of this study, dependent on the type of sample available,

and the amount and purity of mtDNA required.

Isolation of mtDNA.--During the early phase of the study, mtDNA was

isolated following the procedure developed by Zimmerman et al. (1988)

(Appendix III). Standard sucrose gradient methodology was used to isolate a

relatively pure mitochondrial fraction. The mitochondrial suspension was treated

with DNAase I to reduce background contamination caused by nuclear DNA.

Following incubation and pelleting of the mitochondria, the action of DNAase was

arrested by treating the mitochondria with Proteinase K and SDS (sodium dodecyl

sulphate). Extraneous proteins were extracted through a series of treatments with

buffered phenol, phenolxhloroform, chloroform, and hydrated ethyl ether. The

mtDNA was then ethanol precipitated and used in subsequent restriction enzyme

digests. Fragment profiles from these digests were visualized by electrophoresis in

0.7% and 1% agarose gels. Gels were stained in ethidium bromide (0.5 Mg/ml) for

15 min. Excessive fluorescence was removed from the gel medium by destaining

27

in 1 mM MgS04 for up to one hour. Resultant gels were visualized under

ultraviolet light (300 nm) and photographed with Polaroid T-55 film. This

technique was employed when several samples from a population were being

tested to determine levels of intraspecific polymorphism.

Purified mtDNA for restriction mapping was isolated employing a

modification of the techniques of Lansman et al. (1981) (Appendix IV). The

procedure involved homogenization of the tissue and isolation of a fairly purified

mitochondrial fraction. The mitochondria were then lysed and subjected to

cesium chloride (CsCl) density gradient ultra-centrifugation which separated the

covalently closed, circular mtDNA from nuclear DNA (nDNA), RNA, protein,

and glycogen. The DNA was visible under UV illumination as fluorescent bands,

and the bands of mtDNA were removed with a hypodermic needle. For samples

of rare or difficult-to-obtain species, the nuclear DNA was also retained. The

DNA was prepared for restriction analysis by removing the ethidium bromide

through 1-butanol extractions, followed by dialysis against TE (0.01 M Tris-HCl,

0.5 mM EDTA, pH 8.0) to remove the CsCl (Maniatis et al., 1982).

A rapid alkaline-lysis technique, adapted from the procedures of Tamura

and Aotsuka (1988) (Appendix V) was implemented when small quantities of

mtDNA from several individuals from a population were under investigation to

determine species identities from populations suspected of containing sympatric

individuals of cryptic species.

Total genomic DNA was isolated for mtDNA analysis from samples of

28

Xenomys nelsoni following a modification of Maniatis, et al. (1982) (Appendix VI).

These samples consisted of approximately 0.3 g of muscle tissue for which other

isolation techniques are inappropriate.

Restriction analvsis.--The following 17 restriction endonucleases, purchased

from Stratagene (La Jolla, CA), New England Biolabs (Beverly, MA), and Gibco

BRL (Gaithersburg, MD), were used in the analysis: Apa I, Ava I, Bam HI, Bgl I,

Bgl II, Bsp 106, BstE II, ZfcfN I Dra I, Eco RI, Eco RV, Hinc II, Kpn I, Pst I, Pvu

II, Sal I, and Stu I. Single and double restriction digestions were carried out on

approximately 0.05 /xg of DNA in a total volume of 100 jliI, according to the

manufacturer's recommended temperature and buffer composition.

Following digestion, the mtDNA was precipitated in three volumes of ethanol

and 30 /il of 3 M sodium acetate, pH 5.2 (Maniatis et al., 1982) and dried in vacuo

in a Savant Speed Vac™ (Savant Instruments, Inc., Farmingdale, NY). Following

reconstitution with sterile H 2 0 and carrier dye (0.25% bromophenol blue, 0.25%

xylene cyanol, 15% Ficoll-type 400 in water) (Maniatis et al., 1982), the mtDNA

fragments were separated by molecular weight using agarose gel electrophoresis.

Electrophoresis was carried out on 0.7%, 1.0%, or 1.2% agarose gels (Gibco BRL,

Gaithersburg, MD) overnight at 32 volts or for approximately 6 hours at 65 volts

in 1 X TBE (0.089 M Trisma, 0.089 M Boric acid, 0.002 M EDTA, pH 8.0).

Three different size standards were used throughout the study to obtain estimates

of DNA fragment sizes. Lambda phage DNA digested with Hind III, a Hind

29

III/Eco RI double digest, and a DNA Analysis Marker System (Gibco BRL,

Gaithersburg, MD; cat. no. 4401SA) was run on each gel as a molecular weight

standard.

Visualization of mtDNA.--MtDNA fragments were visualized through

modifications of Southern hybridization (Southern, 1975) with recommendations

by DuPont (Boston, MA) for their GeneScreenP/ws™ nylon (Appendix VII).

Denatured DNA was transferred onto Magna NT™ nylon (Micron Separations

Inc.) and then immolilized by UV (254 nm) crosslinking.

Evan Hermel (Southwestern Medical University, Dallas, Texas) kindly

provided the cloned mitochondrial genome of Mus domesticus (New Zealand

Black Strain) which was used as a mtDNA probe. The mtDNA genome is

contained in four pUC18 plasmids, with mtDNA fragment sizes of approximately

7.2, 5.0, 2.7, and 1.0 kilobase (kb) pairs. Purified recombinant DNA was isolated

according to the technique described by Tanaka and Weisblum (1975) (Appendix

VIII).

During the course of the study, two different probe labeling procedures

were employed. Initially, the four mtDNA probes, along with the molecular size

standard, were labeled with 32P-dCTP, 3000 Ci/mmol (DuPont, Boston, MA) in a

random primed labeling reaction (Boehringer-Mannheim, Germany) (Appendix

IX) . During the latter part of the study, The Genius™ non-radioactive

Digoxigenin labeling system (Boehringer-Mannheim, Germany) was employed

30

(Appendix IX). The Genius™ system, in conjunction with Lumiphos™ 530

(Lumigen, Inc., Detroit, MI) provided a safer and more rapid method of

visualizing DNA restriction fragments with equal or greater sensitivity than that

obtained using 32P-labeled probes. In both cases, hybridization was carried out

overnight at 65° C in a shaking waterbath or Hybridizer™ Hybridization Oven

(Techne, Inc., Princeton, NJ). Filters were then washed and prepared for

visualization, as outlined in Appendix IX for the specific method being employed.

The filters were then exposed to x-ray film (X-OMAT AR, Eastman Kodak

Company, Rochester, NY) from 2-48 hours depending on probe system used.

Filters visualized with 32P-labeled probes employed a Cronex™ Quanta III

intensifier (DuPont, Boston, MA) to improve band visualization.

Restriction site mapping.~The restriction sites produced by each enzyme for a

mtDNA sample were mapped with regard to position relative to one another

based on the results of double restriction digests. Restriction site positions were

denoted based on a conserved Bgl I site which was designated as "0.0". Fragments

of approximately 0.25 kilobase pairs (kbp) were resolved with the techniques

employed. Due to limitations in the accuracy of determining fragment sizes,

restriction site positions were determined within an estimated range of 0.2 kbp.

Restriction sites were considered to be homologous on the different maps if the

sites mapped within a 0.2 kbp region for the taxa in question. Side-by-side

comparisons of double restriction digests for different species were used when

31

necessary to confirm the homology of map positions.

Allozvme electrophoresis.-Tissues (liver and muscle) were ground in

double-distilled water, centrifuged at 1,000 x g for 10 min. and stored at -80° C.

Starch gels are prepared as 12% suspensions of hydrolyzed starch (1.25:1; Sigma

Chemical Company, St. Louis, MO: Electrostarch Company, Madison, WI).

Electrophoretic techniques followed Selander, et al. (1971), Ayala, et al. (1974),

and Bohlin and Zimmerman (1982).

Proteins representing 24 presumptive structural gene loci were examined as

follows: superoxide dismutase (E.C. 1.15.1.1; SOD), two peptidases, alanine-1-

leucine (E.C. 3.4.11 or 13; P-ALL-1, P-ALL-2 and 1-valyl-l-leucine P-VLL-1, P-

VLL-2), malic enzyme (E.C. 1.1.1.40; ME-1, ME-2), esterase (E.C. 3.1.1.1 ;EST-1,

EST-2, EST-3), a-glycerophosphate dehydrogenase ( E.C. 1.1.1.8; a-GPD-1),

xanthine dehydrogenase (E.C. 1.2.1.37; XDH-1), lactate dehydrogenase (E.C.

1.1.1.27; LDH-1 and LDH-2), phosphoglucomutase (E.C. 2.7.5.1; PGM-1, PGM-2),

6-phosphogluconate dehydrogenase (E.C. 1.1.1.44; 6-PGD), isocitrate

dehydrogenase (E.C. 1.1.1.42; IDH-1 and IDH-2), malate dehydrogenase (E.C.

1.1.1.37; MDH-1 and MDH-2), sorbitol dehydrogenase (E.C. 1.1.1.14; SDH-1),

creatine kinase (E.C. 2.7.3.2; CK-1, CK-2), and hexokinase (E.C. 2.7.11; HK-1).

Alleles were designated alphabetically in order of decreasing mobility, with the

most anodally migrating allele designated "A". In multi-locus systems, the most

anodally migrating protein was designated 1, the second most 2, etc.

32

Computations of allelic frequencies and genetic variation measures were

made using BIOSYS-1 (Swofford and Selander, 1981). Rogers' (1972) genetic

distance was calculated for all paired combinations of populations and species to

determine genetic similarities among Neotoma. A phenogram summarizing

genetic similarities was constructed using the unweighted pair-group method

(UPGMA) clustering procedure using NTSYS-pc (Rohlf, 1988).

Phvlogenetic analysis of data.-Restriction sites generated by the

seventeen restriction endonucleases were coded as presence/absence data and

subjected to phylogenetic analysis using PAUP Version 3.0 (D. L. Swofford,

University of Illinois). Restriction sites can be assigned to three catagories in a

cladistic sense: autapomorphic sites representing sites unique to a single

population or species, pleisiomorphic sites occurring in all populations or species,

and synapomorphic sites present in more than two of the populations representing

shared derived characters. Autapomorphic characters are of value in studies

addressing population structure, but in systematic analyses they only contribute to

increasing branch lengths of cladograms. Invariant, pleisiomorphic characters

convey no information regarding the pattern of cladogenesis. In all analyses

presented here, uninformative autapomorphic and pleisiomorphic characters were

ignored in the data set.

Three different character-type schemes were employed to determine which

analysis and its underlying assumptions are the most appropriate for analyzing this

33

mtDNA data set. Standard analyses were run which considered the restriction

sites as freely reversible Wagner characters. Under this scenario, a gain or loss of

a restriction site has an equal probability of occurrence. This scheme has been

the traditional method employed in phylogenetic analyses dealing with a broad

array of character forms (Wiley, 1981).

Dollo parsimony criteria were also invoked to determine if alternate

phylogenies would be developed if differential character weighting schemes were

employed. Dollo parsimony has been recommended for restriction site data due

to the asymmetry in the probabilities of losing existing restriction sites versus

gaining a new site at a specific location (DeBry and Slade, 1985). Thus, under a

Dollo parsimony scheme, a restriction site is allowed to arise only once during the

course of evolution, however it may be lost as many times as neccessary such that

monophyletic arrangements of the taxa are achieved.

A "Generalized Parsimony" approach was also employed in the data

analyses of the restriction site data. By using the STEPMATRIX option of

PAUP, restriction site gains were given a higher weight than site losses, such that

parallel loss and gain-loss events are preferred over parallel gains and loss-regains

(Templeton, 1983). This procedure avoids some of the prohibitions employed in

strict Dollo parsimony methods (DeBry and Slade, 1985). The forward

transformation 0 = = > 1 was weighted at a factor 3.0, while reversions,

1 = = > 0, were assigned a weight of 1.0. These weighting coefficients correspond

to character weighting schemes suggested for restriction site data by Swofford

34

(1991) and Albert et al. (1991).

Several standard options were selected for use in the PAUP analyses of the

data. Each phytogeny reconduction was performed using the HEURISTICS

option with TREE BISECTION-RECONNECTION (TBR) branch swapping and

ACCTRAN optimization. Heuristic tree searches begin by obtaining the initial

tree by stepwise addition until all taxa have been included. The tree is then

subjected to branch swapping which attempts to find the shortest trees. The TBR

swapping procedure bisects the tree yielding two disjointed subtrees and then

reconnects the subtrees by joining a pair of branches, each from one tree.

ACCTRAN optimization maximizes character-states by preferring reversals to

parallelisms (Swofford and Maddison, 1987). The order taxa input into all tree

building analyses including the bootrapping procedures was varied to avoid

possible bias introduced by the inadvertent association of groups of taxa.

Confidence limits were determined on the various phytogeny

reconstructions by employing the BOOTSTRAP algorithm (Felsenstein, 1985).

This process involved resampling the data set, with replacement, producing a

series of new data sets each of the same size as the original data set. Different

characters were eliminated from each resampling routine, with other characters

being represented more than once. The newly derived data sets were then

analysed cladistically using the three procedures descibed above. Bootstrapping

was conducted in conjunction with the standard options described previously and

produced a majority-rule consensus tree for each analysis. Bootstrap parameters

35

employed throughout the analyses consisted of a random number seed of 2441

and 100 iterations. The output of this analysis produced a listing of how many

times out of 100 replications a particular clade was produced in the analysis. This

method resulted in a robust measure of the level of confidence that can be placed

on a phytogeny produced in the particular analysis.

Three Goodness-of-Fit statistics were calculated by PAUP for each

phylogenetic tree produced in the analyses. These measures are generally

functions of the level of resolution of the consensus tree. The Consistency Index

(CI), ranging from near 0 to 1, provides a measure of how well a particular tree

explains the data, with trees that describe their respective data sets best scoring

closer to 1 (Kluge and Farris, 1969). The Homoplasy Index (HI) calculated by

PAUP is the Homoplasy Excess Ratio Maximum (HERM) developed by Archie

(1989). This index ranges from 0 to 1, with values of 1 representing data sets that

are devoid of identical character transformations evolving independently

(homoplasy). Farris (1989) developed a Retention Index (RI) which describes

how well a character set fits the consensus tree produced. Data sets which fit the

resultant cladogram poorly have retention indices approaching 0. Uninformative

characters contribute an undefined component to the retention index.

Until recently, the use of cladistics to construct phylogenetic trees from

electrophoretic data has been controversial, since there are no generally accepted

methods of data transformation for allele frequencies as continuous characters

(Buth, 1984; Swofford and Berlocher, 1987). For the allozyme data, character

36

coding which treated the presence or absence of each allele as a binary character

was employed. This data set was then subjected to Wagner parsimony analysis

using PAUP employing the same options described for the earlier analyses. These

data were also analysed using the approach of Swofford and Berlocher (1987)

(FREQPARS) which accounts for the frequencies of the various alleles. This

method appears to negate the difficulties of various other parsimony methods of

constructing cladograms from electrophoretic data.

Phenetic analysis of data.-Distance matrices were constructed consiting of

pairwise comparisons of the estimated level on nucleotide sequence divergence (6

of Nei and Tajima, 1983) for the mitochondrial genome, and Rogers Modified

Distance for the allozyme data set for the 13 taxa included in this study.

Phenograms were constructed using the unweighted pair-group method (UPGMA)

clustering procedure of NTSYS-pc (Rohlf, 1988).

A statistical test for the presence of a molecular clock was conducted by

subjecting the 6 matrix obtained from the mitochondrial DNA restriction sites to

two analyses, FITCH and KITSCH, of the PHYLIP program (Felsenstein, 1986).

FITCH fits a tree using the Fitch-Margoliash method allowing the branch lengths

to be unconstrained. KITSCH, by contrast, assumes that an evolutionary clock is

in effect, holding the branch lengths from the root to each terminal taxon equal,

which, in effect, means that the expected amount of evolution in any lineage is

proportional to the elapsed time (Felsenstein, 1986). Both analyses yielded sum

37

of squares estimates for the phylogenies which were used to calculate an F

statistic. A statistically significant F value would suggest the presence of a

molecular clock.

Congruence of mitochondrial and nuclear genome differentiation was tested

by subjecting the distance matrices produced by the phenetic analysis (Table 5) to

Mantel Analysis (Mantel, 1967) with a matrix correlation coefficient (approximates

a Normalized Mantel Statistic Z) greater than 0.9 representing significant

correlation.

CHAPTER III

RESULTS

General.-Thirty-seven unique mitochondrial haplotypes were found among

the thirteen woodrat taxa analyzed. Mitochondrial DNA restriction mapping

produced a total of 192 characters across all taxa, 141 representing

synapomorphies and 51 representing phylogenetically uninformative characters

(autapomorphies). Approximately 38 restriction sites were mapped for each

species or species variant identified. Appendix VII contains the data matrix

generated from the restriction site mapping procedure and includes a table of

mtDNA restriction sites identifying the location of each site and the corresponding

restriction endonuclease.

Phvlogenetic analysis of the Neotoma floridana species-group.-Four

nominal taxa, N. floridana, N. magister, N. micropus, and N. albigula, included in

the N. floridana species group (see Table 2) were subjected to cladistic analysis

using Ototylomys phyllotis and Xenomys nelsoni as outgroups. Also included were

samples of N. phenax and N. cinerea in a preliminary investigation into the

relationship of subgenera in the cladogenic pattern of speciation of Neotoma.

Seventeen unique mitochondrial haplotypes were observed among N. floridana, N.

38

39

micropus, N. albigula, and N. magister (samples 1-16, and 31 in Appendix VI).

Bootstrapped consensus trees produced by PAUP under the three

character-type criteria were concordant (Figures 8 and 9). Members of the N.

floridana species group formed a well supported clade on each tree, with

bootstrap estimates ranging from 95 to 100 percent. Although Wagner and Dollo

parsimony methods produced consistant trees, the robustness of the bootstrap

estimates derived from the Wagner consensus tree were lower, as a whole, than

those obtained from the Dollo analysis. Tree topologies differed between the

Wagner and Dollo methods primarily in the placement of two distinct N. albigula

clades. The Generalized Parsimony method produced a polytomous grouping of

the entire N. floridana group with little resolution regarding species phylogenies.

Within the N. floridana group, there was a consistent dichotomy between

populations of the white-throated woodrat, N. albigula. The two forms, hereafter

referred to as eastern and western N. albigula, represent populations of white-

throated woodrats from east or west of the Rio Grande River, respectively.

Specimens referable to the two distinct haplotypes, collected on opposite sides of

the river in Sierra and Socorro Counties, New Mexico, and in the vicinity of El

Paso, Texas, confirmed the absence of introgression between the two

mitochondrial haplotypes. Within the clade containing eastern N. albigula, a

dichotomy was found between samples referable to two subspecies, N. a. warreni

and N. a. albigula. Populations of N. albigula sampled from Chihuahua and

Sonora, Mexico are placed on individual branches seperated from the clades

40

100 100

100

Figure 8. Bootstrapped consensus trees produced by employing Wagner

(above) and Dollo (below) parsimony methods for the Neotoma floridana species

group. Values at the nodes represent the number of times the taxa associated by

that node were affiliated out of 100 iterations.

41

w w w w w

Figure 9. Bootstrapped consensus tree produced by employing the

Generalized Parsimony criteria described in the text for the Neotoma floridana

species group. Values at the nodes represent the number of times the taxa

associated by that node were affiliated out of 100 iterations.

42

containing the eastern or western N. albigula groupings in all three analyses

performed. Dollo parsimony methods placed the two samples in an unresolved

clade outside of the clade containing western N. albigula.

Five unique haplotypes were found among populations of N. micropus

representing the highest level of intraspecific variation observed in this study. The

N. micropus clade grouped together at high levels of confidence ranging from 90

to 99 percent depending on the bootstrap analysis performed. The N. micropus

clade was collapsed by both the Dollo and Generalized Parsimony methods, and

confidence in the branching pattern was nonsignificant in the Wagner analysis.

A clade containing N. floridana and N. magister was formed in 100 percent

of the bootstrap replications in all three analyses. Samples of N. floridana from

Texas, Oklahoma, and Kansas formed a separate clade excluding N. magister in

93-96 percent of the bootstrap replications. Neotoma micropus was placed as a

sister taxon to the clade containing N. floridana and N. magister in both the

Wagner and Dollo Parsimony analyses.

Goodness-of-fit statistics were calculated for the three consensus trees

(Table 3), and, based on overall measures of fit, the Dollo phylogeny

reconstruction is the most robust representation of the data. The Dollo method

produced a cladlogram with the highest Homoplasy (HI=0.527) and Retention

(RI=0.897) indices,. Although homoplasy is high in this data set at approximately

43 percent, the stringent character assumptions of the Dollo method may

effectively reduce the impact of homoplasious data in the phylogeny

43

reconstruction. This is especially true with a group of closely related taxa such as

the N. floridana species group, assuming that there has been insufficient time for

very much convergent evolution. The constraint on convergent site gains with

regard to restriction map data, however, may cause certain taxa to be linked by

chance if such gains occur due to the differential probabilities of these changes in

specific enzyme recognition sequences (Templeton, 1983).

Phvlogenetic analysis of the N. lepida species group.-Fifteen populations

belonging to two nominal taxa, N. lepida and N. devia, included in the N. lepida

species group (see Table 3), were subjected to cladistic analysis using Ototylomys

phyllotis and Xenomys nelsoni as outgroups. Also included were samples of N.

phenax and N. cinerea in a preliminary investigation into the relationship of

subgenera in the cladogenic pattern of speciation of Neotoma. Ten unique

mitochondrial DNA haplotypes were observed among the 15 populations

examined (samples 17-26 in Appendix VI).

Members of the N. lepida group formed a significant clade with concordant

topologies in all three analyses employed (Figures 10 and 11). Bootstrap

estimates of 98 to 100 percent were obtained for the group using 100 bootstrap

iterations. A significant dichotomy was revealed between populations of N. lepida.

The western U. S. coastal and Baja California populations, referred to here as the

intermedia-type, form a discrete clade removed from remaining populations of N.

lepida. The remaining populations of N. lepida form a sister taxon to N. devia,

44

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grouping with N. devia in 96-99 of the bootstrap replications in the three

procedures used. The intermedia-type individuals may well represent a distinct

species which is a sister taxon to the N. lepida-N. devia clade.

Goodness-of-fit statistics were calculated for the three consensus trees

(Table 3), and, based on overall measures of fit, the Dollo phylogeny

reconstruction is the most robust representation of the data. Tree length

measurements and Consistency Index values are not reliable measures of fit in the

analysis of the N. lepida group due to the effect that the differing numbers of taxa

play on these measures. The Homoplasy and Retention indices, however, are not

affected by these parameters. The level of homoplasy detected in the analysis of

the N. lepida group is higher than that revealed from the N. floridana group data,

ranging from 53 to 59 percent. The high values of the Retention Indices,

however, support the topologies produced as representative assessments of

cladogenesis for the group.

The Dollo method also produced the consensus tree which comes closest to

representing a meaningful biogeographical distribution of the mtDNA haplotypes

for the N. lepida group. The sample designated "N. intermedia 5" represents a

population collected on Santa Margarita Island off the coast of Baja California

Sur, Mexico. Although there was support for the inference that the island form

has differentiated from the mainland forms, this conclusion can not be drawn since

samples from the adjacent mainland were not obtained. The biogeographical

arrangement of the remaining N. intermedia samples was not well supported by

46

\ rt <X • * /

/ / / / / / / / / / / / / /

/ J $

Figure 10. Bootstrapped consensus tree produced by employing Wagner

(above) and Dollo (below) parsimony methods for the Neotoma lepida species

group. Values at the nodes represent the number of times the taxa associated by

that node were affiliated out of 100 iterations.

47


Generalized Parsimony criteria described in the text for the Neotoma lepida

species-group. Values at the nodes represent the number of times the taxa

associated by that node were affiliated out of 100 iterations.

48

the bootstrap consensus trees produced by any of the methods.

The Generalized Parsimony analysis of the N. lepida group also included

taxa from the subgenus Neotoma, N. mexicana, N. stephensi, and N. fuscipes, in a

preliminary assessment of the validity of the inclusion of the N. lepida group in

this subgenus. Members of the subgenus Neotoma, N. mexicana and N. stephensi,

formed a clade associated with N. phenax of the subgenus Teonopus, which was

removed from members of the N. lepida group. Members of the N. floridana

group were purposely omitted from this analysis to reduce the possibility of

confusion caused by homoplasy. These taxa were included in later analyses

conducted on the entire genus. Neotoma fuscipes was placed in a clade with N.

cinerea of the subgenus Teonoma.

Phvloeenetic assessment of the Genus Neotoma.-The relationships of the

subgenera and species groups within the genus Neotoma were cladistically

analyzed using mtDNA restriction site data and allozyme data. The latter were

coded with alleles serving as binary characters and using FREQPARS which

utilized allele frequencies (Swofford and Berlocher, 1987). The mtDNA

restriction site and binaiy coded allozyme data sets were also combined in a final

analysis to generate a phylogeny for the group. Fourteen OTU's were created

from the set of 35 mtDNA haplotypes found among the eleven nominal woodrat

taxa by collapsing nonsignificant or zero branch lengths. This procedure, in effect,

49

masks intraspecific variability without affecting species level resolution. Ototylomys

phyllotis and Xenomys nelsoni served as outgroups for the analysis.

The genus Neotoma formed a monophyletic group using Wagner and

Generalized Parsimony methods which was supported by bootstrap estimates of 96

and 92 percent, respectively (Figures 12 and 13). The Dollo parsimony procedure

did not support monophyly of the genus as a whole, but provided resolution of the

N. floridana, N. mexicana, and N. lepida species-groups, with bootstrap estimates

of 95, 92 and 83 percent, respectively, validating these subdivisions. The Dollo

phylogeny generated the lowest values for all three goodness-of-flt statistics (Table

4).

A major topological difference was detected within the N. floridana group

in the three analyses conducted with respect to the placement of N. micropus.

When taken in the context of the entire genus, N. micropus was placed in the

clade containing members of the N. albigula complex, which in turn was a sister

clade to the N. floridana-N. magister clade. This topology is supported by

bootstrap estimates of 93-96 percent.

In all phylogenies developed, a strong association is made between N.

fuscipes and N. cinerea and between N. stephensi and N. mexicana. Neotoma

cinerea is assigned to the subgenus Teonoma, while N. fuscipes was originally

assigned to the subgenus Homodontomys (Goldman, 1910), which was later

synonomized with the subgenus Neotoma. Placement of N. fuscipes and N. cinerea

in a monophyletic lineage was supported best by Wagner and Generalized

50

f f f f f >

i , A

Figure 12. Bootstrapped consensus tree produced by employing Wagner

(above) and Dollo (below) parsimony methods for the genus Neotoma. Values at

the nodes represent the number of times the taxa associated by that node were

affiliated out of 100 iterations.

51

/AAKKKf////S////


Generalized Parsimony criteria described in the text for the genus Neotoma.

Values at the nodes represent the number of times the taxa associated by that

node were affiliated out of 100 iterations.

52

parsimony procedures yielding bootstrap confidence estimates of 96 and 92

percent, respectively. The Dollo procedure placed the clade containing N. fuscipes

and N. cinerea as a sister clade to the outgroup species, Ototylomys phyUotis and

Xenomys nekoni. This phylogeny is not statistically supported, with boostrapping

grouping these taxa 27 times out of 100 iterations.

N. stephensi was originally placed in the desertorum group of the subgenus

Neotoma which included the desert woodrats (currently referred to as the N.

lepida group) (Goldman, 1910). Burt and Barkalow (1942) removed N. stephensi

from association with the N. lepida group based on major differences in the bacula

described from the representative taxa, but they did not assign the species to any

specific group. The bootstrapping procedure placed a 92 to 100 percent

confidence level on the placement of N. stephensi as a sister taxon to N. mexicana

within the N. mexicana group. Placement of the N. mexicana group within the

subgenus Neotoma was also supported by the three analyses. However,

bootstraping placed less confidence in this association with values of 72, 31, and 71

percent for the Wagner, Dollo and Generalized parsimony procedures,

respectively. The lowest bootstrap value was obtained from the Dollo analysis and

should not be considered as an indication of the strength of this association due to

the overall weakness of the Dollo method for phylogeny reconstruction at this

taxonomic level.

In both of the supported phylogenies, the N. lepida group was placed as a

distinct clade removed from the subgenus Neotoma suggesting that this group may

53

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54

represent a discrete subgenus. This assessment can be made due to the

placement of N. phenax of the subgenus Teonopus as a sister taxon to the

monophyletic group containing members of the subgenus Neotoma. The

association of N. phenax with the subgenus Neotoma is strong, with bootstrap

levels of 89 and 91 percent reported for the Wagner and Generalized Parsimony

methods. The phylogeny presented by the Dollo analysis, however, places N.

phenax as a separate clade well removed, albeit weakly, from the subgenus

Neotoma. Due to the stringency imposed by the Dollo assumptions, a small

proportion of the data set which consists of rare restriction sites shared with other

taxa, such as members of the N. lepida clade and possibly the N. cinerea-N.

fuscipes clade, would effectively remove N. phenax from association with the

subgenus Neotoma.

A full character matrix consisting of mtDNA restriction sites and binary

coded allozyme data was subjected to a phylogenetic analysis using Wagner

Parsimony criteria and bootstrap confidence level determination. The resultant

consensus cladogram (Figure 14) provides a topology consistent with cladograms

produced by Wagner and Generalized Parsimony methods on the mtDNA set

alone. The topology, however, does not agree with those produced through

Wagner analysis on binary coded allele data or allozyme frequency data analyzed

with FREQPARS (Figure 15). Although the randomization and resampling

procedures of the bootstrap analysis should remove any bias rendered by either

character form, the ratio of 141 mtDNA characters : 55 allozyme characters after

55

combined data set.

Phenetic analysis of the genus Neotoma.-Of the 25 presumptive gene

products resolved electrophoretically, three (PGM-2, MDH-2, CK-1) were

monomorphic for the same allele in all specimens examined. Of the remaining 22

putative gene loci, 14 varied within species (EST-1, EST-2, ALL-2, a-GPD-1,

VLL-1, VLL-2, 6-PGD-l, PGM-1, IDH-1, IDH-2, ME-2, CK-2, XDH-1, SDH-1),

and eight varied only among species (EST-2, ALL-1, MDH-1, ME-1, LDH-1,

LDH-2, HK-1, SOD-1) the latter three loci contributing an unique allele only in

Ototylomys phyllotis (Appendix VI).

Phenetic analysis of Rogers' genetic distance (Table 5, Figure 16) yielded

two main clusters. A large cluster containing the members of the genus Neotoma,

but excluding N. fuscipes, was divided into two subclusters. A small subcluster

containing N. floridana and N. magister was separated from the remaining taxa at

a genetic distance of 0.54. The larger subcluster was subdivided into a grouping

containing members of the N. albigula complex, including N. micropus, another

grouping containing N. stephensi, N. mexicana, and N. cinerea, and a single branch

with only N. phenax. Placement of N. micropus with members of the N. albigula

complex was concordant with the results of the mtDNA restriction site analysis of

the entire genus. However, the placement of N. cinerea among members of the

subgenus Neotoma was unsupported by other evidence.

A small cluster containing members of the N. lepida group was separated

from the remaining members of the genus with a genetic distance of 0.59.

56

¥/////£&.//// (f ^ ^ w ^

Figure 14. Bootstrapped consensus tree produced from mtDNA restriction

sites and binary coded allozyme data subjected to Wagner Parsimony criteria.

Values at the nodes represent the number of times the taxa associated by that

node were affiliated out of 100 iterations.

57

/ / / / / / / / / / / A ' / / (f ^ ^ ^ ^ ^ f *• *•

KKKKKKKK&&KK&

Figure 15. Bootstrapped Wagner consensus tree (above) produced from

binary coded allele data. Values at the nodes represent the number of times the

taxa associated by that node were affiliated out of 100 iterations. Cladogram

based on allozyme frequency data produced by FREQPARS (below).

58

Differentiation within the N. lepida group reported by the allozyme analysis does

not conform to the pattern developed in the mtDNA restriction site analysis but

does coincide with the karyotypic evolution reported by Mascarello (1978).

Neotoma fucipes was placed on an independent branch, its relationship being

unresolved by the UPGMA analysis.

Phenetic analysis of the estimated level of nucleotide sequence divergence

(<5 of Nei and Tajima, 1983; Table 5) based on pairwise comparisons of mtDNA

restriction sites shared between nominal taxa and well differentiated populations

of the genus Neotoma shows strong topological concordance with the cladograms

produced from mtDNA restriction site data (Figure 17).

The overall structure of the phenogram describes two clusters, one

containing members of the N. lepida group, and the other containing the

remainder of the genus and the two outgroup taxa, Ototylomys phyllotis and

Xenomys nelsoni. The N. lepida group cluster is removed from the remaining taxa

at an estimated sequence divergence of approximately 25 percent. The placement

of this species cluster well outside that of the remaining taxa is in agreement with

the Dollo parsimony analysis on the mtDNA restriction site data. However,

inclusion of Ototylomys phyllotis and Xenomys nelsoni within the subgenus Neotoma

cluster is inconsistant with the cladistic analysis.

Measures of sequence divergence support the placement of N. phenax

closer to members of the subgenus Neotoma. Within the subgenus Neotoma

cluster, two discrete subclusters were produced at a level of sequence divergence

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N. albigula CH.

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N. stephensl

N. mexicana

N. clnerea

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N. florldana

N. mag/ster

N. leplda

N. Intermedia

N. devia

N. fusel pes

Ototy/omys phyllotis

o.o

60

Figure 16. Phenogram produced from UPGMA cluster analysis of allozyme

data for 14 Neotoma taxa and outgroup species based on Rogers' genetic distance

derived from analysis of 25 presumptive structural gene loci.

61

of 18 percent; these represent the N. floridana and N. mexicana species-groups.

Within the N. floridana species group cluster, N. micropus grouped with members

of the N. albigula complex, in agreement with the results of the phenetic allozyme

analysis and cladistic mtDNA restriction site analysis. Neotoma fuscipes and N.

cinerea group together in a cluster which contains the two outgroup taxa, which is

separated from the larger woodrat cluster at an estimated 24 percent sequence

divergence.

A statistical test for the presence of a molecular clock (Felsenstein, 1986)

was conducted by subjecting the S matrix obtained from the mitochondrial DNA

restriction sites to two analyses, one assuming the presence of a molecular clock

and one without a clock present. A nonsignificant variance ratio, F = 0.455 (p

> > 0.50), between the sums of squares calculated by FITCH and KITSCH

analyses of PHYLIP, was determined for the the full S matrix. Lack of support

for a molecular clock was also obtained from the analysis of a subset of the S

matrix targeting taxa considered to be in the sugenus Neotoma, F = 0.484 (p »

0.50).

Different rates of evolution are evident in the phylogenetic tree produced

under least squares criteria using the FITCH analysis of PHYLIP in which the

branch lengths represent expected amounts of change between the ancestors and

the terminal taxa (Figure 18). The branch lengths, which represent the estimated

number of nucleotide substitutions (6), are similar within specific clusters of

closely related taxa, suggesting the presence of locally active molecular clocks.

62

i i 0.30 0.25 0.20 0.15

Sequence Divergence

N. floridana

N. magister N. micropus N. albigula £ N. albigula W. N. Albigula Ch. N. stephensi N. mexicana N. phenax N. fuscipes N. cinerea Xenomys Ototylomys N. /epic/a N. devia N. /. intermedia

n i 1 0.10 0.05 0.0

Figure 17. Phenogram produced from UPGMA cluster analysis of 14

Neotoma taxa and two outgroup species using the estimated level of nucleotide

sequence divergence (5 of Nei and Tajima, 1983) based on mtDNA restriction site

data.

63

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64

Major branch length and, hence, rate differences were observed on branches

leading to major clusters of taxa, such as the N. lepida group, N. mexicana group,

or N. phenax, which supports the contention of cohesivness among taxa grouped

into higher taxonomic catagories, i.e. subgenera, and species-groups. Members of

the N. lepida group are well removed topologically and separated by considerable

distance from members of the subgenera Neotoma and Teonopus, again suggesting

that this group is a distinct subgenus. Within the subgenus Neotoma cluster, there

is support for the designation of two major species groups, the N. floridana and N.

mexicana species groups.

Mantel Analysis conducted on the independent distance matrices compiled

from the allozyme and mtDNA restriction site data (Table 5) resulted in a

significant correlation, r = 0.907 (^[random Z > observed Z] =0.004), between the

matices. This high degree of relationship between the data sets infers that

concordant patterns of evolution can be expected from the analysis of both

mtDNA and allozyme data for this group of organisms. Differences observed in

the topological models developed from these data sets are artifacts or peculiarities

of the specific data sets or pairs of taxa being compared.

CHAPTER IV

DISCUSSION

Relationships within the N. floridana species-group and factors contributing

to present distributions.-The levels of differentiation presented here between

members of the N. floridana species group fall within the ranges that have been

reported for other mammalian taxa using comparable approaches (see Table 1.,

Table 5., Zimmerman et al., 1978). The estimated level of sequence divergence

between N. floridana and N. magister found here (2.2%) is less than half of that

reported by Hayes (1990) for these two taxa (5.2%). The discrepancy between

these two estimates may be coincidental with the location of specimens examined

in this study, choice of restriction enzymes employed, or the calculation of this

estimate by Hayes. Hayes reported 5.2% as a maximum estimate of sequence

divergence between N. floridana and N. magister, with "weighted" averages of

sequence divergence between the two taxa ranging between 3.2 - 4.8%. A sample

from Kansas was represented within the lower range of these values which biases

the magnitude of discrepency with regard to the present study. Hayes (1990) was

unable to determine the relationship of N. magbter to the subclades of putative N.

floridana, with one of the possible arrangements suggesting a close relationship

between N. magbter and western populations of N. floridana.

65

66

Relationships between other members of the N. floridana species group (N.

floridana, N. micropus, and N. albigula) are not as straightforward. In mtDNA

analyses based solely on members assigned to this species group, N. micropus was

placed as a sister taxon to the N. floridana-N. magister clade, in agreement with

earlier allozyme and immunoelectrophoretic studies (Birney, 1973; Zimmerman

and Nejtek, 1977; Shipley, et al., 1989). When these taxa were analysed in the

context of the entire genus, N. micropus was placed in the clade containing the

members of the N. albigula complex, which in turn was a sister clade to the N.

floridana-N. magister clade. This arrangement was supported by the allozyme data

presented herein as well.

Difficulty in the placement of N. micropus may be in part due to the large

intraspecific polymorphism present in the species. High levels of chromosomal

polymorphism was reported for N. micropus, a characteristic not reported for

other members of the genus (Baker and Mascarello, 1969; Baker et al., 1970).

Zimmerman and Nejtek (1977), however, reported N. micropus as having the

lowest proportion of polymorphic loci per population but highest individual

heterozygosity of the three species of Neotoma they analysed. Five unique

mtDNA haplotypes were found in this study, representing the highest degree of

mitochondrial polymorphism reported for this genus. Although estimated levels of

sequence divergence (5) between the five N. micropus haplotypes ranged from

0.002 to 0.011, the pattern of cladogenesis produced for these haplotypes did not

present a meaningful biogeographic pattern. Polymorphism within N. micropus

67

may have been the result of random fragmentation caused by habitat restrictions

during the most recent glacial advances during the Wisconsin ( Van Devender, et

al., 1987) which allowed for differentiation among the isolated populations but was

insufficient to result in reproductive isolation. Fragmentation of this nature

increases the degree of polymorphism among mtDNA lineages resulting in a

paraphyletic condition existing for an extended number of generations following

reunion of the isolated populations (Neigel and Avise, 1986; Riddle and

Honeycutt, 1990).

Hybridization revealed by morphological, allozyme, and karyotypic data

between N. micropus and N. albigula has been reported at locations at the

northern limits of both species' ranges in southeastern Colorado and western

Oklahoma (Birney, 1976; Huheey, 1973). Hybrids were also collected at three

localities in the Texas panhandle and one location in the Davis Mountains (Long-

X Ranch, Jeff Davis Co., TX) during this study. However, over larger parts of

their ranges where these two species are sympatric, their genetic integrity appears

to be maintained by habitat separation. Neotoma micropus occurs primarily in flat,

semi-arid plains in plant associations of yucca-, cactus-, or mesquite-grassland

(Schmidly, 1977; Dalbey, 1980). Although N. albigula can often be found in

similar habitats, especially in the western-most parts of its range, it is most

commonly found in rocky habitats and arroyos associated with juniper and yucca

(Finley, 1958; Huheey, 1972; Schmidly, 1977).

Neotoma floridana appears to hybridize with N. micropus at a single locality

68

in northern Oklahoma where the two species are sympatric (Birney, 1973;

Spencer, 1968) and has been induced to hybridize in the laboratory (Birney, 1973).

Birney (1976) described this observation as a case of stasipatric distribution (Key,

1968), where impaired fecundity of hybrids rather than ecological competition is

the major factor preventing broad species overlap. At a sympatric locality in

south-central Texas, Dalbey (1980) failed to support any evidence for hybridization

between these two species. Based on these lines of evidence, greatest support is

placed in associating N. micropus with the N. albigula complex. Due to the lack of

significant hybridization and introgression, as reported here and by Huheey (1972),

the specific integrity of both species should be maintained.

The sharp dichotomy between the forms of N. albigula has not previously

been reported. The estimated level of sequence divergence between the eastern

and western N. albigula clades of 3% is comparable to the values presented for

the cryptic species N. floridana and N. magister and other closely related species

(Table 1).

Zimmerman and Nejtek (1977) reported polymorphism at several allozyme

loci between populations of N. albigula, which, when taken into a geographic

perspective, suggested a limitation of gene flow between some of the populations

sampled. The presence of alternate allelic forms at six loci (Appendix VI) and

two distinct mtDNA haplotypes in the region of El Paso, Texas, indicates this

region is an area of sympatry between the two genetic forms.

Geographically, the eastern and western N. albigula forms are separated

69

along the Rio Grande River in New Mexico, with the exception of the area of El

Paso, Texas, where both forms are found sympatrically. In northern New Mexico

and Colorado, the two forms appear to be restricted by the San Juan Mountains

of the continental divide to the west, and the Sangre de Cristo Range to the east

of the Rio Grande. White-throated woodrats are absent from the San Luis Valley

of southcentral Colorado and adjacent northern New Mexico (Finley, 1958;

Armstrong, 1972; Findley et al., 1975). It is feasible, that the Rio Grande River

may have been an early factor contributing to the segregation of the eastern and

western N. albigula forms following an episode of geographic isolation initiated by

changing climatic patterns during the Pleistocene (Flint, 1971; Hays et al., 1976).

The Rio Grande River and its basin consisted of deep fissure canyons and

large pluvial lakes at various times throughout the Pleistocene (Baldridge and

Olsen, 1989; Flint, 1971). The actual course of the river and position of the river

bed have changed several times in the river's history since the Pliocene. During

the late Pliocene through early Pleistocene, the primary drainage of the river's

headwaters in Colorado and northern New Mexico was deflected into the Pecos

River drainage from a point south of the Sangre de Cristo Mountains, feeding into

the ancestral Rio Grande at the present day Amistad Reservoir (Figure 19).

Drainage from the more southerly mountains in New Mexico flowed through the

Rio Grande floodplain draining into large pluvial lakes in northern Chihuahua

(Belcher, 1975). During this period, the Rio Conchos served as a major drainage

of the Sierra Madre Occidental and regions of the Mexican Plateau, leading into

70

the ancestral Rio Grande at present day Ojinaga, Chihuahua (Belcher, 1975).

The Rio Grande-Pecos-Rio Concho river assemblage may have acted as a

partial barrier to a northerly dispersal of N. albigula forms, if regions of the

southern Bolson de Mapimi and Mexican Plateau served as isolating refugia

during full glacial advances. These regions reportedly consisted of habitats ranging

from papershell pinyon and juniper to Chihuahuan desert scrub during the

Wisconsin, with N. albigula reported as the most abundant contributor to fossil

woodrat middens in the area (Van Devender, 1990; Van Devender and Burgess,

1985). Meyer (1973) concluded that the vegetation of this region has been stable,

with little change along the mountain slopes. Fossil middens recorded pinyon-

juniper-oak woodlands for the Hueco Mountains region of Texas suggesting

relatively stable climates in this area for at least 30,000 years (Van Devender,

1990; Van Devender et al, 1987).

During later periods of the Pleistocene, the opening of the canyon at El

Paso allowed the river to flow in its present course towards the confluence with

the Rio Conchos. At various times during the Pleistocene, however, the

southward course of the river was deflected between the eastern and western sides

of the Franklin Mountains north of El Paso (Belcher, 1975) (Figure 20). It is

feasible to suggest that the presence of both the eastern and western N. albigula

forms in the El Paso area is a result of fluctuations in the course of Rio Grande

with respect to its orientation with the Franklin Mountains. This form of

transplantation across a partial barrier has been suggested as a mechanism for

71

kilometers

Figure 19. Proposed ancestral drainage pattern of the Rio Grande River

during the late Pliocene through early Pleistocene.

72

other mammals (Kennerly, 1963; Russell, 1968).

The genetic data for the distinct forms of N. albigula are supported by

numerous morphological studies conducted over the past 20 years. Multivariate

analyses performed on cranial measurements from large, geographically diverse

samples of N. albigula yielded broad ranges of variability attributed to geographic

variation (Birney, 1976; Rogers and Schmidly, 1981). A priori sorting of localities,

with respect to mitochondrial haplotypes obtained from specimens used in this

study, allowed for a reevaluation of the geographic variation reported by Rogers

and Schmidly (1981), attributing most of the variation they observed to a genetic

dichotomy.

In the area near El Paso, Texas, habitat differences between the two N.

albigula forms and the sympatric N. micropus were observed. Neotoma micropus,

as stated previously, occurs on mesquite-grassland plains, as did individuals having

the western N. albigula mtDNA haplotype. No evidence of hybridization or

mtDNA introgression was detected among the twenty-six specimens sampled from

this area. The presence of N. albigula in this habitat is concordant with its typical

habitat in Arizona (Vorhies and Taylor, 1940). The sympatric relationship

between these two species has been reported previously for this area (Schmidly,

1977). Of the twenty-six specimens collected on the mesquite-grassland in this

study, only five were N. micropus. This agrees with the report of Ederhoff (1971),

that N. albigula was abundant in this habitat, while N. micropus was rare. Also it

possibly suggests that some level of competition exists between the western N.

73

Rio Grande River Current

Early Pleistocene

klfomeCrs

Figure 20. Proposed variations in the drainage pattern of the Rio Grande

River in the vicinity of the Franklin Mountains during the Pleistocene.

74

albigula form and N. micropus. East of the El Paso area, N. micropus is abundant,

and the western N. albigula form is absent. Six samples of the eastern N. albigula

mtDNA haplotype were collected along a rocky arroyo in the foothills of the

Franklin Mountains north of El Paso, however, samples of N. micropus were not

collected at this locality. The presence of N. micropus in the grassland region of

the Rio Grande floodplain may be a positive factor contributing to the

maintainance of microallopatry between the eastern and western N. albigula forms

in New Mexico.

The distribution of the eastern and western mtDNA haplotypes is also in

concordance with fossil data presented by Harris (1984) for New Mexico and

Chihuahua. Diagnostic measurements of the ml segregate by locality among the

fossil materials, while a Holocene reference sample shows broad variability. Fossil

information from the El Paso, Texas area, dated as stadial or early Holocene,

reveals intermediate distributions among the measurements, suggesting that both

forms of N. albigula were present in this region at the close of the Pleistocene.

In addition to the two forms of N. albigula discussed above, samples

collected from northwestern Chihuahua and western Sonora introduce additional

variation. Twenty-two N. albigula and three N. micropus were collected near

Janos, Chihuahua, in primarily mesquite-grassland habitat. The majority of the N.

albigula were collected among boulders of an isolated rock outcropping located on

the plains. The N. albigula from this locality and one specimen captured north of

75

Guaymas, Sonora, formed a distinct subclade usually associating with the western

mtDNA haplotype of N. albigula (Figures 12 and 13) when analysed in the context

of the entire genus. Analysis of the N. floridana species group independently,

however, resulted in the placement of these two populations as separate clades,

basal to the entire species group. An estimated percent sequence divergence

ranging from 4.4 - 6.7% with the western and eastern N. albigula haplotypes,

respectively, poses a question regarding the distinctiveness of these forms. This

level of divergence is higher than that reported between the two N. albigula forms

and N. floridana and N. magister. Although the Chihuahuan sample is referable to

N. a. albigula, it differs markedly from samples collected approximately 100 km

north in New Mexico and Arizona. The Sonoran sample, referable to N. a.

venusta, associates most closely with the Chihuahuan sample, not with N. a.

venusta samples from western Arizona.

The degree of differentiation among the populations of N. albigula

described here was not appparent in the previous taxonomic treatment of this

group by Hall and Genoways (1970) based solely on mensural characteristics.

Anderson (1969) first described a rearrangement in the distribution and taxonomy

of N. albigula from Chihuahua acknowledging the existence of two distinct forms

meeting along the Rio Conchos in northeastern Chihuahua. Specimens east of the

Rio Conchos, were allocated to N. a. durangae which Anderson (1969; 1973)

stated was clearly intermediate to N. a. albigula and N. micropus. The distribution

of N. micropus in Chihuahua is limited to regions of the Rio Grande floodplain

76

and mesquite grasslands north and west of the Rio Conchos. The sympatric

association of the N. albigula-type woodrats from northwestern Chihuahua with N.

micropus is concordant with the association described between N. mkropus and

the western N. albigula form. A collection of N. micropus from near Janos,

Chihuahua, suggests a possible range extention southwesterly by N. micropus since

the time of Anderson's survey work during the late 1950's and 1960's (Anderson,

1972). Westward expansion by N. micropus likely will be limited by the Sierra

Madre Occidental, as it forms the southern extensions of the Rocky Mountains in

southwestern New Mexico.

Ecologically, records of N. albigula from east of the Rio Conchos in

Chihuahua (Anderson, 1969), Coahuilla (Baker,1956), and San Luis Potosi

(Dalquest, 1953) report this form to be found in association with rocky outcrops

and caves and in vegetative associations ranging from cactus and mesquite to oak

and yellow pine (Baker, 1956). Neotoma micropus is not found in close association

with the white-throated woodrats in these regions. The ecological scenario in

northeastern Mexico corresponds closely to the relationship found between the

eastern N. albigula form and N. micropus in the southwestern United States.

Although specimens from the northeastern states of Mexico were not available for

inclusion in this study, ample evidence is present to suggest that the populations of

N. albigula found east and south of the Rio Conchos-Rio Grande confluence in

Mexico should be assigned to the same species as the eastern N. albigula form.

The distinctiveness of N. a. warreni described by Rogers and Schmidly

77

(1981) is supported by the mtDNA data presented here. Neotoma a. warreni,

represented by specimens collected from the Texas panhandle and southeastern

Colorado, formed a distinct subclade associated with members of the eastern N.

albigula group (Figures 8 & 9). Close examination of the distribution of the

respective mtDNA haplotypes is warranted to properly determine if a contact

zone exists between the two forms.

Relationships within the N. lepida species-group and factors contributing to

present distributions.-The desert woodrats of the N. lepida species group formed

a discrete monophyletic group independent of remaining members of the subgenus

Neotoma to which they have traditionally been assigned. The distinctiveness of

this group, however, is not restricted to the mtDNA and allozyme data presented

herein. Studies of the baculum (Burt and Barkalow, 1942) revealed a marked

divergence in structure of N. lepida-type bacula from the remaining members of

the genus Neotoma, not only in terms of its dimensions, but also in the presence

of a curved baculum, which is unique to the desert woodrats, leading these authors

to suggest that subgeneric recognition for the N. lepida group may be in order.

Although Hooper (1960:10) agreed with the suggestion of Burt and Barkalow

(1942:290) in his treatise on the glans penes of members of the genus Neotoma

and allied genera, he did not pursue the matter further. In an extensive,

morphologically based phylogenetic analysis of the Neotomine-Peromyscine

rodents, Carleton (1980) also reported that N. lepida was patristically highly

78

differentiated from other Neotoma, but not to the extent that Hodomys alleni

exhibited, which he considered generically distinct from Neotoma. Although

recognizing the problem existed, Carleton (1980) was not able to comment on the

differentiation within the N. lepida group, since his study material was restricted to

samples from Baja California and California referrable to the N. I. intermedia-type.

Chromosomal studies of N. lepida revealed a 2N = 52 karyotype for the

species, with marked variability in the autosomal arm number in populations east

and west of the Colorado River in Arizona and California (Mascarello and Hsu,

1976). Variations in allozymes, differentially stained chromosomes, penile

morphology, and cranial characters analysed by Mascarello (1978) indicated two

chromosomal races representing cryptic species occurred on opposite sides of the

Colorado River. Mascarello (1978) elevated N. I. devia to specific rank, assigning

desert woodrat populations east and south of the Colorado River in Arizona to

this species, whose status is currently accepted (Vaughan, 1990).

The specific status of N. devia, however, has been questioned based on a

morphometric analysis of desert woodrat populations in Arizona and eastern

California (Hoffmeister, 1986). One of Hoffmeister's (1986:411) main criticisms

concerned the lack of specimens from northern portions of Arizona and adjacent

Utah in Mascarello's (1978) analysis. My analysis included specimens from the

Little Colorado River area in Coconino Co., Arizona, south of the Grand Canyon,

and areas of Arizona and southwestern Utah north of the Colorado River, in

addition to localities east and west of the Colorado River in western Arizona and

79

adjacent California. The results of mtDNA and allozyme analyses of these

specimens are in concordance with the findings of Mascarello (1978). Desert

woodrats east and south of the Colorado River in Arizona are referable to N.

devia. Genetic subdivision within N. devia, based on the mtDNA analysis, does

agree in part with Hoffmeister's (1986) recognition of a subspecific distinction in

desert woodrats in southwestern Arizona. Specimens from Yuma and Mohave

counties, Arizona, differed from Coconino Co. specimens of N. devia with an

estimated sequence divergence (S) of 0.008. Although the exact range and zone

of contact between these two haplotypes was not addressed by this study, I

recommend that N. lepida auripila, as designated by Hoffmeister (1986), be

retained as a subspecies of N. devia, with the approriate taxonomy being N. devia

auripila.

The bacula and glans penes of western coastal and Baja California

populations, referred to as N. /. intermedia-type, possess significantly different

characters compared to those of inland desert woodrats (Mascarello, 1978; D.

Huckaby, pers. comm.). Although allozymic markers verified the distinctiveness of

this form, Mascarello (1978) did not address the issue in his study. The degree of

differentiation between the intermedia-type and inland N. lepida, as determined by

the mtDNA and allozyme data presented here, are of sufficient magnitude to

suggest that the intermedia-type represents a distinct species which is a sister taxon

to a clade containing N. lepida and N. devia (Figure 10 and 11).

The estimated levels of sequence divergence (6) between N. lepida and N.

80

devia based on mtDNA restriction site analysis was 2.5 percent, a level consistent

with that reported for other cryptic species within the genus Neotoma (Table 5).

Populations of N. intermedia differ from the other two desert woodrat species by

an average estimated sequence divergence of 6.2 percent, which is similar to the

degree of divergence seen between other woodrat species and well defined species

of Peromyscus (De Walt, 1991) and Onychomys (Riddle and Honeycutt, 1990).

The pattern of differentiation within the N. lepida group reported by the

allozyme analysis does not conform to the consensus pattern developed in the

mtDNA restriction site analysis but does coincide with the karyotypic evolution

reported by Mascarello (1978). Phylogenetic analysis of the allozyme data yielded

two topologies dependant on the method of analysis employed. Wagner

Parsimony analysis resulted in a topology for the N. lepida group in which N. devia

and N. intermedia occur on a clade that has N. lepida as a sister taxon. Analysis of

the same data set using FREQPARS provided a cladogram in which N. lepida and

N. intermedia occur together on a clade with N. devia as a sister taxon (Figure 15).

This phylogenetic representation is concordant with the phenetic analysis of the

allozyme data using UPGMA clustering (Figure 16).

The discrepency between the results of the allozyme and mtDNA analyses

may be an artifact related to the uncoupled nature of evolution between the

nuclear and mitochondrial genomes. The relatively robust degree of mtDNA

sequence divergence between N. intermedia and the other two desert woodrats

would suggest that the separation of populations leading to the N. intermedia stock

81

from ancetral N. lepida-type woodrats occurred prior to the speciation event which

separated N. devia from the remaining N. lepida stock. This scenario is supported

by morphological evidence which provides a distinct division between the N.

intermedia-type and N. lepida-typt glandes (Mascarello, 1978; D. Huckaby, pers.

comm.). A later subdivision of the proto-AT. lepida stock giving rise to the present

N. lepida and N. devia would allow karyotypic, allozyme, and mtDNA evolution to

occur in isolation, resulting in distinctive karyotypic modification, possibly resulting

in genie rearrangement in N. devia, while N. lepida retained karyotypic and some

degree of allozymic similarity with N. intermedia. The degree of mtDNA evolution

between these two species is consistant with that recorded for the other

arrangements of cryptic species among Neotoma.

Varying levels of support for this scenario can be extracted from the fossil

and paleoecological records. Woodrat middens have been used extensively by

paleoecologists to determine changes in floristic assemblages in southwestern

North America induced by the cycles of glacial advance-retreat during the

Pleistocene (Spalding et al., 1990; Van Devender, 1985; 1987; 1990; Mead et al.,

1983; Wells, 1976). Although the botanical material obtained from fossilized

woodrat excretia and middens has been thoroughly analysed, considerable neglect

has been paid to the Neotoma fossils also present in the materials.

The lower Colorado River Valley may have provided a complete or

periodic barrier to the gene flow in desert woodrats at various times throughout

the Pleistocene. This area, reported to be the most arid region in North America,

82

remained as a relatively arid region throughout the Pleistocene and served as a

desert core region for xerophytic plant species such as Larrea divaricata, whose

sparse distribution characterizes the area today (Cole, 1986). The arid lowlands

surrounding the head of the Sea of Cortez would have been greatly expanded

during such periods of the Pleistocene when sea levels decreased by approximately

120 meters. The Colorado River itself may have served as an initial barrier to

vicariant distribution and may have been responsible for the segregation of N.

intermedia from early N. lepida-stock. The modern Colorado River drainage

pattern became established during the latter part of the Pliocene and has

continued to the present (Wilson, 1967). Early drainage patterns of the ancestral

Colorado River did not include the southerly draining component which currently

separates Arizona and California. However the xeric entrenchment of this region

was extreme following the establishment of the current drainage pattern (Wilson,

1967).

Plant assemblages determined from woodrat midden materials suggest

communities to the north and east of the lower Colorado Valley resembled more

typically Great Basin and Mohave assemblages, including pinyon-juniper on

mountain ranges, while the majority of the Sonoran succulents and subtropical

plants were limited to refugia further south in Sonora (Van Devender, 1987). The

absence of these succulents and an increased area of aridity in the Colorado

Valley, dating from the Wisconsin through Holocene, suggests the presence of

effective vicariance that barriers alternately segregated desert woodrat populations

83

several times throughout the Pleistocene.

The direct fossil record of the N. lepida group is sketchy. Neotoma lepida

(sensu lato) has been reported in the fossil record from Arizona at more than

30,000 YBP (Mead et al., 1983). Records from the Picacho Peak area in

southeastern California date from the early Holocene (Cole, 1986). Specimens

from Nevada and New Mexico are reported from the mid-Rancholabrean (Kurten

and Anderson, 1980). Midden samples from near Catavina, Baja California

(Figure 21) record the presence of N. lepida (N. intermedia ?) from the mid-

Pleistocene (Wells, 1976). An interesting record of N. lepida is reported by Harris

(1984) from Jiminez Cave in southeastern Chihuahua (Figure 21). This record,

being far removed from any modern N. lepida population, is of late Pleistocene

origin, possibly suggesting a Wisconsin refugium for desert woodrats in the

Chihuahuan desert. The question to be addressed, however, is: Which species of

desert woodrat is represented by the sample? Harris (1984) reports that a

modern reference sample of "N. lepida" from California and Baja California differs

significantly in ml length from the Chihuahuan sample. This result is not

surprising since his reference sample would be referable to N. intermedia, which

has other diagnostic mensural characteristics that differ from other members of

the N. lepida group. No significant difference was found in ml length between

modern samples of N. lepida and N. devia collected in this study (mean = 2.98

mm, range 2.72-3.12, n = 17), however, the careful examination of additional

specimens of these species may uncover a distinction. A conscientious effort on

84

the part of paleontologists is needed to reevaluate the Neotoma fossils obtained

from the abundant midden sites to clarify the evolutionary history for the N. lepida

and other woodrat species.

Intrageneric relationships within the Genus Neotoma and an evaluation of

taxonomic subdivisions within the group.-Monophvletic status for the genus

Neotoma was supported by the mtDNA and allozyme analyses. The relationships

among the taxa, however, differ in several aspects from the taxonomic

arrangements suggested by Goldman (1910), Burt and Barkalow (1942), Hooper

(1960), and Hall (1981). The mtDNA cladogenesis for species of Neotoma

corresponds in some cases to subgeneric alignments proposed from data based on

differentially stained chromosomes (Koop et al., 1985) and phylogenetic

relationships based on morphological characters (Carleton, 1980).

Neotoma fuscipes and N. cinerea formed a monophyletic lineage removed

from members of the subgenus Neotoma. The relationship of N. fuscipes,

currently a member of the nominate subgenus, to members of Neotoma or

Teonoma was denied by Goldman (1910), who isolated N. fuscipes in the subgenus

Homodontomys. Burt and Barkalow (1942) later synonymized Homodontomys with

Neotoma, but retained the subgenus Teonoma for N. cinerea on the basis of

bacular specialization.

Neotoma fuscipes and N. cinerea are united by two unique chromosomal

synapomorphies (Koop et al., 1985) and five distintive morphological features

(Carleton, 1980). These two species differ with regard to diploid chromosomal

85

4 -

Catavina ^

Jimenez Cave

kilometers

0 200 400

-40

— 30

Figure 21. Geographic location of critical fossil sites for the Neotoma

lepida species group referred to in text.

86

number (2N=56 for N. fuscipes and 2N=54 for N. cinerea) from the typical

2N=52 reported for most other species of Neotoma (Baker and Mascarello, 1969;

Lee and Elder, 1977). Estimated degree of sequence divergence (6) based on

mtDNA data between the two species was 7.9 percent, a level comparable to the

amount of differentiation between the N. floridana and N. albigula species

complexes, but greater than values between closely related cryptic species and

sister taxa (Table 5).

Phenetic analysis of the mtDNA data presents a different, but explainable,

relationship between N. fuscipes and N. cinerea and the remaining taxa (Figure

17). Neotoma fuscipes and N. cinerea group together in a subcluster containing the

outgroup taxa (Ototylomys phyllotis and Xenomys nelsoni) for the cladistic analysis.

The phenogram is in agreement with the cladograms produced from phylogenetic

analysis of restriction site maps suggesting a basal relationship of N. fuscipes and

N. cinerea to the remaining members of the genus Neotoma. The relatively close

sequence relationship between the four taxa in this subcluster probably reflects the

retention of several pleisiomorphic characters that have been lost by the remaining

species of Neotoma.

Koop et al. (1985) presented two alternative suggestions concerning the

taxonomic treatment of N. cinerea and N. fuscipes: placement of N. cinerea into

the subgenus Neotoma or placement of both N. cinerea and N. fuscipes into the

subgenus Teonoma. The low level of resolution provided by the chromosomal

analysis prevented the authors from making any definitive taxonomic conclusions.

87

Based on the results of the allozyme and mtDNA analyses presented herein,

chromosomal analysis of Koop et al. (1985), and morphological evidence provided

by Carleton (1980), the arrangement of N. fuscipes in the subgenus Teonoma with

N. cinerea is warranted. The placement of N. cinerea into the subgenus Neotoma

is not supported based on the phylogenetic position that N. cinerea and N. fuscipes

hold with regard to other subgenera within the genus and unique morphological

and genetic characters which distinguish this group.

The placement of N. stephensi in association with N. mexicana is somewhat

surprising. Neotoma stephensi was originally placed in the desertorum group of the

subgenus Neotoma, which included the desert woodrats (currently referred to as

the N. lepida group) (Goldman, 1910). Burt and Barkalow (1942) removed N.

stephensi from association with the N. lepida group based on major differences in

the bacula described from the representative taxa, but did not assign the species

to any specific group. The status of N. stephensi was reviewed in studies of the

baculum (Hoffmeister and de la Torre, 1959) and morphology of the glans

(Hooper, 1960), and subspecies underwent taxonomic revision by Hoffmeister and

de la Torre (1960). These studies resulted in the suggestion that N. stephensi

should be placed between N. mexicana and N. phenax (Hooper, 1960).

Phylogenetic and phenetic analyses of allozymes and mtDNA restriction sites are

concordant with the suggestion of Hoffmeister and de la Torre (1959) and Hooper

(1960), placing N. stephensi as a sister taxon to N. mexicana. Cladistic analysis of

the chromosomes of these two species and N. phenax was unable to provide any

88

resolution concerning the position of these taxa with regard to other members of

the subgenus Neotoma (Koop et al., 1985). The mtDNA analysis, however, is in

agreement with the placement of N. phenax close to N. mexicana and N. stephensi.

Neotoma phenax is placed as a sister taxon to the clade containing the members of

the subgenus Neotoma, with the most basal members of that clade represented by

N. mexicana and N. stephensi. The retention of the monotypic subgenus Teonopus

with N. phenax as its sole species is recommended on the basis of the genetic data

presented herein and morphologic and karyotypic distinctions reported previously

(Merriam 1903; Burt and Barkalow, 1942; Baker and Mascarello, 1969).

The degree of genetic and morphological differentiation from other species

groups within the subgenus Neotoma exhibited by the members of the N. lepida

group warrants the recognition of a distinct subgenus for these taxa. The desert

woodrats form a discrete clade that is positioned basally to members of the

subgenus Neotoma and N. phenax, but not as distantly removed as are N. cinerea

and N. fuscipes. Collaborative research currently underway should result in a

taxonomic revision of this group of woodrats and additional information

concerning the species' limits and geographic ranges of its members.

The allozyme data, analysed phenetically and cladistically, are not

concordant (Figures 15 and 16). Agreement is also lacking between either of

these analyses and results of the mtDNA restriction sites data analyses. Major

topological discrepancies between the allozyme analyses primarily involve the

placement of N. phenax, N. fuscipes, and N. cinerea. Phenetic analysis of Rogers'

89

genetic distance results in N. fuscipes being placed on an independant branch, with

N. cinerea occurring in a subcluster with N. mexicana and N. stephensi. Neotoma

phenax is also placed on an independent branch but is associated with members of

the N. albigula complex, N. mexicana species-group, and N. cinerea. Although the

resultant topologies produced by the phenetic analyses show some measures of

similarity to the cladograms produced from the phylogenetic analyses, the use of

clustering methods using measures of genetic distance to infer phylogenies is not

justified since the assumption of an evolutionary clock cannot be assumed for

these taxa (Michener and Sokal, 1957; Felsenstein, 1984).

Cladistic analysis of the allozyme data set resulted in N. phenax being

placed in a clade with the N. lepida group. The placement of N. cinerea and N.

fuscipes differs between FREQPARS and Wagner Parsimony analyses (Figure 15).

FREQPARS placed N. fuscipes in its a clade branching basally to the remaining

Neotoma taxa, with N. cinerea on a branch sharing a common ancestor with

members of the subgenus Neotoma but excluding the N. lepida group. Wagner

Parsimony places N. fuscipes and N. cinerea on a clade containing N. floridana, N.

magister, N. mexicana, and N. stephensi. However bootstrap estimates for this

topology are nonsignificant, suggesting little credence should be placed in the

topology produced by this method (Figure 15).

CHAPTER V

CONCLUSIONS

Summary of taxomomic suggestions for the genus Neotoma.-Based on the

phytogenies developed using allozyme and mtDNA characters in this study, and

information concerning fossil, morphological, and genetic relationships among

members of the genus Neotoma, several suggestions relevant to the taxonomy and

evolutionary relationships of these organisms can be made.

Individuals currently assigned to N. albigula may well be represented by at

least two cryptic species. Populations of white-throated woodrats found to the

east and north of the Rio Grande River and east of the Rio Conchos in Mexico

represent a distinct species possibly referrable to N. leucodon Merriam. A strong

conviction for this taxonomic rearrangement cannot be made until critical

specimens from north eastern Mexico are examined. Populations currently

assigned to N. a. warreni would retain subspecific status under this proposed taxon.

The status of white-throated woodrats from western Chihuahua and Sonora is still

questionable. Degrees of genetic differentiation reported in this study between

samples of white-throated woodrats from western Mexico and populations from

Arizona, New Mexico, and Utah exceed the levels of differentiated exhibited by

other woodrat taxa and other rodent species assemblages reported in the

90

91

literature. Taxonomic considerations for this complex cannot be made at this

time. Firm geographic distributions of the genetically differentiated forms have

not been obtained to sufficiently describe biogeographic patterns that may be

responsible for incipient speciation in this group.

Based on the broad spectrum of evidence presented, the desert woodrats of

the N. lepida species-group represent a distinct lineage of at least subgeneric

status. Nomenclatoral consideration will be presented in a revisionary publication

on this group currently in preparation. Within this assemblage, the desert

woodrats of Baja California, Mexico and the coastal deserts of California

represent a distinct species, well differentiated from the inland, Mohave Desert /

Great Basin form, N. lepida. This species is assignable to N. intermedia Rhoads.

Neotoma stephensi should be considered as a member of the N. mexicana

species group. Although the vast majority of the range of N. mexicana was not

sampled during this study, some level of differentiation is evident between the

northern populations occurring in the United States and isolated populations in

more southern Mexico (Jalisco). Further study of the N. mexicana complex is

warranted especially at the southern terminus of the species' range in Central

America to assess the genetic continuity of this taxon and its relationship with N.

chrysomelas, also assigned to this species group.

Based on the results of the allozyme and mtDNA analyses presented

herein, chromosomal analysis of Koop et al. (1985), and morphological evidence

provided by Carleton (1980), the arrangement of N.fuscipes in the subgenus

92

Teonoma with N. cinerea is warranted. These taxa share a clade which is

positioned basally to the remaining members of the nominate genus. Although

the evidence provided by the various data sets is strong, a more stringent test of

the relationship between N. cinerea and N. fuscipes, and their evolutionary ties to

the other Neotoma would be possible if samples of Hodomys alleni and Nelsonia

neotomodon could be included.

APPENDIX I

SPECIMENS EXAMINED

93

94

APPENDIX I

SPECIMENS EXAMINED

(Museum acronyms described in Chapter II)

Neotoma micropus

TEXAS: Cottle Co., 12 mi. N Paducah (34°10'N 100°18'W) (1) CM.

El Paso Co., 6.75 mi N, 26.5 mi. E El Paso, 3950 ft. (31°51'N

106°03'30"W) (5) CM.

Foard Co., 3 mi. N Crowell (34°03'N 99°44'W) (1) CM.

Hood Co., 19.3 mi. SE Granbury (C. Russell Ranch) (4) CM; 17.5 mi. SE

Granbury (Fort Spunky) (1) CM.

Jack Co., 8 km SW Jacksboro (33°07'N 98°15'W) (10) CM.

Jeff Davis Co., 4.5 mi. S, 7.5 mi. E Kent (Long-X Ranch) (31°00'N

104°05'W) (2) CM.

Johnson Co., 4 km E Nemo (Fry Ranch) (4) CM.

Palo Pinto Co., 1 mi. W Palo Pinto (1) CM.

Taylor Co., 1.1 mi.S, 3.7 mi. W Merkel (32°37'N 100°05'W) (4) CM; 2.5mi. N,

1.8 mi. E Merkel (32°32'N 99°59'W) (.1) CM; 3 mi. N Merkel (32°31'N

10Q°01'W) (12) CM.

NEW MEXICO: Soccoro Co., Seviletta National Wildlife Refuge (21) MSB.

95

MEXICO: Est. Chihuahua, 3 km S, 14 km E Pancho Villa, west of Loma los

Ratones, 1500m (30°50'N 108°30'W) (2) MSB.

Neotoma albigula (sensu lato)

ARIZONA: Cochise Co., 3 mi. N, 1 mi. E Portal, 4600 ft. (31°57'N

109°07'30"W) (11) CM; 5mi. N Portal, 4600 ft. (31°59'N

109°07'30"W) (5) CM.

Coconino Co., 25 mi. NE Flagstaff, 5000 ft. (J) CM; 38 km N, 23 km E

Flagstaff, Coconino National Forest, 4500 ft. (2) CM.

Graham Co., Coronado National Forest, 21.5 mi. N, 5.5 mi. E Wilcox,

4200 ft. (32°33'N 109°45'W) (9) CM.

Mohave Co., 9 km S, 30 km W Kingman (near Oatman), 3000 ft.

(35°07'N 114°23'W) (3) CM.

COLORADO: Los Animas Co., Pinyon Canyon Maneuver Site, 16 mi. E

Thatcher, 4600 ft. (8) CM.

NEW MEXICO: Catron Co., 0.25 mi. S, 2.5 mi. W Mogollon, Gila National

Forest, 6800 ft. (33°23'N 108°50'W) (7) CM.

Dona Ana Co., 18 km W Newman (32°01'N 106°30'W) (4) CM.

Grant Co., Gila National Forest, 32km S, 18 km W Silver City, 6100 ft.

(32°30'N 108°30'W) (6) CM.

Sierra Co., 8 mi E Truth or Consequences (3) CM.

Socorro Co., 34 mi. S, 20 mi. W Socorro, R4W,T9S Sec 16 (2) MSB; 32mi. 5

26 mi. W Socorro, R5W, T8S Sec 4 (5) MSB.

96

TEXAS: El Paso Co., 6.75 mi. N, 26.5 mi. E El Paso, 3950 (31°51'N

106°03'30"W) (4) CM; 7.8 mi. N, 28 mi. E El Paso, (31°52'30"N

106°03'30"W) (3) CM; 8.5 mi. N, 28 mi. E El Paso, (31°53'N

106°02'30"W) (6) CM.

Foard Co., 3 mi. N Crowell (34°03'N 99°44'W) (1) CM.

Hardeman Co., Copper Breaks State Park, 13.2 mi. S, 1 mi. E Quanah

(34°07'N 99°44'W) (10) CM.

Jeff Davis Co., Long-X Ranch, 3 mi. S, 5 mi. E Kent (1) CM.

Moore Co., 9.5 mi. S, 18 miE Dumas (35°44'N 101°40'W) (1) CM.

Randall Co., Palo Duro Canyon State Park, 2.5 mi. S, 15.6 mi. E Canyon

(34°56'N 101°39'W) (8) CM.

UTAH: San Juan Co., Comb Wash, 4 mi. S, 10 mi. W Blanding (7) CM.

MEXICO: Est. Chihuahua, 3 km S, 14 km E Pancho Villa, west of Loma los

Ratones, 1500m (30°50'N 108°30'W) (10) MSB; 6 mi. SW V.

Ahumada (3) UV.

Est. Sonora, 1 mi. NNW San Carlos (1) MSB.

Neotoma albigula/micropus Hybrids

TEXAS: Foard Co,., 3 mi. N Crowell (34°03'N 99°44'W) (JVP 1846) (1) CM.

Jeff Davis Co., Long-X Ranch, 3 mi. S, 5 mi. E Kent (JVP 1937) (1) CM.

Moore Co., 9.5 mi. S, 18 mi E Dumas (JVP1695) (i) CM.

Neotoma floridana

KANSAS: Butler Co., 1 mi. W Potwin along Whitewater River (2) CM.

97

OKLAHOMA: Marshall Co., 3.6 mi. S, 2.3 mi- E Kingston (3) CM.

TEXAS: Cooke Co., 0.7 mi. S, 3.9 mi. E Valley View, 650 ft. (32° 29'N

97°06'W) (17) CM.

Dallas Co., 5.6 mi. SW Dallas (2) CM.

Denton Co., 2 mi. N, 2.8 mi. W Aubrey (33°20'N 97°02'W) (5) CM; 2.8 mi.

N, 6.5 mi. E Sanger, 630 ft. (33°23'30"N 97° 03'W) (7) CM.

Neotoma magister

WEST VIRGINIA: Hampshire Co., Nathaniel Mountain Public Hunting and

Fishing Area, 7.5 mi. S, 8.7 mi. W Augusta, 2880 ft. (39°H'N

78°47'30"W) (2) CM.

Hardy Co., 4 mi. N, 0.7 mi. E Bean Settlement, 3010 ft. (39°10'30"N

78°49'W) (7) CM.

Neotoma mexicana

COLORADO: Archuleta Co., 1.5 mi. S, 2 mi. W Chromo, 7500 ft. (37°05'N

106°50'W) (2) CM.

Costilla Co., 1 mi. S, 2 mi. W San Luis, 8000 ft. (37°15'N.

105°27'W) (5) CM.

Las Animas Co., Pinyon Canyon Maneuver Site, 1.2 mi. S, 20.4 mi. E

Thatcher, 5000 ft. (37°36'N 103°45'W) (5) CM.


Forest, 6800 ft. (33°23'N 108°50'W) (5) CM; 1.5 mi. N, 0.75 mi. E

Luna, 7200 ft. (33°51'N 108°50'W) (8) CM.

98

TEXAS: Jeff Davis Co., Long-X Ranch, 8.2 mi. S, 7.8 mi. E Kent, 5620 ft.

(30°58'N 104°08'W) (4) CM.

MEXICO: Est. Durango, 18 mi. W Durango, 7000 ft. (2) UV.

Est. Jalisco, 8 mi. W Cocula, 5800 ft. (2) UV.

Neotoma pkenax

MEXICO: Est. Sonora, 8 mi NNW San Carlos, 500 ft. (28°20'N

111°10'W) (2) MSB.

Neotoma cinerea

COLORADO: Archuleta Co., 1.5 mi. S, 2 mi. W Chromo, 7500 ft. (37°05'N

106°50'W) (2) CM.

Conejos Co., Rio Grande National Forest, 12 mi. W Antonito, 8700 ft.

(37°04'N 106°12'W) (5) CM.

Costilla Co., 1 mi. S, 2 mi. W San Luis, 8000 ft. (37°15'N

105°27'W) (1) CM.

UTAH: Grand Co., Castle Valley, 12 mi. NE Moab (2) CM.

Neotoma stephensi

ARIZONA: Coconino Co., 25 mi. NE Flagstaff, 5000 ft. (<5) CM.


Forest, 6800 ft. (33°23'N 108°50'W) (2) CM; 1.5 mi. N, 0.75 mi. E

Luna, 7200 ft. (33°51'N 108°50'W) (2) CM.

Neotoma devia

ARIZONA: Coconino Co., 25 mi. NE Flagstaff, 5000 ft. (4) CM; 38 km N, 23 km E

99

Flagstaff, Coconino National Forest, 4500 ft. (2) CM; 2 mi. S,

7 mi. E Marble Canyon (2) CM.

Mohave Co., 9 km S, 30 km W Kingman (near Oatman), 3000 ft.

(35°07'N 114°23'W) (3) CM.

Yuma Co., 50 km S, 7 km E Quartzsite, 3600 ft. (33°13'N

114°10'W) (4) CM.

Neotoma lepida

CALIFORNIA: Imperial Co., 7 mi. S, 3 mi. W Palo Verde, 270 ft. (33°27'N

114°45'W) (3) CM.

Riverside Co., 4 km N, 4.5 km W Desert Hot Springs, 1600 ft.

(33°59'N 116°32'45"W) (2) CM.

San Bernardino Co., 3 km N, 3 km W Yucca Valley, 4000 ft. (340ll'N

116°W) (2) CM; Cima Volcanic Fields, 7 km S, 28.5 km E

Baker (20) LACM.

UTAH: Cane Co., 32 mi. NNW Bigwater (2) CM.

Garfield Co., 8 mi. E Escalante (2) CM.

Washington Co., 13 km N, 6 km W St. George, 4000 ft. (37°12'N

113°38'W) (2) CM; 8 km S, 13 km E Hurricane, 4500 ft.

(37°06N 113°09'W) (5) CM.

Neotoma intermedia

CALIFORNIA: Los Angeles Co., Rancho Palos Berdes, Palo Verde Hills, 1/8 mi. E

of Marineland entrance, 135 ft. (7) LACM.

100

Riverside Co., 13 km S, 3 km W Palm Desert,elev 4000 ft. (Base of

Sugarloaf Mtn.) (33°35'N 116°26'W) (2) CM.

MEXICO: Est. Baja California, 90 km S Mexicali (31°52'N 115°10'W) (5) CM.

Est. Baja California Sur, 12 km S Santa Rosalia, 1000 ft. (27°14'N

115°10'W) (4) CM; Isla Santa Margarita, 2 km N, Naval Base (5) CM.

Neotoma fuscipes

CALIFORNIA: Orange Co.,2 mi. N, 16 mi. E El Toro, Trabuco Canyon

(2)LACM.

San Bernardino Co., 1 km W Morongo Valley, Dry Morongo Creek,

2800 ft. (34°03'N 116°37'W) (2) CM.

Ventura Co., Los Padres National Forest, Pine Mountain, T6N R24W NE

1/4 Sec 1 (2) LACM; Los Padres National Forest, 1/4 mi. W Blue

Point Campground (3) LACM.

Xenomys nelsoni

MEXICO: Est. Jalsico, 6 km E Chamela (J) TTU; 5 km S Chamela (J) TTU; 6

km SE Chamela (1) TTU.

Ototylomys phyllotis

MEXICO: Est. Campeche, 10 km N El Refugio (3); 18 km S X-Kanha (2)

ASNHC.

APPENDIX II

KARYOTYPING PROCEDURES

101

102

APPENDIX II

KARYOTYPING PROCEDURES

1. Remove femurs immediately after sacrificing and flush marrow with 0.075

M KC1 (@37°C) into a centrifuge tube.

2. Agitate to break up marrow and increase volume to approximately 6ml

with the KC1.

3. Add one drop of 0.005% Colchicine and incubate at 37°C for 32 min.

4. After 31 min. add approximately 1ml of Carnoy's Fixative (3:1

methanol:glacial acetic acid).

5. Centrifuge at 1500 rpm for approximately 1 min. to pellet cells.

6. Decant supernatant and add 1ml fixative.

7. Agitate to break up pellet and add approximately 3ml fixative.

8. Centrifuge at 1500 rpm for 1 min.

9. Repeat steps 6-8 three times.

10. After final spin resuspend cells in 2ml fixative.

11. Prepare three slides for analysis and freeze remaining cell suspension in a

cryo vial in liquid nitrogen. (Slides may be flamed for test slides, must be

air dried for banding.)

103

12. Stain test slides with Giemsa Stain (two quantities of Giemsa Buffer to one

part Giemsa Stain stock soln.) for 2-3 minutes.

13. Rinse slide with a stream of dH20 and allow to drain.

Solutions:

0.075M KC1 (0.56g KC1 aliquoted into microfuge tubes)

0.005% Colchicine

Carnoy's Fixative: 3:1 :: Methanol: Glacial Acetic Acid

Giemsa Stain Stock Soln.

Giemsa Buffer:

0.05g NaH2P04

0.09g Na2HP04

100ml dH2Q

Note : To increase the mitotic index of specimens that are to be banded,

yeast pretreatment can be used and mitotic inhibitor reduced or eliminated.

Yeast stress method follows Lee and Elder (1980) : Inject yeast solution (3g yeast

: 2g dextrose : 12ml water) at a dose of O.lml/lOg body weight. After 12-48H

inject 0.05-0. lml Colchicine interperitoneally, incubate 10 min prior to continuing

with the procedure presented above.

APPENDIX III

MITOCHONDRIAL DNA ISOLATION TECHNIQUES

104

105

APPENDIX Ilia

RAPID ISOLATION TECHNIQUE FOR mtDNA1

Isolation of Mitochondria

1. Homogenize tissues, approximately 2g, in 15ml 0.24M Sucrose/EDTA, pH

7.4.

2. Layer homegenate over 20ml 0.34M Sucrose/EDTA, pH 7.4 in a 50-ml

centrifuge tube.

3. Centrifuge layered homogenate at 700 x g for 15 min. to remove nuclei and

debris, decant supernatant into new 50-ml centrifuge tube. Discard debris.

4. Repeat 700 x g centrifugation for 15 min. Decant supernatant into new 50-ml

tube.

5. Centrifuge supernatant at 6800 x g for 15 min. Pellet contains mitochondria.

Decant and discard supernatant.

6. Carefully suspend pellet containing mitochondria in 2ml of 0.24M

Sucrose/EDTA, pH 7.4.

7. Layer mitochondrial suspension over Sucrose step gradient (5ml of 1M

Sucrose-5mM EDTA-lOmM Tris over 3ml 1.5 M Sucrose-5mM EDTA-lOmM

Tris, pH 7.5). This is carried out in 5/8" x 3" Polyallomer tubes.

8. Centrifuge at 37,000 x g at 4°C in TI70.1 fixed angle rotor for 45 min.

106

9. Remove tubes from rotor and place in ultra-cold freezer for 20 min.

10. Using tube slicer cut tube above mitochondrial band (Mitochondria will band

out at the interface between the 1.0 and 1.5M sucrose) and pull frozen

core out of tube. Carefully slice out mitochondrial band and place in 6.0ml

of Soln..A (0.3M Sucrose-lOmM MgCl2-0.15% Bovine Serum Albumin 20mM

Tris HC1, pH 7.5).

11. Centrifuge at 6800 x g for 15 min. Discard supernatant.

NOTE - Steps 6 through 10 can be omitted when isolating mtDNA from

heart and kidney tissues. Replace 2.0ml of 0.24M sucrose in step 6 with 6ml of

0 .24 . sucrose and continue with step 11.

Isolation of mtDNA

1. Disperse mitochondrial pellet in 2.0ml of Soln. A.

2. Add 40^1 DNase I stock (final concentration 0.05 mg/ml). DNase I stock

solution is lmg/ml in 0.15M NaCl-50% glycerol and stored at -20°C.

3. Add lOjul RNase A stock (Maniatis et al., 1982). RNase stock solution is

lOmg/ml of lOmM Tris-HCl, pH 7.5-15mM NaCl heated to 100°C for 15 min

and allowed to cool slowly to room temperature. Store at -20°C.

4. Incubate at 37°C for 30 min.

5. Add 4.0ml Soln. A and centrifuge at 6800 x g for 15 min.

6. Discard supernatant. Resuspend pellet in 1.0ml of Soln. B (0.005M EDTA-

0.01M Tris, pH 8.0).

107

7. Add 30/Ltl of stock Proteinase K solution (20mg/ml in H 2 0) and incubate 5

min at room temperature.

8. Add 25jul of 10% SDS and incubate 10 min at 37°C to lyse mitochondria.

9. Transfer solution into a 6ml tube and centrifuge at 10,000 x g for 10 min to

remove mitochondrial membranes. Supernatant is a solution containing

mtDNA.

10. Decant supernatant into 6-ml polypropylene tube. Extract Proteins with an

equal volume of buffered phenol and centrifuge for 2 min at 10,000 x g.

11. Decant upper aqueous layer into a new 6-ml centrifuge tube and extract

proteins with an equal volume of phenolxhloroform (24:1

chloroform:isoamyl alcohol, pH 7.6). Centrifuge for 2 min at 10,000 x g.

Repeat this step until no protein appears at interface of aqueous and

phenolxhloroform layers.

12. Decant upper aqueous layer and extract additional lipids with an equal

volume of chloroform (24:1 chloroform:isoamyl alcohol, pH 7.6).

Centrifuge for 2 min at 10,000 x g.

13. Decant upper aqueous layer and extract organics with an equal volume of

hydrated ethyl ether (1:1 water:ether) and centrifuge for 2 min at 10,000 xg.

14. Remove ether layer under suction (top layer). Bottom layer contains clean

mtDNA.

15. Precipitate mtDNA 30 min in ulta-cold freezer at -80°C in 3 volumes of

anhydrous ethanol.

108

16. Centrifuge at 10,000 rpm for 20 min to pellet mtDNA.

17. Decant ethanol and wash pellet with 1ml 70% ethanol to remove residual

salts.

18. Carefully remove 70% ethanol under suction and vacuum evaporate

ethanol in vacuo.

19. Resuspend pellet in an appropriate amount of TE Buffer and store frozen

at 20°C until used for restriction analysis.

RECIPES

0.24M Sucrose/ ImM EDTA. pH 7.4

85.6g Sucrose

0.373g EDTA

800ml H 2 0

Adjust pH with NaOH to 7.4 and dilute to 1 liter.

0.34M Sucrose/ ImM EDTA. pH 7.4

116.4g Sucrose

0.373g EDTA

800ml H 2 0

Adjust pH with NaOH to 7.4 and dilute to 1 liter.

109

Solution A (0.3M Sucrose-lOmM MgCl2-0.15% BSA-20mM Tris)

20.54g Sucrose

0.40g MgCl2

0.30g BSA

0.48g Tris

160ml H2O

Adjust pH to 7.5 with HC1 and dilute to 200ml.

Solution B (U005M EDTA-0.01M Tris)

0.37g EDTA

0.24g Tris

160ml H 2 0

Adjust pH to 8.0 with HC1 and dilute to 200ml.

Condensed from Zimmerman et al. (1988)

110

APPENDIX Illb

l ISOLATION OF PURIFIED mtDNA

1. Mince tissues (up to lOg) and homogenize in 2 - 3ml MSB-Ca+2 per gram

of tissue.

2. Add 0.2M EDTA to a final concentration of lOmM (150/il per 3ml

solution).

3. Centrifuge at 700 x g for 5 min at 4°C.

4. Decant supernatant into a fresh 50ml centrifuge tube making sure not to

disturb the debris pellet and repeat spin as in step 3.

5. Decant supernatant into Oakridge Tube and centrifuge at 20K x g for 20

min at 4o C to pellet mitochondria.

6. Decant supernatant and resuspend pellet in 6ml of MSB-EDTA Repeat

centrifugation as in step 5.

7. Decant supernatant and resuspend pellet in 3ml of STE. Add 0.375ml of

10% SDS. Incubate 5 min at room temperature.

8. Alloquot 3.85g CsCl for each sample into Beckman 0.5" X 2" UltraClear™

ultracentrifuge tubes.

9. Add mtDNA suspension and 0.2ml of ethidium bromide (10 mg/ml in

STE). Mix contents well by inversion.

10. Adjust weight to equality using a lg/ml CsCl solution and top each tube

with a layer of mineral oil.

I l l

11. Centrifuge at 36,000 rpm at 20°C for 44-48 hours in a Beckman 50.1

swinging bucket rotor.

12. Observe tube under ultraviolet light (300 nm) to detect bands of DNA,

RNA, glycogen and proteins. Mitochondrial DNA band appears

approximately 0.5 cm below the large nuclear DNA band.

13. Remove DNA bands by puncturing the tube with a syringe and carefully

extracting the DNA.

14. Remove EtBr with at least 3 extractions with of 1-Butanol, followed by one

extraction with 1 volume ethyl ether. Heat at 70°C for about 10 min after

decanting the ether to insure that all ether is extracted.

15. Pipet DNA solution into a 1.5ml microfuge tube and seal with a layer of

dialysis membrane. Insert the inverted tube into a foam float and dialyze

in TE buffer, changing buffer twice on the first day and once on the

following day.

16. DNA is ready for use or may be concentrated by ethanol precipitation.

RECIPES

MSB IU21M Mannitol. 0.07M Sucrose. 0.05M Tris-HCl. pH 7.51

Mannitol 7.65g

Sucrose 4.79g

Tris 1.21g

H 2 0 160ml

adjust pH to 7.5 and bring volume to 200ml

112

MSB-Ca+2 fMSB + 3mM CaCM

As above with 0.088g CaCl2

MSB-EDTA rMSB + 0.01M EDTA. pH 7.51

As above with 0.744g EDTA

STE r O . l M N a d 0.05M Tris-HCl. Q.01M EDTA. pH 8.01

NaCl 1.16g

Tris 1.21g

EDTA 0.74g

H 2 0 160ral

adjust pH to 8.0 and bring volume to 200ml

Adapted from Lansman et al. (1981).

113

APPENDIX IIIc

ISOLATION OF mtDNA BY ALKALINE LYSIS1

1. Homogenize 0.15-0.3g of liver in 1ml of chilled homogenizing buffer (0.25M

Sucrose-lOmM EDTA-30mM Tris-HCl, pH 7.5).

2. Transfer the homogenate to a chilled 1.5ml microfuge tube and centrifuge at

setting "2" (approx. 2000 x g) in a Savant High Speed centrifuge for 5 min at

4°C.

3. Recover supernatant and recentrifuge at 10,000 x g for 10 min at 4°C,

thereby pelleting the mitochondria.

4. Discard supernatant and resuspend mitochondrial pellet in lOmM Tris-EDTA

(pH 8.0) buffer, containing 0.15M NaCl and lOmM EDTA. Total volume

should be 50/il.

5. Add lOOjiil of freshly prepared 0.18M NaOH containing 1% SDS. Vortex

briefly and incubate on ice for 5 min.

6. Add 75jul of ice-cold potassium acetate (3M K-5M acetate). Vortex mixture

and incubate on ice for 5 min.

7. Centrifuge mixture for 5 min at 10,000 x g at 4°C. Recover supernatant and

extract proteins with an equal volume of phenol-chloroform. Mix well by

vortexing.

8. Extract organic solvents with an equal volume of water-saturated ether.

Remove upper ether layer.

114

9. Add 2-3 volumes of anhydrous ethanol and centrifuge for 15 min at room

temperature and 10,000 x g.

10. Decant ethanol and wash resulting DNA pellet with 1ml of 70% ethanol.

Centrifuge at 10,000 x g for 5 min to repellet the DNA. Discard 70% ethanol.

Dry pellet briefly (2 min) in vacuo.

11. Resuspend pellet in 15-20/xl of TE buffer. If DNA is to be stored, treat with

ljul of DNase-free RNase prior to storage.

Adapted from Tamura, K. and T. Aotsuka (1988).

115

APPENDIX Illd

ISOLATION OF TOTAL GENOMIC DNA

1. Mince tissue very fine using a scalpel or razor blade.

2. Transfer tissue into 1ml of Solution B (0.005M EDTA-0.01M Tris, pH 8.0) in

a 15ml conical centrifuge tube. Vortex the mixture briefly, 5 seconds, then

wash down the sides of the tube with 1ml of Soln. B.

3. Add 30jul of 20% SDS and 70/ul of Proteinase K (20 mg/ml in H20).

4. Incubate for 5 hours in a shaking waterbath at 50°C.

5. Centrifuge at 3000 x g for 10 min to sediment any undigested particles.

6. Decant supernatant into a 6-ml polypropylene tube. Extract Proteins with an

equal volume of buffered phenol and centrifuge for 2 min at 10,000 x g.

7. Decant upper aqueous layer into a new 6-ml centrifuge tube and extract

proteins with an equal volume of phenolxhloroform (24:1 chloroform:isoamyl

alcohol, pH 7.6). Centrifuge for 2 min at 10,000 x g. Repeat this step until no

sediment appears at interface of aqueous and phenolxhloroform layers.

8. Decant upper aqueous layer and extract additional lipids with an equal volume

of chloroform (24:1 chloroform:isoamyl alcohol, pH 7.6). Centrifuge for 2

min at 10,000 x g.

9. Decant upper aqueous layer and extract organics with an equal volume of

hydrated ethyl ether (1:1 watenether) and centrifuge for 2 min at 10,000 x g.

10. Remove ether layer under suction (top layer). Heat at 70°C for about 10 min

116

after decanting the ether to insure that all ether is extracted.

11. Precipitate DNA with an equal volume of 1-Propanol in -80°C for 30 min.

Centrifuge for 20 min at 10,000 x g at 4°C to recover DNA.

12. Wash DNA pellet with 70% ethanol and dry in vacuo for 5 min.

13. Resuspend pellet in 1ml of sterile H 2 0 or TE and store frozen until used for

restriction analysis.

APPENDIX IV

SOUTHERN TRANSFER

117

APPENDIX IV

SOUTHERN TRANSFER

1. Electrophorese DNA that has been cut with restriction enzymes an 0.7, 1.0

or 1.2% agarose gel. (65v = 5.5 to 6 hrs., 32v = 1 1 hrs. for 1.0% gels)

2. Cut wells off of the gel and trim if neccessary. Mark size standard lane by

clipping the anodal corner of the gel at the with a razor blade.

3. Place gel in Denaturation soln. (0.4M NaOH, 0.6M NaCl) on shaker at

room temp, for 30 min.

4. Soak gel in Neutralization soln. (1.5M NaCl, 0.5M Tris, pH 7.5) on shaker

at room temperature for 30 min.

5. Cut the nylon transfer membrane (Magna NT™, MSI, Inc.) to the size of

the gel and cut corner to match marking on gel. Wet nylon in ddH20 and

then soak in 10X SSC for approximately 1 min.

6. Layer three sheets of blotting paper (Whatman 3MM) which are cut 7cm

longer than the gel on a platform so that the ends contact a reservoir of

10X SSC. Place the gel such that it will be oriented DNA side up on the

platform.

7. Place the nylon membrane on the gel making sure to align the origin of the

gel with the edge of the membrane. Carefully remove any bubbles by

rolling a glass rod over the filter. Place three to five layers of blotter paper

cut to the size of the gel on top of the filter making sure to remove all air

118

119

and bubbles. Layer approximately six inches of blotting pads or paper

towels on top. Place a glass plate over the stack. Weight the assembly

with a 500-1000ml flask of water and allow it to blot overnight.

8. Remove the paper towels and blotter paper. Label the DNA-side of the

membrane with pencil or permanent ink.

9. Crosslink the DNA onto the membrane by exposing it to UV (254nm) light

for 3 min at a distance of 10cm.

APPENDIX V

LABELING OF PROBES AND HYBRIDIZATION

120

APPENDIX V

LABELING OF PROBES AND HYBRIDIZATION

Preparation of probes (32P-dCTP and Digoxigenin labeling)

1. Prepare probe or oligonucleotide for labeling (i.e. with mtDNA probes in

pUC 18 digest from l-3^g of probe with Hinf II for 30 min to linearize

plasmid with insert).

2. To prepared probe add 2/zl of hexanuclucleotide primers and 2/il of dNTP

mixture and adjust volume to a total of 19/zl. For 32P-dCTP labeling, dNTP

mixture lacks dCTP. Radioactive nucleotide must be added independently.

3. Heat-denature mixture for 10 min at 98°C and then place immediately on

ice.

4. Spin down mixture briefly and add 1/xl Klenow enzyme, mix well then spin

down briefly.

5. Incubate at 37°C for at least 45 min to allow random priming reaction to

effectively label probes.

6. Probes can now be diluted and stored for later use (dilute to 75^1).

Radioactively-labelled probes must be measured on a liquid scintillation

counter such that proper amounts of probe can be used during

hybridizations.

121

122

Hybridization with 32P-dCTP-labelled probes

1. Preheat hybridization solution (see below) to 65oC for 10-15 min. Add

0.58g NaCl and heat for an additional 10-15 min at 65°C.

2. Place membrane in a thermal sealable hybridization bag and seal the edge.

Make a .small hole in the corner and pipet the hybridization soln. (10-15ml)

into the bag. Remove as much of the air as possible and reseal the corner.

Incubate at 65°C in shaking water bath for 30 min.

3. Prepare the probe mixture as follows:

To 500/xl of TE, add 100/il of salmon sperm DNA (lOOjug/ml) and the

equivalent of 106 counts per min. of each probe. Denature the probes at

95°C for 10 min and place immediately on ice until used.

4. Using a syringe, inject the probe mix into the hybridization bag, remove all

air bubbles, and reseal.

5. Hybridize at 65°C in a shaking water bath overnight.

6. Decant the radioactive hybridiztion solution into an approved liquid waste

container and perform three 5-min washes on the membranes in

approximately 500ml of 2X SSC at room temperature with mild agitation.

7. Wash the membrane once in 500ml of 2X SSC, 1% SDS in a 65°C in a

shaking water bath for 30 min.

8. Rinse the nmembranes breifly with 2X SSC, blot the filter with a piece of

absorbent paper, and then place in a resealable plastic bag. Do not allow

the nylon to dry completely if planning to reprobe.

123

9. Detect DNA restriction patterns by autoradiography. Expose film for 24

hrs. to 10 days, depending on the quantity of DNA used, using an

intensifier screen at -80°C.

** For alternate washing schemes see Micron Separations Inc. information manual

Probe removal

1. Wash membranes in 0.4N NaOH at 42°C for 30 min.

2. Wash for 30 min in probe removal soln. (0.1X SSC, 0.1% SDS, 0.2M Tris-

HC1, pH 7.5).

3. Check membrane with a minimonitor to determine if any radioactivity

remains. If necessary, repeat steps 1 and 2.

4. Prehybridize and hybridize membranes as before.

Hybridization Solution

7ml H 2 0

2ml 50% dextarn sulfate

lml 10% SDS

Add 0.58g NaCl prior to use.

124

Hybridization and detection with Digoxigenin-labelled probes1

1. Prehybridize filters in hybridization solution at 65°C for 1 hour. Volume of

hybridization solution used will depend on the number of filters to be

probed and apparatus used.

2. Preparation of probes: combine 5-10/xl of each probe and 200^1 H 2 0 in a

1.5-ml tube and heat-denature at 98°C for 10 min.

3. Place immediately on ice to cool then centrifuge briefly before use.

4. Hybridize overnight at 65°C with agitation.

Hybridization Solution

5XSSC

1% w/v blocking reagent1 (10g/l)

0.1% w/v N-lauroylsarcosine sodium salt (lg/1)

0.02% w/v SDS (0.2g/l)

Post-hybridization treatment

1. Wash filters using the following schedule for a fairly high stringency

treatment:

3 X 5 min @ room temp, in 2X SSC

1 X 30 min @ 65°C in 2X SSC/1% SDS

(50ml 20X SSC+425ml H20+25ml 20X SDS)

2. Equilibrate filter in Buffer A (see below) for 1 min.

3. Block nylons for 3 hours in Buffer B (see below) at room temp with

agitation.

125

4. Dilute anti-DIG/AP conjugate 1:5000 (i.e. 12jul Vial 8 + 60ml Buffer B).

5. Incubate filter for 1 hour or more in anti-DIG soln at room temp while

agitating.

6. Wash filter 2 X 15 min in Buffer A at room temp while shaking.

7. Equilibrate filter in Buffer C (see below) for 2 min.

BUFFER A

12.1 lg Tris

8.77g NaCl

900ml H 2 0

pH 7.5 dilute to 1L

BUFFER B (2% w/v Blocking Agent in Buffer A)

200ml Buffer A

4g Blocking Agent

BUFFER C

12.11g Tris

5.85g NaCl

10.17g MgCI2

pH 9.5 adjust to 1L

126

Lumiphos™ 530 visualization

1. Place filter DNA side up on a piece of clear acetate. Do not allow filter to

dry.

2. Prewarm aliquotted Lumiphos™ 530 to room temp, and apply 100/xl to the

DNA side of the filter.

3. Using another piece of acetate cover filter such that the Lumiphos™ 530

is distributed evenly across the membrane. Use a glass rod to roll solution

between acetate sheets.

4. Seal margins of acetate and incubate at 37°C for 30 min to allow light

emission to reach steady state.

5. Expose film for required amount of time... this depends on the

concentration of DNA that is on the filter.

Filter stripping for reprobing

1. Do not allow filter to dry before removing probe.

2. Rinse membrane in sterile distilled H 20.

3. Incubate membrane in 0.2 N NaOH, 0.1% SDS at 37°C for 30 min with

agitation.

4. Wash membrane in 2X SSC. Filter can now be reprobed.

I This is a modified protocol from the Boehringer Mannheim Genius™

DIG DNA labeling and detection kit (Cat. No. 1093 657) using Lumiphos™ 530

(B. M. Cat No. 1275 470).

APPENDIX VI

ALLELE FREQUENCIES FOR NEOTOMA SAMPLED

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APPENDIX VII

CHARACTER STATE DATA, RESTRICTION SITE POSITIONS,

AND SPECIMEN LOCALITY CODES

134

APPENDIX VII

CHARACTER STATE DATA, RESTRICTION SITE POSITIONS,

AND SPECIMEN LOCALITY CODES

Character state matrix generated from restriction site analysis of 35 Neotoma OTUs and two outgroup taxa, Xenomys nelsoni and Ototylomys phyllotis. Specific restriction sites detailed in accompanying table.

CHARACTER

N. floridana 1 N. floridana 2 N. magister N. micropus 1 N. micropus 2 N. micropus 3 N. micropus 4 N. micropus 5 N. albigula El N. albigula E2 N. albigula E3 N. albigula E4 N. albigula W1 N. albigula W2 N. albigula W3 N. albigula Ml N. albigula M2 N. lepida 1 N. lepida 2 N. lepida 3 N. devia 1 N. devia 2 N. intermedia 1 N. intermedia 2 N. intermedia 3 N. intermedia 4 N. intermedia 5 N. mexicana 2 N. mexicana 3

10000010000001110010000100000010001010010011000100 10000010000001110010000100000010001010010011000100 10000010000001110010000100000010001010010011000100 10100010000001110010000100100010001000000010000000 10100010000001110010000100100010001000000010000000 10100010000001110010000100100010001010000010000000 10100010000001110010000100100010001000000010000000 10100010000001110010000100100010001000000010000000 10011010000001110010000100100000001010000010010000 10011010000001110010000100100000001010000010010000 10011010000001110010000100100000001000000010010000 10011010000001110010000100100000001000000010010000 10001010000001110010000100100000001000000011000000 10001010000001110010000100100000001000000011000000 10001010000001110010000100100000001000000011000000 10001010000001110000000101000000000000000011000000 10001011000001000011000100000000001000000010000000 10000011110100000000110001010000000100000011100000 10000011110100000000110001010000100100000011100000 10000011110100000000110001010000000100000011100000 10000011110100000000110001010000000000000001100000 10000011110100000000110001010000000100000001100000 10000011011100000000010001000000000001000001100000 10000011010100000000011001000000000001000000100000 10000011011100000000010001000000000001000001100000 10000011011100000000010001000000000001000001100000 10000011011100000000010001000000000001000001100000 10000011000011000110000000000000010000000010000100 10000011000001100110000000010000000000000010001100

135

136

N. stephensi N. phenax N. fuscipes 1 N. fuscipes 2 N. cinerea Xenomys Ototylomys N.floridana 1 N. floridana 2 N. magister N. micropus 1 N. micropus 2 N. micropus 3 N. micropus 4 N. micropus 5 N. albigula El N. albigula E2 N. albigula E3 N. albigula E4 N. albigula W1 N. albigula W2 N. albigula W3 N. albigula Ml N. albigula M2 N. lepida 1 N. lepida 2 N. lepida 3 N. devia 1 N. devia 2 N. intermedia 1 N. intermedia 2 N. intermedia 3 N. intermedia 4 N. intermedia 5 N. mexicana 1 N. mexicana 2 N. mexicana 3 N. stephensi N. phenax N. fuscipes 1 N. fuscipes 2 N. cinerea Xenomys Ototylomys

10000101000011000010000000000001000000000010001000 11000011000001000010000100011000000000100100000010 10000011000001000000000001000100011000100000000000 10000011000001000000000001000100011000100000000000 11000011000001000000001100000100000000101000000001 00000011100001000000001101000001000000100000000001 10000011000000001000100111000101000000100000000001 01100010000010000001000010000011000000100011000001 01100010000010000001000010000011000000100010000001 01000110000010000001000010001011000000100010100001 00100110010000000001000010001011001000100110000000 00100110010000000001000010001011001000100110000000 00100110010000000001000010001011001000100110000000 00100110010000000001000010001011001000100110000000 00100110010000000001000010001011001000100110000000 00100011010010000001000010101011000000100110000000 00100011010010000001000010101011000000100110000000 00100011010010000001000010101010000000100110000000 00100011010010000001000010101010000000100110000000 00100011010010000001000010101011000000100110000000 00100011010010000001000010001011000000100110000000 00100011010010000001000010001011000000100110000000 00100011001010000001000010001001000000100100000000 00100011000010000001000010101001000000100110000000 10010100001000000000100100001001000010100000000010 10010100001000000000100000001001000010100000000010 10010100001000000000100100001001000010100000000010 10000100001000000000100100011001000010100000000010 10010100001000000000100100011001000010100000000010 00010100101000000000100000010001000000100000000010 00010100101000000000100000010001000000100000000010 00010100101000000000100000010001000000100000000010 00010100101000000000010000010001000000100000000010 00010100101000000000100000010001000001000000000010 00000000000010000101001010001001100101100100000000 00000000000010000101001010001001100101100100000000 00000000000010000101000010001011100101100100000000 00001000000010000001000010001011000100100100000001 00100000101010100001000000001100000000100110000101 00000001000000011011010000001001110000000000010000 00000001010000011011010000001001110000000000011000 00000001000000011000000000001001010000000011000001 00000001000100000000000000001001000000001011000000 00000001000100000000000001000001000000010010000000

137

N. floridana 1 N. floridana 2 N. magister N. micropus 1 N. micropus 2 N. micropus 3 N. micropus 4 N. micropus 5 N. albigula El N. albigula E2 N. albigula E3 N. albigula E4 N. albigula W1 N. albigula W2 N. albigula W3 N. albigula Ml N. albigula M2 N. lepida 1 N. lepida 2 N. lepida 3 N. devia 1 N. devia 2 N. intermedia 1 N. intermedia 2 N. intermedia 3 N. intermedia 4 N. intermedia 5 N. mexicana 1 N. mexicana 2 N. mexicana 3 N. stephensi N. phenax N. fuscipes 1 N. fuscipes 2 N. cinerea Xenomys Ototylomys N. floridana 1 N. floridana 2 N. magister N. micropus 1 N. micropus 2 N. micropus 3 N. micropus 4

00000000001100000000000100100000010001000000001000 00000000001000000000000100100000010001000000001000 00000000001100000000000000100000010001000000001000 00000000001100000010100000100000010000100000000000 00000000001100000010100000100000010000100000000000 00000000001100000010100000100000010000100000001000 00000000001100000010100000100000010000100000000000 00000000001100000010100000100000010000100000000000 00000000001000000001100000000000010001000000000000 00000000001000000001100000000000010001000000000000 00000000001000000001100000000000010001000000000000 00000000001000000001100000000000010001000000000000 00000000001100000000100000100000010001010000000000 00000000001100000000100000100000010001010000000000 00000000001100000000100000100000010001010000000001 00000000001000000000100000100000010001000000000000 00000100001100000000100000000000010001000000000001 00100000100001000000000100010000010000000011010000 00100000101001000000000100010000010100010011010000 00100000100001000000000100010000010000000011010000 00100000000011000000000000010000000100010001010000 00100000000011000000000000010000010000000001010000 01000000001001001000000000010000000000000001000010 01000000001001001000000000000000000000000001000110 01000000001001001000000000010000000000000001000110 01000000001001001000000000010000000000000001000110 01000000001001001000000000010000010000000000000110 00100000001000010101000010000000011000000100001000 00100000001000010101000010000010011000000100001000 00010000001000010101000010000010011000000100001000 01100000001000010101000010001000010000000000001000 00000000010000000000000000000001010000001101000000 00001000001001000100011001000000111000000000000000 00001000001001000100011001000000111000000000000000 00000000001001010000001001000000011000000000000000 00000010001011100000000000000000110011000100100000 10000001001011000000000000000100100000000000100000 000000001000000001010000000000010010001000 000000001000000001010000000000010010001000 000100001100000001010000100000000010001000 100000011010000000010000010000001000101001 100000011010000000010000010000000000101001 100000011010000000010000000000001000101001 100000011010000000010000010000000000101001

138

N. micropus 5 N. albigula El N. albigula E2 N. albigula E3 N. albigula E4 N. albigula W1 N. albigula W2 N. albigula W3 N. albigula Ml N. albigula M2 N. lepida 1 N. lepida 2 N. lepida 3 N. devia 1 N. devia 2 N. intermedia 1 N. intermedia 2 N. intermedia 3 N. intermedia 4 N. intermedia 5 N. mexicana 1 N. mexicana 2 N. mexicana 3 N. stephensi N. phenax N. fuscipes 1 N. fuscipes 2 N. cinerea Xenomys Ototylomys

100000011010000000010000010000000000101001 110010011000000000000000010000001100100001 110010011000000000000000010000001100100001 110010011000000000000010000000001101100001 110010011000000000000010000000001101100001 100100111001000000010010010000001100100001 100100111001000000010010010000001100100001 100100111001000000010010010000001100100001 001000110001000000000010010000000101100000 100000110001000001000010010000001101100000 001100000000100001011000001010000100000100 001100000000100001011000001010000100000100 001100000000100001011000001010000100000100 001000000000100001011000001010000100000100 011010000000100001011000001010000100000100 001000000000100001011000001010000000000100 001000000000100001010000001010000000000100 001000000000100001011000001010000000000100 001000000000100001011000001010000000001100 000000000000100001011000001000000000000000 000000000000010000000010000000000100000010 000000000000010001000010000000000100000010 000000000000010001000010000000001100000010 000000000000010001000000000100000000000010 000000000000001001100110010001000100010100 100001111000000101000000001010000000100000 100001111000000101000000001010000000100000 100000100000000001000000001000000000000000 100100100000010001001011001000000100010100 100100100000000011000010000000100100000100

139

Character description of restriction site location mapped for a conserved Bgl I site which is character 1.

RACTER ENZYME MAP POSITION fkM 1 Bgl I 0.0 2 4va I 0.05 3 Dra I 0.20 4 Bam HI 0.30 5 Stu I 0.40 6 Eco RI 0.40 7 Bsp 106 0.60 8 Bst NI 0.70 9 Bam HI 0.80 10 Stu I 0.95 11 Ava I 0.95 12 Dra I 1.05 13 BstE II 1.00 14 Hinc II 1.00 15 Bst NI 1.20 16 Eco RV 1.50 17 Dra I 1.30 18 4va I 1.40 19 Apa I 1.60 20 Eco RI 1.50 21 Apa I 1.10 22 i/mc II 1.70 23 Dra I 1.90 24 Ava I 2.00 25 Pst I 1.90 26 Pvu II 2.20 27 Eco RV 2.50 28 Stu I 2.50 29 Dra I 2.50 30 BstE II 2.40 31 Bam HI 2.50 32 Dra I 2.80 33 Hinc II 2.95 34 Stu I 3.05 35 Bam HI 3.10 36 Eco RI 3.05 37 Ava I 3.40 38 Bgl I 3.40 39 Eco RI 3.30 40 Ava I 3.40

140

CHARACTER ENZYME MAP POSITION (kb) 41 Bst NI 3.70 42 Pst I 3.60 43 Stu I 3.80 44 Ava I 3.90 45 Bst NI 3.90 46 Bgl I 4.00 47 Bam HI 4.00 48 Eco RV 4.20 49 Eco RI 4.20 50 Apa I 4.25 51 Pvu II 4.30 52 Bst NI 4.40 53 Pvu II 4.50 54 Hinc II 4.40 55 Bst NI 4.70 56 Bam HI 4.70 57 Hinc II 4.80 58 Eco RV 4.90 59 Stu I 4.90 60 BstE II 5.10 61 Eco RV 5.30 62 Ava I 5.40 63 Eco RI 5.50 64 Kpn I 5.50 65 BstE II 5.80 66 Bst NI 5.70 67 Apa I 5.90 68 Stu I 5.90 69 Hinc II 6.00 70 Dra I 6.00 71 Bgl II 6.00 72 Eco RI 6.15 73 Pvu II 6.20 74 Stu I 6.30 75 Ava I 6.30 76 Dra I 6.50 77 Eco RI 6.40 78 Eco RI 6.60 79 Bam HI 6.80 80 Bst NI 7.00 81 Hinc II 7.30 82 Bst NI 7.30 83 Eco RV 7.40

141

CHARACTER ENZYME MAP POSH 84 Stu I 7.50 85 Eco RI 7.50 86 Hinc II 7.70 87 Eco RV 7.80 88 Stu I 7.90 89 Bgl II 8.00 90 Dra I 8.00 91 Apa I 8.10 92 Bam HI 8.20 93 Pst I 8.30 94 Eco RI 8.70 95 Eco RV 8.90 96 Hinc II 8.70 97 Eco RV 8.80 98 Bgl II 8.90 99 Hinc II 9.00 100 Bam HI 9.20 101 Apa I 8.85 102 Pvu II 9.60 103 BstE II 9.50 104 Stu I 9.40 105 Stu I 9.70 106 Eco RI 9.60 107 Kpn I 9.70 108 Bst NI 9.10 109 Bst NI 9.90 110 Bam HI 9.90 111 Eco RI 10.00 112 Eco RI 10.40 113 Ava I 10.00 114 Apa I 10.20 115 Hinc II 10.20 116 Bst NI 10.20 117 Stu I 10.40 118 Pvu II 10.60 119 Pst I 10.70 120 Hinc II 10.70 121 Dra I 10.80 122 Bst NI 10.90 123 Bam HI 11.00 124 Eco RV 11.20 125 Dra I 11.10 126 Hinc II 11.90

142

CHARACTER ENZYME MAP POSIT 127 Pvu II 11.20 128 Eco RI 11.40 129 Apa I 11.30 130 Ava I 11.50 131 BstE II 11.50 132 Stu I 10.80 133 Eco RV 11.60 134 Stu I 11.60 135 Hinc II 11.90 136 Eco RV 12.00 137 Bam HI 11.90 138 Pvu II 12.00 139 Apa I 12.00 140 Eco RV 12.10 141 Bsp 106 12.00 142 Eco RI 12.30 143 Bgl II 12.10 144 Dra I 12.40 145 Ava I 12.50 146 Hinc II 12.50 147 Bst NI 12.60 148 BstE II 12.70 149 Bsp 106 12.80 150 Bgl I 12.90 151 Dra I 13.00 152 Bam HI 13.00 153 Eco RV 13.00 154 Bst NI 13.30 155 Bam HI 13.05 156 Bam HI 13.60 157 Dra I 13.50 158 Stu I 13.60 159 Apa I 13.90 160 Eco RI 13.90 161 Eco RV 13.90 162 BstE II 14.00 163 Stu I 14.00 164 Bst NI 14.10 165 Ava I 14.10 166 Apa I 14.10 167 Bsp 106 14.10 168 BstE II 14.40 169 Pvu II 14.50

143

CHARACTER ENZYME MAP POSITION (kb)

170 Eco RI 14.60 171 Pvu II 14.80 172 Hinc II 14.70 173 Dra I 15.00 174 Pvu II 15.10 175 Apa I 15.18 176 Bst NI 15.20 177 Hinc II 15.30 178 Dra I 15.40 179 Bam HI 15.50 180 Bgl II 15.60 181 Bsp 106 15.50 182 Dra I 15.60 183 Bst NI 15.80 184 Dra I 15.90 185 Stu I 16.00 186 Bgl II 16.10 187 Hinc II 16.20 188 Eco RI 16.10 189 Bgl II 16.30 190 Bst NI 16.30 191 Dra I 16.40 192 Pst I 16.40

144

SPECIMEN LOCALITY CODES

N. floridana 1 N. floridana 2

N. micropus 1 N. micropus 2 N. micropus 3 N. micropus .4 N. micropus 5

N. albigula El N. albigula E2 N. albigula E3 N. albigula E4 N. albigula W1 N. albigula W2 N. albigula W3 N. albigula Ml N. albigula M2

N. lepida 1 N. lepida 2 N. lepida 3

N. devia 1 N. devia 2

all populations from Texas populations from Oklahoma and Kansas

Texas: Jack and Taylor cos. Texas: all remaining localities Mexico: Chihuahua Texas: Jeff Davis Co. New Mexico

Texas: Jeff Davis Co.; New Mexico: Sierra Co. New Mexico: Dona Ana Co. Colorado: Las Animas Co. Texas: Randall and Hardeman cos. Arizona: Coconino Co.; Utah: San Juan Co. Texas: El Paso Co. Arizona: Remaining localities; New Mexico Mexico: Chihuahua Mexico: Sonora

Utah: all localities California: San Bernardino Co.,Cima Volcanic Fields California: all remaining localities

Arizona: Coconino Co. Arizona: Mohave and Yuma cos.

N. intermedia 1 N. intermedia 2 N. intermedia 3 N. intermedia 4 N. intermedia 5

N. mexicana 1 N. mexicana 2 N. mexicana 3

N. fuscipes 1 N. fuscipes 2

Mexico: Baja California Sur, Santa Rosalia Mexico: Baja California Sur, Isla Santa Margarita California: Riverside Co. Sugarloaf Mtn. California: Los Angeles Co. Mexico: Baja California, Mexicali

Texas; Colorado; New Mexico Mexico: Durango Mexico: Jalisco

California: Orange and Ventura cos. California: San Bernardino Co.

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Anderson, S. 1972. Mammals of Chihuahua Taxonomy and Distribution. Bulletin

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Armstrong, D. M. 1972. Distribution of mammals in Colorado. Monograph of

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