MOLECULAR SYSTEMATICS OF THE GENUS Neotoma

BASED ON NUCLEAR DNA SEQUENCES FROM

INTRON 2 OF THE ALCOHOL DEHYDROGENASE GENE

by

LISA KAY LONGHOFER, M.S.

A THESIS

IN

ZOOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved

Chairperson of the>86^5mittee

Accepted

Dean of the Graduate School

December, 2004

ACKNOWLEDGMENTS

I would like to express thanks to The National Institutes of Health

(DHHSAI4135-01 to R. Bradley) who provided fimding that was instrumental to

performing this research. I also would like to thank the Department of Biological

Sciences at Texas Tech University for all their support and help in many different aspects

of my graduate career. I would like to extend my gratitude to Natural History Museums

and their associated curators of mammals for tissue loans that were crucial to the success

of this project, these include: The Natural Science Research Laboratory at the Museum

of Texas Tech University (Dr. Robert J. Baker); and The Museum of Southwestern

Biology, University of New Mexico (Dr. Terry Yates).

I am thankful for the constant assistance and guidance provided by my committee

members throughout this project: Dr. Robert Bradley, Dr. Robert Baker, and Lou

Densmore. I would also like to thank Dr. Nancy Mclntyre for setting in on my defense

on such short notice. I would like to thank and extend my utmost gratitude to my

committee chair. Dr. Robert Bradley for providing mentorship and helping in the

completion of this work in a timely fashion that was crucial in earning this Master's

Degree as well as the next educational step in my life.

I am grateful and indebted to all of my fellow graduate students and

undergraduate students of Texas Tech University for their assistance and fi^iendship

throughout my years as a graduate student. They include former and recent Bradley lab

members: Brian Amman, Dnate' Baxter, Nevin Durish, John Hanson, Francisca

11

Mendez-Harclerode, Michelle Haynie, and John Suchecki. I would also like to thank

members of the Baker and Densmore labs, particularly Steve Hoofer for providing his

expertise in assisting with this work. I especially gratefiil to Brian Amman and John

Hanson for their assistance and knowledge in the Adh project while working in

conjunction with them. Brian Amman and Dnate' Baxter were instrumental in completion

of this project. Their sense humor, crazy conversions, and our constant attacks of

laughter made the whole journey worthwhile which will never be forgotten. I would

especially like to thank Dnate' Baxter, my closest friend, colleague, and roommate.

Without her support, conversations, and positive attitude, it would not have been

possible. I am gratefiil to them all and am looking forward to lasting relationships. Of

course, nothing would have been possible without the constant love and support of my

family, especially my parents Eddie and Judy Longhofer. Then- support, kindness, and

patience during my college career will never be forgotten. I will always be indebted to

you both.

Ill

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

LIST OF TABLES vi

LIST OF FIGURES vii

CHAPTER

I. INTRODUCTION 1

Background and Significance I

Objectives 3

Hypotheses 4

Chapter Summaries 4

Literature Cited 6

n. MOLECULAR SYSTEMATICS OF THE GENUS NEOTOMA BASED ON NUCLEAR DNASEQUENCES FROM INTRON 2 OF THE ALCOHOL DEHYDROGENASE GENE 9

Abstract 9

Introduction 10

Materials and Methods 16

Results 20

Discussion 23

Literature Cited 34

III. CONCLUSIONS 46

IV

Summary 46

Literature Cited 48

LIST OF TABLES

2.1 Average Tamura and Nei (1993) genetic distances (AGO) for selected taxa of Adh clades and Cyt-b species groups examined in this study 40

2.2 Specimens examined and GenBank accession numbers 41

VI

LIST OF FIGURES

1.1 Maximum likelihood tree (GTR+I+G model) based on Edwards and Bradley (2002b) depicting phylogenetic relationships of 76 samples of Neotoma 8

2.1 Strict consensus tree generated from a parsimony analysis of nucleotide sequences obtained from the .4isf/i-1-12 gene 43

2.2 Phylogenetic topology using Bayesian analysis (GTR+I+G model of evolution) obtained from the Adh-X-n gent 44

2.3 Phylogenetic topology using Bayesian analysis (GTR+I+G model of evolution) obtained from the Adh-\-lll CyiochromQ-b genes 45

Vll

CHAPTER I

INTRODUCTION

Background and Significance

The genus Neotoma, commonly referred to as woodrats, is an assemblage of

medium-sized rodents in the New World subfamily Sigmodontinae. Woodrats are

distributed across most of the continental United States, portions of southern Canada,

several Baja Islands, throughout much of Mexico, and into northern Central America

(Hall 1981). Woodrats are abundant, widespread, and diversified as indicated by their

success in adapting to a wide variety of habitats ranging from above the timberline in

mountain regions to costal lowlands and from mesic, redwood forests to the xeric High

Plains and deserts (Finley 1953). Consequently, Zimmerman and Nejtek (1977)

suggested that the genus Neotoma provides an excellent model for studjdng genetic

differentiation among species. Furthermore, woodrats are important because they are

associated with many New World zoonotic viruses and diseases, including arenavirus

(Fulhorstetal. 2001).

Although the genus was 1st revised by Goldman (1910), various systematic

studies have attempted to establish evolutionary relationships among the members of this

group (Burt and Barkalow 1942; Hooper 1960; Bimey 1973; Mascarello and Hsu 1976;

Zimmerman and Nejtek 1977; Koop et al. 1985; Shipley et al. 1990; Hayes and Harrison

1992; Hayes and Richmond 1993; Planz et al. 1996; Edwards et al. 2001; Edwards and

Bradley 2001,2002a, b; Patton and Alvarez-Castaneda in press). Several factors, both

genetic and ecological, have made it difficult to address phylogenetic relationships within

1

Neotoma. Neotoma has been divided into 3 subgenera (Neotoma, Teonoma, Teonopus)

with the subgenus Neotoma containing 22 species whereas subgenera Teonoma and

Teonopus are monotypic. Traditionally, studies of the subgenus Neotoma have used

phenetic approaches to assess similarities and to determine associations among taxa.

Goldman (1910) divided this subgenus into 6 species groups (albigula, desertorum,

floridana, intermedia, mexicana, zxidpennsylvanica). Burt and Barkalow (1942) later

identified 6 species groups (albigula, floridana, fuscipes, lepida, mexicana, and

micropus), although the species groups differed from Goldman's (1910) assessment. Due

to the confroversial recognition of species-groups, there have been several conflicting

conclusions on how to aUgn, organize, and classify taxa within the genus Neotoma

(Goldman 1910; Hall and Kelson 1959; Hall 1981).

Recentiy, Edwards and Bradley (2001,2002a, b) and Edwards et al. (2001) used a

phylogenetic approach with DNA sequences from the mitochondrial DNA cytochrome-/)

gene and divided the subgenus into 4 species groups (floridana, micropus, lepida, and

mexicana). They have recognized 24 species of Neotoma, which were placed into 4

species groups: 1) the N. floridana group including N. albigula, N. floridana, N.

goldmani, and N. magister; 2) the N. lepida group including N. fuscipes, N. lepida, and N.

stephensi; 3) the N mexicana group including N. angustapalata, N chrysomelas, N

isthmica, N. mexicana, and A/, picta; and 4) the N micropus group including A/i micropus

and N. leucodon. However, Edwards and Bradley (2002b) also suggested further studies

to determine ifN. nelsoni, N. palatina, and A . varia belong in the N micropus species

group.

Edwards and Bradley (2002b) conducted an extensive phylogenetic study on the

genus Neotoma using mitochondrial sequences from the cytochrome-/) gene, providing a

phylogeny that could be used for comparisons with subsequent studies (Figure 1.1).

Although Edwards and Bradley (2002b) provided a working hypothesis on phylogenetic

relationships within the genus, their study was based solely on the mitochondrial genome.

Ballard et al. (2002) has questioned whether divergence of mtDNA should be used as the

sole basis for species recognition and cautions about using mtDNA divergence

conclusions for organismal differentiation without additional support from independent

data sets. Avise (1994) also states that mitochondrial variation may not cortelate with

other genomic aspects because of selection, infrogression, poor resolution of the data, or

lineage sorting. Additionally, no study has included more than half of the taxa in the

genus. Although the results of previous studies have provided valuable information,

nuclear independent data sets are needed to obtain an alternative perspective on the

phylogenetic relationships within the genus.

Objectives

The 3 objectives of this study are outlined below:

1.) to use nuclear DNA sequences from intron 2 of the alcohol dehydrogenase

gene 1 (Adh-l-U) to evaluate current hypotheses of phylogenetic relationships

among the members of Neotoma.

2.) to assess the utihty of the Adh-l-I2 gene, and its variability within the genus.

3.) to utilize both nuclear and mitochondrial sequence data in a combined

analysis in order to evaluate phylogenetic relationships among the members of

this group.

Hvpothesis

The hypothesis of Edwards and Bradley (2002b), regarding phylogenetic

relationships among members of Neotoma, will be tested using DNA nucleotide sequence

variation in the nuclear alcohol dehydrogenase gene 1. Specifically, the following

hypotheses will be tested:

1.) The phylogenetic relationships among Neotoma using DNA sequences

from the nuclear alcohol dehydrogenase gene 1 (Adh-1) will be congruent to

the Edwards and Bradley (2002b) phylogeny based on DNA sequences from

the mitochondrial cytochrome b gene.

2.) The genus Neotoma is a monophyletic assemblage in which Hodomys is not a

member.

3.) Hodomys is a valid and distinct genus sister to Xenomys.

4.) The intron 2 ofAdh-l is a variable nuclear marker that provides sufficient

resolution for the phylogenetic assessment of Neotoma.

Chapter Summary

Chapter U examines phylogenetic relationships within the genus Neotoma using

nuclear DNA sequence data from intron 2 of the alcohol dehydrogenase gene 1. In

addition, to determine the phylogenetic utility of this gene, the results obtained from tiie

study are compared to the mitochondrial DNA sequence data from the cytochrome-/)

gene (Edwards and Bradley 2002b). This chapter is written in the form of a manuscript

that will be submitted for publication in the Journal of Mammalogy (authors are L. K.

Longhofer and R. D. Bradley). Consequently, the format of the Journal of Mammalogy

is followed throughout the manuscript for consistency.

Chapter III serves as a review of the results from Chapter II and provides a

summary of major conclusions drawn from the chapter.

Literature Cited

Avise, J. C. 1994. Molecular markers, natural history, and evolution. Chapman and Hall, New York,-511 pp.

Ballard, J. W., B. Chemoff, J. C. Avis. 2002. Divergence of mitochondrial DNA is not cortoborated by nuclear DNA, morphalogy, or behavior in Drosophila Simulans. Evolution 56:527-545.

Bimey, E.G. 1973. Systematicsof 3 species of woodrats. Miscellaneous Publications, Museum of Natural History, University of Kansas 58:1 -173.

Burt, W. H., F. S. Barkalow, Jr. 1942. A comparative study of the baculum of woodrats (subfamily Neotominae). Journal of Mammalogy 23:287-297.

Edwards, C. W. and R. D. Bradley. 2001. Molecular phylogenetics of the Neotoma floridana species group. Journal af Mammalogy 82:791-798.

Edwards, C. W. and R. D. Bradley. 2002a. Molecular systematics and historical phylobiogeography of the Neotoma mexicana species group. Journal of Mammalogy 83:20-30.

Edwards, C. W. and R. D. Bradley. 2002b. Molecular systematics of the genus Neotoma. Molecular Phylogenetics and Evolution 25:489-500.

Edwards, C. W., C. F. Fulhorst, and R. D. Bradley. 2001. Molecular phylogenetics of the Neotoma albigula species group: further evidence of a paraphyletic assemblage. Journal of Mammalogy 82:267-279.

Finley, R. B., Jr. 1953. A new subspecies of wood rat (Neotoma mexicana) from Colorado. University of Kansas Publications, Museum of Natiiral History 5:527-534.

Fulhorst, C. F., R. N. Chan-el, S. C. Weaver, T. G. Ksiazek, R. D. Bradley, M. L. Milazzo, R. B. TesJi,.and M. D. Bowen. 2001. Geographic distribution and genetic diversity of Whitewater Arroyo Virus in the southwestern United States. Emerging Infectious Dise^ases 7:403-407.

Goldman, E. A. 1910. Revision ofthe wood rats of the genus A eoto/wa. North American Fauna 31: 1-124.

Hall, E. R. 1981. The Mammals of North America, 2nd ed. Wiley, New York.

Hall, E. R., and K. R. Kelson. 1959. The mammals of North America. Ronald Press

Co., New York, 2:547-1073+79.

Hayes, J. P., and R. G. Harrison. 1992. Variation in the mitochondrial DNA and the biogeographic history of woodrats (Neotoma) in the eastern United States. Systematic Biology 41:331-344.

Hayes, J. P. and M. E. Richmond. 1993. Clinal variation and morphology of woodrats (Neotoma) of the eastern United States. Journal of Mammalogy 74:204-216.

Hooper, E. T. I960. The glans penis in Neotoma (Rodentia) and allied genera. Occasional Papers of the Museum of Zoology, University of Michigan, 618:l-2{).

Koop, B. F., R. J. Baker, and J. T. Mascarello. 1985. Cladistic analysis of chromosomal evolution within the genus Neotoma. Occasional Paper, The Museum, Texas Tech University 96:1-9.

Mascarello, J. T., and T. C. Hsu. 1976. Chromosome evolution in woodrats, genus Neotoma (RodeRtiz.: Cricetiae). Evolution, 30:152-169.

Patton, J. L., and S. T. Alvarez-Castaneda. in press. Phylogeography of the Desert woodrat, Neotoma lepida, with comments on systematics and biogeographic history. Volume in Honor of Bernardo Villa.

Planz, J. v., E. G. Zimmerman, T. A. Spradling, andD. R. Akins. 1996. Molecular phylogeny of the Neotoma floridana species group. Journal of Mammalogy 77:519-535.

Shipley, M. M., F. B. Stangl, Jr., R. L. Gate, and C. S. Hood. 1990. Immunoelec.trophoretic relationships among 4 species of woodrats (Cricetidae: Neotoma). The Southwestern Naturalists 35:173-176.

Zimmerman, E.G., and M. E. Nejtek. 1977. Genetics and speciation of 3 semispecies of Neotoma. Journal of Mammalogy 58:391-402.

Outgroup Taxa (4) , . ^ _ ^ _ _ Xenomys nelsoni (1)

' ^ Hodomys alleni (2)

N. goldmani Q)

' \

N. albigula (12)

^

>i N. floridana (11)

N. magister (3)

OTQ

J K leucodon (7)

N. micropus (10)

N. lepida complex (7)

fuscipes (4)

N. stephensi (2) A', isthmica (3)

* 3" N. picta (1)

N. mexicana (11)

^ ^ N. cinerea (2)

Figure 1.1 Maximum likelihood tree (GTR+I+G model) based on Edwards and Bradley (2002b) depicting phylogenetic relationships of 76 samples of Neotoma. Roman numerals depict major clades whereas letters A and B depict minor clades. Sample sizes are shown in parentheses.

CHAPTER II

MOLECULAR SYSTEMATICS OF THE GENUS

NEOTOMA BASED ON NUCLEAR DNA

SEQUENCES FROM INTRON 2 OF THE

ALCOHOL DEHYDROGENASE GENE

Abstract

In order to examine phylogenetic relationships in the genus Neotoma, DNA

sequences from infron 2 of the nuclear alcohol dehydrogenase gene 1 (Adh-\-]2) were

obtained from 33 individuals that represented 13 species of the genus Neotoma, 2

ingroups, and 3 outgroups. These data were analyzed using parsimony, likelihood, and

Bayesian methods. Three major clades consistently were recovered in all analyses. The

1 ' major clade (I) contained samples of X nelsoni and H. alleni as sister taxa. The 2"*

major clade (E) included 23 individuals representing 10 species (A . picta, N. isthmica, N

floridana, N. magister, N. micropus, N stephensi, N. leucodon, N. albigula, N mexicana,

and N. goldmani). The 3'* major clade (III) contained 5 individuals representing 3

species (N fuscipes, N cinerea, and N. lepida). Species and relationships within clades I

and III always remained the same. However, the minor clades within the 2^^ major clade

(II) differed slightly in that the parsimony analysis had minor clades A-F and the

likelihood and Bayesian analyses only had minor clades A-D with slightiy different

relationships. Genetic divergences between species of Neotoma averaged 2.16%. The

average genetic distance of Hodomys to subgenus Neotoma was 5% and subgenera

Teonoma to Neotoma was 4%. These results support Hodomys as a separate genus and

the placement of Teonoma as a subgenus within Neotoma. Hodomys was sister to

Xenomys. and A/: cinerea (Teonoma) and A/: fuscipes were sister taxa. In addition, Adh-\-

12 sequences then were concatenated with mitochondrial cytochrome-/) sequences

generated from the same individuals and were analyzed in a total evidence approach.

The combined data resulted in a phylogeny whose topology was very similar to a tree

based only on the cytochrome-/) gene. Although internal relationships were slightly

different, Bayesian support values were high for most relationships, and no support

values were apparent for the cytochrome-/) only tree.

Introduction

Woodrats (genus Neotoma) are medium-sized rodents in North America and are

distributed across most of the continental United States, portions of Southern Canada,

several Baja Islands, throughout much of Mexico, and into northern Central America

(Hall 1981). Determinations of relationships among taxa within this genus are difficult

due to the morphological uniformity among members, their problematic systematic

history, and the absence of a study that includes a comprehensive sample of taxa within

Neotoma. Therefore, Neotoma remains a taxonomically perplexing and systematically

complex group of taxa.

Since Say and Ord (1825) 1 ' erected the genus Neotoma, there has been much

debate on how many subgenera and species groups are included within the genus, and

which taxa represent distinct species. Morphological data (cranial, skeletal, dental, and

10

pelage) were l" used to separate various members into a dichotomy, wherein round-tailed

taxa were assigned to the genus Neotoma and bushy-tailed woodrats were placed into

Teonoma (Gray 1843). Although, Mertiam (1894) made the 1'' preliminary revision

(synopsis of the known members of Neotoma), Goldman (1910) published the most

extensive revision of tiie genus, in which he divided the genus into 3 subgenera

(Neotoma, Homodontomys, and Teonoma) and placed N. alleni as a separate genus

(Hodomys). Hall (1981) divided Neotoma into 4 distinct subgenera (Neotoma, Teonopus,

Hodomys, and Teonoma); consequently relegating Hodomys to a subgenus.

Traditionally, studies of the subgenus Neotoma have used phenetic approaches to

assess similarities and to determine associations among taxa. Goldman (1910) arranged

this subgenus into 6 species groups (albigula, desertorum, floridana, intermedia,

mexicana, andpennsylvanica). Burt and Barkalow (1942) later identified 6 species

groups (albigula, floridana, fuscipes, lepida, mexicana, and micropus), although the

species groups differed from Goldman's (1910) assessment.

Another confroversy has surtounded the placement of Neotoma alleni. Goldman

(1910) placed N. alleni into its own genus, Hodomys. Later, Burt and Barkalow (1942),

based on the examination of bacular morphology, suggested H. alleni be reinstated as a

subgenus of the genus Neotoma; conversely. Hooper (1960) considered A/: alleni to be

most closely related to Xenomys nelsoni using glans penis morphological data. However,

Hall (1981) relegated Hodomys to subgeneric status. Subsequently, Carieton (1980)

agreed v^th Goldman's (1910) assessment and recommended that Hodomys be re-

elevated to its former generic status and suggested that it is most closely related XoX.

11

nelsoni. Musser and Carieton (1993) also recognized Hodomys as a separate genus, sister

to Xenomys.

Finally, much debate has occurred concerning the classification of individual taxa

within the genus. Due to the confroversial recognition of species-groups, there have been

considerable conflicting conclusions on how to align, organize, and classify taxa within

the genus Neotoma (Goldman 1910; Hall and Kelson 1959; Hall 1981). Therefore,

various systematic studies have attempted to establish evolutionary relationships among

the members of this group (Burt and Barkalow 1942; Hooper 1960; Bimey 1973;

Mascarello and Hsu 1976; Zimmerman and Nejtek 1977; Koop et al. 1985; Shipley et al.

1990; Hayes and Harrison 1992; Hayes and Richmond 1993; Planz et al. 1996; Edwards

et al. 2001; Edwards and Bradley 2001,2002a, b; Patton and Alvarez-Castaneda in

press). Several factors, both genetic and ecological, have made it difficult to address

phylogenetic relationships within Neotoma.

Recentiy, Edwards and Bradley (2002b) used DNA sequences from the

mitochondrial cytochrome-/) gene to produced 1 of the l" comprehensive phylogenies for

the genus focusing on 13 species of Neotoma, whereas the majority of previous

systematic studies (Bimey 1976; Zimmerman and Nejtek 1977; Shipley et al. 1990;

Hayes and Harrison 1992; Hayes and Richmond 1993; and Planz et al. 1996) included

only 3 taxa (A': albigula, N floridana, and A/: micropus). Consequently, Edwards and

Bradley (2002b) agreed with Musser and Carieton (1993) in supporting the recognition of

Hodomys as a separate genus and suggested that Neotoma consisted of 3 subgenera

(Neotoma, Teonopus, and Teonoma).

12

Edwards and Bradley (2001, 2002a, b) and Edwards et al. (2001) recognized 24

species of Neotoma, which were placed into 4 species groups: 1) the N. floridana group

including N albigula, N. floridana, N. goldmani, and N. magister; 2) the N. lepida group

including N. fuscipes, N. lepida complex, and A. stephensi; 3) the N. mexicana group

including N angustapalata, N. chrysomelas, N. isthmica, N. mexicana, and N. picta; and

4) the N. micropus group including N. micropus and N. leucodon. However, Edwards

and Bradley (2002b) also suggested further study to determine if A'; nelsoni, N. palatina,

and N. varia belong in the N. micropus species group.

Systematic problems involving sibling and cryptic species (distinguishable only

through genetic analysis) within several groups of woodrats have been addressed over the

past 2 decades. Edwards et al. (2001) elevated N. albigula leucodon to species status,

and Edwards and Bradley (2002a) elevated 2 subspecies within N. mexicana to species

level (A . isthmica and A'; picta). In addition Edwards and Bradley (2001) agreed with

Merriam (1894), Goldman (1910), and Hayes and Harrison (1992) in the recognition of

N. magister as a distinct species instead of a subspecies of N. floridana. Thus, the recent

recognition of these cryptic species (Hayes and Harrison 1992; Planz et al. 1996;

Edwards and Bradley 2001,2002a; Edwards et al. 2001) led to an increase in the

proposed species count to 24 recognized species and questioned previous taxonomy and

systematic relationships among the members of Neotoma.

Although Edwards and Bradley's (2002b) study provides an effective hypothesis

concerning phylogenetic relationships of the complex, there are potential weaknesses

with using the mitochondrial genome for DNA sequence comparisons. Ballard et al.

13

(2002) has questioned whether divergence of mtDNA should be used as the sole basis for

species recognition and cautions about using mtDNA divergence conclusions for

organismal diflferentiation witiiout additional support from independent data sets. Avise

(1994) also states that mitochondrial variation may not correlate well with other genomic

aspects because of selection, introgression, poor resolution of the data, or lineage sorting.

Thus, phylogenies obtained from nuclear data should be used as an independent data set

to test the accuracy of those generated by mitochondrial data due to uniparental

inheritance, gene linkage, retention of ancestral polymorphisms, lineage sorting, and the

gene free-species tree confroversy (Avise 1994). In addition, mitochondrial DNA can

contain misleading information about phylogenetic relationships, especially if

hybridization is a concern (Avise 1994; Bradley and Baker 2001). Given that Bimey

(1973) reported natural hybrids between N. micropus and A. floridana, data from nuclear

sequences are needed to test phylogenetic relationships of the genus Neotoma. Even

though mitochondrial genes have provided substantial resolution concerning biological

species in nature (Bradley and Baker 2001), data from nuclear genes are needed to

represent a more detailed account of genomic diversity due to biparental inheritance of

DNA. Nuclear genes also have been shown to be effective in resolving phylogenetic

relationships within other groups of rodents (Robinson et al. 1997; Flynn and Nedbal

1998; Walton et al. 2000; Adkins et al. 2001; DeBry and Sagel 2001; Huschon et al.

2002).

The primary objective of this study is to test current hypotheses of phylogenetic

relationships among members of Neotoma using DNA nucleotide sequence variation

14

from infron 2 of tiie nuclear alcohol dehydrogenase gene 1 (Adh-\-U; Amman et al.

submitted). The Adh-l stmctural gene consists of 9 exons, 8 introns, and is roughly 13

kb in length (Crab 1989). The choice of tiie Adh-l-I2 region was based on evidence from

protein electrophoreses studies that the alcohol dehydrogenases are highly polymorphic

in rodents and otiier mammals. Bradley et al. (1993, 1998) demonstrated that nucleotide

sequences from the coding region of the Adh-\ gene were species specific. In addition,

Amman et al. (submitted) presented preliminary data demonsfrating that the Adh-l-U

was informative in a phylogenetic analysis of several genera of rodents. In light of this

evidence, it is assumed that infron 2 will have greater variation than of the coding regions

due to fewer evolutionary consfraints acting on introns (Li and Graur, 1991). A 2"* goal

is that the nuclear DNA sequences obtained from the Adh-l-U in this study will be

concatenated in a combined analysis to mitochondrial DNA sequences from cytochrome

b (Edwards and Bradley, 2002b). To address these questions, nuclear DNA sequences

from 33 individuals, chosen from the Edwards and Bradley (2002b) study, will be used to

evaluate the cytochrome-/) phylogeny of Neotoma. Specifically, the following questions

will be examined: 1) are phylogenetic relationships among Neotoma using DNA

sequences from the nuclear alcohol dehydrogenase gene 1 congment to the Edwards and

Bradley (2002b) phylogeny based on DNA sequences from the mitochondrial

cytochrome-/) gene, 2) is the genus Neotoma a monophyletic assemblage with the

exclusion of Hodomys, 3) is Hodomys sister to Xenomys, and 4) does Adh-l-U provide

resolution for the phylogenetic assessment of Neotoma.

15

Materials and Methods

Samples.—The sampling approach for this study utilizes a subset of taxa

examined in Edwards and Bradley (2002b). Thirty-three animals were selected from that

study to represent members of primary clades, geographic variation for wide ranging

taxa, and at least 1 individual for each of the 13 species of Neotoma including: N.

albigula (n = 4), N. cinerea (n = 2), N. lepida (n = 2), N. floridana (n = 4), N. fuscipes (n

= I), N. goldmani (n = 1), N isthmica (n = 2), N. leucodon (n = 2), N. magister (n = 1), N.

mexicana (n = 3), N. micropus (n = 4), A'', picta (n = 1), and N. stephensi (n = 1). H.

alleni and X. nelsoni were included as ingroups. Outgroup taxa included 1 representative

each of Tylomys nudicaudus, Ototylomys phyllotis, and Peromyscus attwateri.

Data collection.—Genomic DNA was isolated from frozen liver tissue using a

Dneasy tissue kit (Qiagen, Valencia, California). Sequences from exon 2 and exon 3

were used from a variety of taxa (Homo, Rattus, and Mus) to constmct PCR primers.

These primers that were anchored in exon 2 and 3 (ExIIF and ExIIIR or ExIIF and 2340-

II~Amman et al. submitted) were used to amplify the entire region ofAdh-\-l2. This

complete Adh-l-U (583 bp) region was amplified using the polymerase chain reaction

(PCR) (Saiki et al. 1988) with the following tiiermal profile: 25-30 cycles of 95°C (30

sec) denaturing, 53°C^ 48°C^ 53°C^ 73 °C ramped at 0.6°C/sec annealing, 73^0 (1

min 30 sec) extension, followed by 1 cycle of 73°C (4 min). Amplifications were

performed in 35^1 reactions using 1.25 U FailSafe PCR Enzyme Mix (Epicentre,

Valencia, California); however, AmpliTaq Gold (5U/lnl) was used in some reactions.

16

PCR products were purified witii silica gel using the QIAquick PCR Purification Kit

(Qiagen, Valencia, Califomia).

PCR amplicons were sequenced using ABI Prism Big Dye Terminator v3.0 ready

reaction mix and an Avant 3100 (PE Applied Biosystems, Foster City, Califomia) with

cycle sequencing conditions of 95 °C (30 sec) denaturing, 50 °C (20 sec) annealing, and

60 °C (3 min) extension. Five primers (ExIIF, ExIIIR, 2340-1,2340-11, 350F, and 350-

Amman et al. submitted) were used in 1-2 ^M concenfrations in the sequencing protocol.

Following 25-30 cycles, reactions were then precipitated in isopropanol.

Sequencher 3.0 software (Gene Codes, Ann Arbor, Michigan) was used to align

contiguous fragments and proof nucleotide sequences. The chromatograms were then

examined to verify all base changes and to determine if heterozygous sites existed.

Clustal X (Thompson et al. 1997) was used for multiple sequence alignments of

nucleotides that were then adjusted and proofed by eye in MacClade 4.0 (Maddison and

Maddison 2000). Sequence alignments produced gaps that represented insertion and

deletion events. These gaps were inserted because of primary homology assessment, and

the fact they often contain historical information suitable for phylogenetic analysis

(Giribet and Wheeler 1999). All DNA sequences generated were deposited in GenBank,

and accession numbers are listed in Table 2.2.

Because the Adh-l-U primers used for amplification were placed in the conserved

coding regions of the gene (exon 2 and exon 3), small fragments of the exon 2 and exon 3

regions were amplified resulting in a 732 bp region of Adh-l including: approximately

17

107 bp of exon 2, 583 bp of infron 2, and 42 bp of exon 3. However, only the 583 bp of

infron 2 sequences were considered in the phylogenetic analyses.

Data analysis—Nucleotide sequence data were analyzed using parsimony,

likeUhood, and Bayesian models with the software package PAUP*4.0blO (Swofford

2002). All nucleotide positions were freated as imordered, discrete characters with 5

possible states: A, C, G, T, and gaps (-). Heterozygous sites were designated using the

accepted lUB polymorphic code. To avoid the subjectivity inherent with differential

weighting, characters initially were weighted equally in the parsimony analysis (Allard

and Carpenter 1996).

Maximum parsimony was performed using equal weighting, and optimal frees

were estimated using the heuristic search method with tree bisection-reconnection branch

swapping and stepwise addition sequence options. Uninformative characters were

excluded from all parsimony analyses. Robustness and nodal support of topologies were

assessed using heuristic bootsfrapping (Felsenstein 1985) with 1,000 iterations and with

Bremer support indices (Bremer 1994), calculated with the Autodecay program (Eriksson

1997).

The HKY+G model of evolution was identified as the simplest and most

appropriate model by the MODELTEST v3.06 software (Posada and Crandall 1998).

This model significantiy fits tiie data better than altemative models. The HKY+G model

subsequently was used in a maximum likelihood analysis (heuristic search option in

PAUP). Parameters for this model included the proportion of invariable sites (0.0000),

18

gamma disfribution shape (y = 0.9822) and base compositions estimated from the data (A

= 0.3220, C = 0.1884, G = 0.1708, and T = 0.3189).

Because tiie HKY+G model was selected for the likelihood analysis, MrBayes

(Huelsenbeck and Ronquist 2001) was used to constmct a separate topology and posterior

clade probabilities. In this analysis, the following parameters were employed: GTR+I+G

model, ssgamma, 4 Markov chains, 2x10^ generations, and sampled every 100*

generation. Following a visual inspection of the likelihood scores, the 1 ' 100 trees were

"burned" to allow free constmction based on stabilized scores.

Pairwise genetic distance analyses were generated using the nucleotide data under

the Tamura-Nei (Tamura and Nei 1993) model of evolution. This model was chosen so

that resulting values could be compared to those of other studies involving woodrats

particularly the cytochrome-/) dataset (Edwards and Bradley 2001; 2002a, b; Edwards et

al. 2001). To obtain values for comparisons within and among species, the mean of the

pairwise comparisons was calculated.

Finally, the resulting nuclear J/z-1-12 sequences were concatenated with the

mitochondrial cytochrome-/) sequences from Edwards and Bradley (2002b) and analyzed

using both prior agreement or conditional data combination (Bull et al. 1993; de Queiroz

1993; Huelsenbeck et al. 1994; Drovetski 2002) and total evidence (Kluge 1989)

approaches. Sequences from each of the data sets were fruncated so that only taxa shared

between studies were analyzed. This resulted in a combined analysis of 32 taxa; 1

sample ofN lepida was eliminated due to the absence of cytochrome-/) data. Sequences

for the cytochrome-/) gene were generated from the same individuals as the Adh-l

19

sequences. MrBayes (Huelsenbeck and Ronquist 2001) was used for Baysian analysis

using the GTR+I+G model of evolution with the parameters as described above for the

Adh MrBayes analysis.

Results

Nucleotide sequences were obtained from the nuclear Adh-l-U locus (583 bp) for

33 individuals representing 6 genera and 18 species. The length of the intron ranged

from 520 bp (N. floridana) to 572 bp (P. attwateri) among taxa due to insertion and

deletion events and resulted in 583 aligned sites. Nucleotide frequencies were: A =

32.2%, C = 18.9%, G = 17.1%, and T = 31.9%. Comparison of nucleotide substitutions

revealed that fransitions were 2.1 times more common than fransversions.

Heterozygosity values ranged from 0 (26 taxa) to 2 (4 taxa) polymorphic sites. Several

insertion and deletion events were present, varied in length, and appeared to be conserved

for different recognized species of woodrats. For example, a 4 bp deletion was present in

all members except Peromyscus; members of the genus Neotoma shared a 4 bp and 1 bp

deletions, respectively; Ototylomys a 11 bp deletion, N. cinerea a 7 bp deletion; N.

floridana attwateri a 48 bp deletion, A. goldmani a 10 bp deletion; Xenomys a 7 bp

deletion; and Ototylomys and Tylomys shared a 9 bp deletion.

Under a parsimony framework, 497 uninformative characters were excluded

resulting in 86 informative characters. Twelve equally most-parsimonious frees were

generated (Length = 125 steps, CI = 0.7600, RI = 0.8701, RC = 0.6613, and HI =

0.2400). A strict consensus free (Figure 2.1) produced 3 major clades. The 1 ' major

20

clade (I) contained samples of Z nelsoni and H. alleni as sister taxa. The 2""* major clade

(II) included 23 individuals representing 10 species (N picta, N. isthmica, N. floridana,

N. magister, N micropus, N. stephensi, N. leucodon, N. albigula, N mexicana, andN.

goldmani). Witiiin this clade, 6 minor clades (A~F), primarily reflecting clusters of

closely related species, were formed which were unresolved relative to each other. Clade

A contained samples of AA picta and N. isthmica. Clade B contained 9 samples

representing N. floridana, N. magister, and N. micropus. This clade (B) placed A':

floridana and N. magister in a group that was sister to N. micropus. One species (N.

stephensi) formed clade C. Clade D contained 6 individuals representing 2 species (N.

leucodon and N. albigula). Clade E contained 3 individuals of AA. mexicana, and N.

goldmani represented the basal clade F. The 3"* major clade (HI) contained 5 individuals

representing 3 species (N. fuscipes, N cinerea, and N lepida) with N. fuscipes and N.

cinerea as sister followed by the addition ofN. lepida. The major clades n and III were

sister, and the V^ major clade (I) was basal. Bootsfrap and Bremer support values for the

1 * (I) and 3" (Id) major clades were high, as were values within these clades. The 2"*

clade (n) had relatively low support (bootstrap = 58%) resulting in the minor clades

being unresolved. However, support within some of the minor clades (B, D, and E) was

moderate to relatively high.

The maximum likelihood analysis based on the HKY+G model of evolution

produced a score of-In L 2,046.42. The likelihood free (not shown) generated was

similar to the topology generated in parsimony and the Bayesian analysis (discussed

below) in that both contained identical major clades and similar minor clades. Clades II

21

and III were sister to each other with clade I joining in a stepwise fashion. However,

clade II only contained 4 minor clades (A-D), and clade D included 10 samples

representing4 species (A . leucodon, N. albigula, N mexicana, andN. goldmani). The A .

albigula-N leucodon clade formed a sister relationship with tiie N. mexicana clade, and

N. goldmani formed tiie basal most member of clade D.

The Bayesian analysis resulted in a free (Figure 2.2) similar to parsimony and

likelihood analyses but with all relationships resolved. Parametiic posterior probabilities

were generated and are shown on tiie free. Three major clades, representing the same

taxa as in the frees generated by parsimony and likelihood, were produced. Clades n and

III were sister followed by the addition of clade I in a stepwise fashion. The minor

differences involved the minor clades within the 2°'* major clade (II) and the placement of

N. goldmani and A': stephensi. Within the 2°' major clade (H), only 4 minor clades (A—

D) were apparent. Clades A and B contained the same individuals as in parsimony and

likelihood, but the clades were joined in a stepwise fashion. Clade D was sister to clade

B and contained 10 samples representing 4 species (N. leucodon, N. albigula, N.

goldmani, and A . mexicana). Another difference was that A . goldmani was basal to the

N. albigula and A . leucodon clade with the N. mexicana clade joining in a stepwise

fashion. Also, Clade C contained A'! stephensi as the basal most member of the 2^^ major

clade (II). All other relationships were identical to previous topologies. Support

(posterior probabilities > 95) was present for major clades I and III as well the minor

clades within the 2°* major clade (II). Clade II had no intemal support for the minor

clades.

22

Genetic distances (Table 2.1) calculated using the Tamura-Nei (Tamura and Nei,

1993) model of evolution for members of the subgenus Neotoma ranged from 0.0000 for

the 2 species ofN. lepida to 0.0504 between N. fuscipes and N. albigula durangae.

Average genetic distance values for comparison of the subgenera within Neotoma to

Hodomys ranged from 0.0403 for Neotoma and Teonoma to 0.0657 for Hodomys and

Teonoma.

The combined data set (sequences for alcohol dehydrogenase and cytochrome b)

that was analyzed using the Bayesian analysis (GTR+I+G) produced a topology (Figure

2.3) similar to that produced in the cytochrome-/) only tree; however, minor differences

in the combined analysis did reflect some Adh-l-U only relationships. For example, N.

cinerea was sister to N fuscipes with a sfrong posterior probability of 98. Also, the

mexicana clade was placed sister to the A': picta and N isthmica clade. All nodes were

sfrongly supported with posterior probabilities > 95 except for 2 subspecies ofN

micropus and the relationship between the A . albigula and N. floridana clades.

Discussion

DNA sequences obtained from the Adh-l-U gene appear to be phylogenetically

informative at various levels in the genus Neotoma. Although insertion and deletion

events were present, they were not problematic for sequence alignment and were often

phylogenetically informative. In addition, heterozygosity was low (11 of 19,239 aligned

sites) and did not appear to affect phylogenetic analyses. The Adh-l intron shows a high

A-T content (64.1%), characteristic of noncoding regions (Li and Graur, 1991). The

23

fransition to fransversion ratio was approximately 2.1:1 and was somewhat higher

compared to other vertebrate infrons. Prychitko and Moore (1997) reported a ratio of

1.24:1 within the Fbg-ll gene in woodpeckers, and Carroll (2002) calculated a value of

1.46:1 in cotton rats. However, Reeder (2003) showed a higher ratio of 3:1 in the Fbg-ll

gene within tribal levels of rodents; the higher value may be explained by the higher

taxonomic ranks examined.

The 13 species of Neotoma formed similar topologies in all analyses. However,

the taxa comprising these clades and their respective alignments differed, depending on

the analysis employed. In every analysis, a clade containing samples of Jt nelsoni and H.

alleni was formed (clade I). The 2 taxa always formed sister relationships and

maintained an intemal position to the remaining clades.

A clade (III) containing samples ofN. picta, N. isthmica, N. floridana, N

magister, N. micropus, N. leucodon, N albigula, N. goldmani, N. mexicana, and N.

stephensi was obtained in each analysis of the Adh sequences although the members of

minor clades varied, 6 minor clades in parsimony and 4 each in the likelihood and

Bayesian methods. Witiiin this clade, the associations of the minor clades (A-F) relative

to each other were unresolved in the parsimony analysis. In all 3 analyses, clade A

contained samples ofN. picta andN. isthmica. Clade B contained samples of A':

floridana, N magister, andN. micropus. Samples ofN. floridana andN. magister

formed a sister relationship, followed by the sister placement of tiie N micropus clade.

Clade C contained only 1 member (N. stephensi). N. stephensi differed in its placement

between analyses. The parsimony and likelihood analyses placed A': stephensi as an

24

intermediate minor clade; however, the Bayesian analysis placed N stephensi as the basal

most member of the 2"* major clade (II). Clades D-F differed between analyses with the

parsimony analysis placing N. leucodon and A . albigula in clade D, N mexicana in clade

E, and A'; goldmani in clade F. However, the likelihood and Bayesian analyses included

these taxa within a single clade (D). Although likelihood and Bayesian analyses placed

the N. albigula-N. leucodon clade sister to the N. mexicana clade, they were not well

supported by posterior probabilify values. A . goldmani was placed as the basal most

member of clade D in the likelihood analysis, but was placed basal to the AA. albigula

clade in the Bayesian analysis.

All 3 analyses produced a 3"* clade (HI) containing N. fuscipes, N. cinerea, and N.

lepida. Clade III and relationships within the clade were sfrongly supported with high

values both in parsimony and the Bayesian analyses.

Average genetic distances (Table 2.2) obtained using the Tamura-Nei (Tamura

and Nei, 1993) model of evolution indicated low levels of genetic divergence but

sufficient to show variation among the taxa. Genetic divergences between species within

the subgenus Neotoma averaged 2.16%. The average genetic distance of Hodomys to

subgenus Neotoma was 5% and subgenera Teonoma to Neotoma was 4%. These results

support the recognition of Hodomys as a separate genus and the inclusion of Teonoma as

a subgenus within Neotoma although genetic distances approached values that were seen

between recognized genera.

The combined data set (alcohol dehydrogenase and cytochrome b) supported most

of the topology and phylogenetic relationships obtained from the cytochrome-/) data.

25

Altiiough tiie combined data set was more heavily weighted by the larger number of

characters supplied by the cytochrome-/) data set, it appears that the alcohol

dehydrogenase gene I was informative and produced similar results as the cytochrome-/)

data, in some cases. Also, the Adh-l-U produced additional support in relationships

obtained from tiie cytochrome-/) data. The Adh-l-U data as well as the combined data set

indicated that the genus Neotoma is a monophyletic assemblage. Hodomys also is well

supported in all analyses (Figures 2.1,2.2, and 2.3) as a separate genus sister to Xenomys.

Traditionally, the subgenus Neotoma has been artanged into species groups to

reflect similarities and relationships among taxa. Goldman (1910) described 6 species

groups (albigula, desertorm, floridana, intermedia, mexicana, and pennsylvanica). Burt

and Barkalow (1942) recognized a different arrangement of species groups (albigula,

floridana, fuscipes, lepida, mexicana, and micropus) and also made remarks on the

constmction of subgenera. Recently, Edwards and Bradley (2002b) provided a revision

of the genus and species groups. Specifically, they acknowledged 4 species groups

{floridana, micropus, lepida, and mexicana). Several other studies (Schwartz and Odum,

1957; Hooper 1960; Bimey, 1973; Mascarello and Hsu 1976; Zimmerman and Nejtek

1977; Carieton 1980; Koop et al. 1985; Shipley et al. 1990; Hayes and Harrison 1992;

Hayes and Richmond 1993; Planz et al. 1996; Edwards et al. 2001; Edwards and Bradley

2001,2002a, b; Patton and Alvarez-Castaneda in press) have provided further data on the

systematics of members of the genus and species groups.

Herein, parsimony, maximum likelihood, and Bayesian analyses and pairwise

distance values based on a nuclear infron sequence provided a means to re-evaluate the

26

members of tiie species groups. Data from this study were used for comparison to tiie

mitochondrial DNA from the cytochrome-/) gene (Edwards and Bradley 2002b) as well

as a combined analysis of tiie 2 data sets to examine the relationships of the 13 species of

Neotoma. The Adh-l-U analyses grouped the taxa into groups that did not directly

support any previous research, but may reflect a combination of the Goldman (1910),

Burt and Barkalow (1942), and Edwards and Bradley (2002b) versions of species groups.

The systematic relationships of these taxa witiiin Neotoma are discussed below.

Goldman's (1910) assessment of the floridana species group (N floridana and N

micropus) was supported in that Adh-l-U placed N floridana and A': micropus as sister

species. Hooper (1960), using morphology of the glans penes, considered A': albigula, N.

floridana, and N micropus to be sibling species. Bimey (1973,1976) agreed and

suggested that woodrats assigned to these 3 groups should be placed in Xhe floridana

species group. However, Edwards and Bradley (2002b) recognized N. albigula and

subspecies N. albigula leucodon as distinct species, and placed N. micropus and N.

leucodon into the micropus group. The Adh-l-U data indicates that N. albigula, N.

floridana, and A'', micropus and N mexicana, N. goldmani, and N. stephensi are members

of a single clade (II). This is in agreement with Zimmerman and Nejtek (1977) in

suggesting that N. micropus forms a sister relationship with N. floridana, whereas N.

albigula forms a sister-taxa relationship with the N. floridana -N. micropus clade. In

addition, the Adh-l-U results agree with Merriam (1894), Goldman (1910), Hayes and

Harrison (1992), and Edwards and Bradley (2001) in the recognition ofN. magister as a

distinct species instead of a subspecies of N. floridana. N. magister is the basal most

27

member of the N floridana clade. Although Edwards and Bradley (2002b) and Bimey

(1976) suggested that the floridana species group include A . albigula and N goldmani,

the Adh-l-U Bayesian analysis supports Hall (1955) and Rainey and Baker (1955) in the

placement ofN. goldmani to be a member of the albigula group and sister to the N.

mexicana clade. Adh-l-U also does not support the recognition of A . leucodon as a

distinct species from N. albigula. Although Edwards et al. (2001) and Planz et al. (1996)

reported N. leucodon as a distinct species from N albigula, the alcohol dehydrogenase

data placed samples ofN. leucodon within the N albigula clade with an average genetic

distance of 0.0054.

Burt and Barkalow (1942) l" referted to N. lepida and N. goldmani in the lepida

group. Recently, Edwards and Bradley (2002b) assigned A . lepida, N. fuscipes, and N

stephensi to the lepida group; however, the Adh-l-U data showed that N. lepida, N

fuscipes, and A . cinerea composed clade HI in all analysis and N. stephensi alone

represented minor clade C within clade 11. N fuscipes and N. cinerea were sister taxa

and were highly supported with bootsfrap value of 100 and posterior probability of 100.

A': lepida joined in a stepwise fashion that was also highly supported with bootstrap value

of 97 and posterior probability of 100. N. lepida showed an average pairwise distance of

2.9% to other members of the subgenus Neotoma. Previously, Edwards and Bradley

(2002b) recommended that subgenus Teonoma (N cinerea) may be a separate genus due

to its placement as the basal clade of Neotoma and its high genetic distance value of

17.2% to the subgenus Neotoma. Although the genetic distance (4%) approached values

that were seen between recognized genera (5%) in the Adh-l-U data, it is probably best

28

to retain Teonoma as a subgenus within Neotoma, until a member of Teonopus (tiie 3"*

subgenus in Neotoma) can be analyzed. Goldman (1910) placed A': fuscipes in a newly

erected subgenus Homodontomys and considered it to be the lone member of the fuscipes

group. The Adh-l-U data showed that the subgenus Neotoma and N. fuscipes were

highly divergent with an average genetic distance value of 4.4%. This level of genetic

divergence was higher than the previously mentioned subgenus Teonoma and may

warrant realignment of this species.

N. isthmica and N. picta were placed in a clade separate from N. mexicana

(average genetic distances = 0.0194) but may not be distinct from each other (average

genetic distances = 0.0084). The 3 Adh-l-U analyses placed the N. picta and N isthmica

clade paraphyletic to the N mexicana clade. These data support Edwards and Bradley

(2002a, b) in the recognition ofN. picta and AA. isthmica as distinct species; however,

Edwards and Bradley (2002b) placed N. isthmica and N picta sister to N. mexicana in the

mexicana group. Burt and Barkalow (1942) and Hooper (1960) using morphological

characteristics of bacula and gland penes suggested that A/: mexicana was aligned closely

toN. albigula, N. floridana, andN. micropus. Adh-l-U also supports the close alignment

of these species by placing them in the same major clade (H) and also included AT.

goldmani and N. leucodon. N. mexicana was shown to be a sister to A'', albigula, and the

basal member of clade (II).

The genetic distance values for the Adh-l-U differed dramatically from the

cytochrome-/) values in that tiie cytochrome-/) genetic distances were much higher (by

about 4 times) on average. This was expected as nuclear genes are predicted to evolve at

29

a slower rate tiian mitochondrial genes (need reference...). However, the 2 data sets did

show a similar pattern in dividing genera, subgenera, and species. The Adh-l-U average

genetic distance (Table 2.1) for comparisons within subgenus Neotoma was 2.16%.

However due to tiie high divergent values of subgenus Neotoma to N. lepida, N. fuscipes,

and N. cinerea clade (3.7%), the average genetic distance within the subgenus, excluding

of N. fuscipes, N. cinerea, and N. lepida, was a low 1.9%. The average genetic distance

for the cytochrome-/) data in comparisons within the subgenus Neotoma was 11.24%.

Hodomys to subgenus Neotoma was 5% for alcohol dehydrogenase and 19.1% for

cytochrome b. The average genetic distance between genera Hodomys and Xenomys was

4.3% and 16.4%, respectively. Whereas, the average genetic distance comparisons

between subgenera Teonoma and Neotoma was 4% for Adh and 17.2% for the

cytochrome-/) data. The nuclear genetic distance between Teonoma and Hodomys was

6.6% and 22.7% for the mitochondrial value. Consequently, both had similar ranging

pattems placing Hodomys as a distinct genus and Teonoma in a subgeneric status.

Teonoma also only had a Adh-l-U genetic divergent value of 2.5% with N. fuscipes.

However, the Adh-l-U gene did provide evidence that N. fuscipes may be a distinct

subgenus or genus with a genetic distance of 4.4% to subgenus Neotoma.

As a result from the phylogenetic analyses, Adh-l-U does not support the species

groups (floridana, micropus, lepida, and mexicana) recognized in Edwards and Bradley

(2002b). Consequently, Adh-l-U may have failed in being phylogenetically informative

at higher taxonomic levels (species groups) but was successful in distinguishing 3 distinct

genera and members within species. This may be due to the lack of informative

30

characters (86), and the possibility that nuclear J/?-1-12 may be evolving at too slow of a

rate for phylogenetic relationships to be informative at higher levels. Carroll (2002) used

tiie nuclear Fgb-I7 infron and also obtained few informative characters (86) with

divergent values only from 0.72% to 1.8% for species of cotton rats (genus Sigmodon).

This suggests a rate of evolution for the nuclear Adh-l-U to be slower (approximately Vi

the rate) than the cytochrome-/) gene in Neotoma (Edwards and Bradley, 2002b).

Although this study does provide confidence that Adh may be an informative nuclear

marker, additional nuclear and mitochondrial data sets are needed to further evaluate the

Adh-l-U utility.

The combined data set (alcohol dehydrogenase and cytochrome b) was well

supported by Bayesian posterior support values of > 95. Consequently, the combined

topology supported most of the phylogenetic relationships obtained from the cytochrome-

b likelihood analysis, which may result from the combined data set being more heavily

weighted by the larger number of characters supplied by the cytochrome-/) data set.

Minor differences existed in the inclusion of subgenus Teonoma (N. cinerea) in the

lepida group and the placement of A'', stephensi; however, 5 major clades were retained.

In addition, clade probability values were increased at all nodes except for 1 that joined

N. albigula to N. floridana and another between 2 subspecies ofN micropus. The 1st

major clade (I) contained 2 minor clades (A and B). Clade A contained N. floridana

sister to N. albigula with N. goldmani joirdng in a stepwise fashion, the same taxa as the

recogpized floridana species group (Edwards and Bradley 2002b). Clade B contained N.

micropus and N. leucodon as sister taxa and supported the elevation ofN. leucodon to

31

species level; tiiese results supported Edwards and Bradley's (2002b) composition of the

micropus species group. The 2nd major clade (fl) contained the same 3 species (N

lepida, N fuscipes, and A . cinerea) as the Adh-l-U only data with identical relationships.

The cytochrome-/) data, however, suggested that only N lepida, N. fuscipes, and N.

stephensi were included in clade H; thus, Edwards and Bradley (2002b) designated these

3 species as the members of the lepida species group. Consequently, they placed N.

cinerea in its own clade (IV) basal to Neotoma. The current sttidy argues for the

inclusion ofN. cinerea within the N. lepida-N. fuscipes clade based on the nuclear Adh-l-

12 and combined data sets. The 3rd major clade (III) placed A': mexicana sister to the A':

picta-N. isthmica clade, again supporting Edwards and Bradley (2002b) in their

cytochrome-/) topology and the recognition of the mexicana group. The 4th major clade

(IV) for the combined analysis only contained N. stephensi whereas Edwards and Bradley

(2002b) clade IV only contained N cinerea. Herein, all 3 data sets supported the

recognition of Hodomys as a distinct species sister to Xenomys. As a result, the combined

data set of Adh-l-U and cytochrome b, for the most part, supports the recognized species

groups (floridana, micropus, lepida, and mexicana) in Edwards and Bradley (2002b).

However, the combined data set, the Adh-l-U topologies, and the genetic distances

wartant the placement ofN. cinerea (subgenus Teonoma) within the N lepida-N. fuscipes

clade. In addition, this study argues for the subgeneric realignment ofN lepida and N

fuscipes within the genus Neotoma. Additionally, more informative data sets are needed

to further assess the phylogenetic relationships within the genus Neotoma. Subsequently,

the results from the combined data set as well as Adh-l-U data is encouraging toward the

32

;oal of generating reliable phylogenetic relationships from nuclear genes, and also the

eliabilify of combining data sets to produce comprehensive trees and relationships that

ire verified by high support values.

33

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38

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39

'able 2.1. Average Tamura and Nei (1993) genetic distances (AGD) for selected taxa of Adh lades and Cyt-b species groups examined in this study.

^axon Adh Taxon Cyt-b

Vithin N. floridana clade 0.0028

WitiuiL N. micropus clade 0.0012

f/iMn N. albigula clade 0.0089

A' ithin N. mexicana clade 0.0072

A' ithin A': picta-N. isthmica 0.0084

;lade

Within A . lepida, N. fiiscipes, 0.0450

md A' cinerea clade

V. floridana versus A . micropus 0.0175

N. floridana versus N. albigula 0.0205

N. floridana versus N. mexicana 0.0191

N. micropus versus N. albigula 0.0253

N. micropus versus N. mexicana 0.0238

N. albigula versus N. mexicana 0.0189

N. mexicana versus N. picta- 0.0194

N. isthmica

Within subgenus Neotoma 0.0216

Within genus Hodomys 0.0000

Within subgenus Teonoma 0.0072

Subgenus Neotoma versus 0.0513

genus Hodomys

Subgenus Neotoma versus 0.0403

subgenus Teonoma

Genus Hodomys versus 0.0657

subgenus Teonoma

Within N. floridana species group

Within N. micropus species group

Within N. mexicana species group

Within N. lepida species group

N. floridana species group versus

N. micropus species group

A'; floridana species group versus

mexicana species group

N. floridana species group versus

N. lepida species group

N. micropus species group versus

mexicana species group

N. micropus species group versus

N. lepida species group

A . mexicana species group versus

N. lepida species group

Within subgenus Neotoma

Within genus Hodomys

Within subgenus Teonoma

Subgenus Neotoma versus

genus Hodomys

Subgenus Neotoma versus

subgenus Teonoma

Genus Hodomys versus

subgenus Teonoma

0.1084

0. 0954

0.0957

0.0854

0.1282

0.1378

0.1443

0.1264

0.1390

0.1334

0.1124

0.0000

0.0194

0.1909

0.1719

0.2236

40

Table 2.2. Specimens examined and GenBank accession numbers.

Species

Genus Neotoma Subgenus Neotoma

N. albigula

N. albigula albigula

N. albigula durangae

N. albigula laplatensis

N. floridana attwateri

N. floridana floridana

N. floridana osagensis

N floridana osagensis

N. fuscipes

N. goldmani

N. isthmica

N isthmica

N. lepida

N. lepida

N. leucodon

N. leucodon

N. magister

N. mexicana mexicana

N. mexicana scopulorum

Museum ID#

TK77931

TK74854

NK17583

NK50148

TK52109

NK64089

TK27751

TK28244

TK83707

TK28315

TK93257

TK93296

TK77284

TK77285

TK48594

TK49716

NK64158

TK90038

TK51346

Adh-l-U Accession #

AY817648

AY817649

AY817651

AY817650

AY817638

AY817637

AY817639

AY817640

AY817632

AY817656

AY817630

AY817631

AY817633

AY817634

AY817644

AYS 17643

AY817641

AY817645

AY817647

Cyt-b Accession #

AF376477

AF186103

AFl 86804

AF 186808

AF186819

AF294335

AF294341

AF186818

AF376479

AFl86829

AF329079

AF305567

AF307835

(imavailable)

AFl 86809

AFl86806

AF294336

AF294346

AFl86821

41

Species

N. mexicana scopulorum

N. micropus

N. micropus

N. micropus

N. micropus

N. picta

N. stephensi

Subgenus Teonoma N cinerea

N. cinerea

Genus Hodomys H. alleni

Genus Ototylomys 0. phyllotis

Genus Peromyscus P. attwateri

Genus Tylomys T. nudicaudus

Genus Xenomys X. nelsoni

Museum 1D#

TK78350

TK31643

TK77270

TK84556

TK84557

TK93390

TK77928

NK36287

NK56291

TK45042

TK101714

TK23396

TK41551

TK19559

Adh-l-U Accession #

AY817646

AY817652

AY817653

AY817654

AY817655

AY817629

AY817642

AY817635

AY817636

AY817627

AY817624

AYS 17626

AYS 17625

AYS 17628

Cyt-b Accession #

AF294345

AFl86822

AF376474

AFl86826

AFl 86827

AF305569

AF308867

AFl86799

AFl 86800

AF186801

(unavailable)

AFl55384

AF307839

AF307838

42

O. phyllotis (V) T. nudicaudus (1)

"""i*. attwateri (X) H. alleni (!) X. nelsoni (1) N. picta (1)

A'; isthmica

K flpridma (4)

magister (1)

N. micropus (4)

N. stephensi (1) N. leucodon (1) N. albigula (1) N. leucodon (1)

K albigula (3)

N. mexicana (3)

N. goldmani (I) N. fuscipes (I)

N. cinerea (2)

N. lepida (1)

Figure 2.1. Strict consensus tree generated from a parsimony analysis of nucleotide sequences obtained from the Adh-l-U gene. Bremer support values appear below branches and bootsfrap values (> 50) appear above branches. Roman numerals depict major clades while letters A—F depict minor clades as defined in text. Sample sizes are shown in parentheses. O = Ototylomys, T = Tylomys, P = Peromyscus, X = Xenomys, H = Hodomys, and N = Neotoma.

43

-H-100

100

5 changes

{

•• O. phyllotis (1) ••~ '^~~~"~' T. nudicaudus (1) '~~~~~~~~~^~^~" P. attwateri (1)

T. 100 I H. alleni (I) ^ ~ ~ ~ ~ " X. nelsoni (I)

N. picta (1)

N. isthmica (2)

N. floridana (4)

N. magister (1)

N. micropus (4)

99

III

99| N. leucodon (1)

8S lOOi -^

iiooPL L

N. albigula (\) N. leucodon (1)

N. albigula (3)

N. goldmani (1)

N. mexicana (3)

96 '; ; • *6- ^ D r " TV. leucod

lodLf -^ |iooi— >-

iV. stephensi (\) 100 I ^- fuscipes (1)

100 100 •T _ ^ ^ - cinerea Q.)

lOOj "1 " - ^ J " i V . lepida (2)

Figure 2.2. Phylogenetic topology using Bayesian analysis (GTR+I+G model of evolution) obtained from the Adh-l-U gene. Posterior probabilities are shown near nodes. Roman numerals depict major clades while letters A-D depict minor clades as defined in text. Sample sizes are shown in parentheses. O = Ototylomys, T = Tylomys, P = Peromyscus, X = Xenomys, H = Hodomys, and N = Neotoma.

44

100

O. phyllotis (1) T. nudicaudus (1)

P. attwateri (1)

V 100 1 H. alleni (I) X. nelsoni (1)

N. lepida (1) N. fuscipes (1)

100

N. stephensi (1)

100 ( N. picta (1)

99p- ^ ^ i f/im/ca (2)

f" _r^ N. cinerea(2) OTQ

O

e

V 2 ^

A'l mexicana (3)

A . leucodon (2)

ora ?5.-

^

85

N. micropus {4)

N. goldmani (1)

l o o T " "^

L T ^ iV. albigula l O o l r - (4) l o o l - ^

jQQJ ^' magister (1)

<<5

>

50 changes

9iLj Wj lOolj-J (4)

96*-^

floridana

I: a

orq ' •1 o s

9 6 * - ^ ^

Figure 2.3. Phylogenetic topology using Bayesian analysis (GTR+I+G model of eyolution) obtained from the ^(ife-l-I2/CytQchrome-Z) genes. Posterior probabilities are shown near nodes. Roman numerals depict major clades while letters A & B depict minor clades as xiefined in text. Sample sizes are shown in parentheses. O = Ototylomys, T = Tylomys, P = Peromyscus, X = Xenomys, H = Hodomys, and N = Neotoma.

45

CHAPTER III

CONCLUSIONS

The following is a review of results and a summaty of major conclusions drawn

from tiie main data chapter (II) of the thesis. The data chapter (fl) has been written as a

separate manuscript and will be submitted for publication in the Joumal of Mammalogy.

Consequentiy, the format of the Joumal of Mammalogy was followed throughout this

manuscript for consistency.

Summary

DNA sequences from infron 2 of the nuclear alcohol dehydrogenase gene 1 (Adh-

l-U) were obtained from 33 individuals that represented 13 species of the genus

Neotoma, 2 ingroups, and 3 outgroups. These data were analyzed using parsimony,

likelihood, and Bayesian methods. Three major clades consistently were recovered in all

analyses. The 1st major clade (I) contained samples of X nelsoni and H alleni as sister

taxa. The 2nd major clade (U) included 23 individuals representing 10 species (A': picta,

N. isthmica, N. floridana, N. magister, N. micropus, N. stephensi, N leucodon, N.

albigula, N. mexicana, andN. goldmani). The 3rd major clade (EI) contained 5

individuals representing 3 species (N fuscipes, N. cinerea, and N. lepida). Species and

relationships within clade I and HI always remained the same. However, the minor

clades within the 2nd major clade (II) differed slightly in that the parsimony analysis had

minor clades A-F and the likelihood and Bayesian analyses only had minor clades A-D

46

(floridana, micropus, lepida, and mexicana) recognized in Edwards and Bradley (2002).

Genetic divergences within the subgenus Neotoma averaged 2.16%. The average genetic

distance of Hodomys to subgenus Neotoma was 5% and subgenera Teonoma to Neotoma

was 4%. These results support Hodomys as a separate genus as well as Teonoma as a

subgenus within Neotoma. Hodomys was sister to Xenomys, and A cinerea (Teonoma)

and N. fuscipes were sister taxa. hi addition, Adh-l-U sequences then were concatenated

with mitochondrial cytochrome-/) sequences generated from the same individuals and

were analyzed in a total evidence approach. This combined data set resulted in a

phylogeny whose topology was very similar to a tree based on the cytochrome-/) gene.

Although intemal relationships were slightly different, Bayesian support values were

higher for most relationships than in the cytochrome-/) only tree.

In conclusion, combined analyses of nuclear and mitochondrial DNA sequence

data result in the best provisional phylogeny for this group. It appears that the genus

Neotoma is a monophyletic assemblage, as described above. However, additional nuclear

and mitochondrial data sets are needed to fiirther evaluate the utility of Adh-l-U.

47

Literature Cited

EDWARDS, C.W. AND R D.BRADLEY. 2002. Molecular systematics of the genus Neotoma. Molecular Phylogenetics and Evolution 25:489-500.

48

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