MOLECULAR SYSTEMATICS OF PEROMYSCUS

MOLECULAR SYSTEMATICS OF PEROMYSCUS AND ALLIES

BASED ON NUCLEAR AND MITOCHONDRIAL DNA SEQUENCES

by

BRIAN RICHARD AMMAN, B.S., M.S.

A DISSERTATION

IN

ZOOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

DOCTOR OF PHILOSOPHY

Approved

Robert D. Bradley Chairperson of the Committee

Robert J. Baker

Richard E. Strauss

Clyde Jones

David J. Schmidly

Accepted

John Borrelli

Dean of the Graduate School

May, 2005

ii

ACKNOWLEDGMENTS

Funding for this study was provided by NIH (DHHS A141435-01 to Robert D.

Bradley). Tissue loans were made available by R. J. Baker and the Natural Science

Research Laboratory, Museum Texas Tech University, C. William Kilpatrick and the

University of Vermont, and Duke S. Rogers and Brigham Young University. I would

like to thank the members of my committee, Drs. Robert J. Baker, Clyde Jones, David J.

Schmidly, and Richard E. Strauss for their guidance during this project and through all

aspects of my graduate experience. I would especially like to thank major advisor Dr.

Robert D. Bradley for his patience and guidance through my graduate career and

throughout this and other projects. Also, I would like to thank Dr. Bradley for his

friendship and willingness to accept a novice into his lab and show him the ropes.

I would also like to thank my colleagues and friends Brooke D. Baxter, Dr. Darin

S. Carroll, Nevin D. Durish, John D. Hanson, Francisca Mendez-Harclerode, Michelle L.

Haynie, Dr. Steven R. Hoofer, Lisa K. Longhofer, L. Rex McAliley, Serena A. Reeder,

and John R. Suchecki for their extremely valuable input and assistance, and especially for

their friendship and camaraderie during the hard times.

I would like to acknowledge my parents, Jane E. Garnett, L. Richard Amman, and

Ona M. Amman, for their support and encouragement throughout my graduate career. I

would like to extend my most heartfelt gratitude and love to my wife Colleen for her

unwavering support and encouragement these many years and to my boys, Brendan and

Liam, for giving me the inspiration to press on when I wanted to stop.

iii

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii

ABSTRACT iv

LIST OF FIGURES v

CHAPTER

I. INTRODUCTION 1

II. MOLECULAR SYSTEMATICS OF PEROMYSCUS AND ALLIES BASED ON NUCLEAR AND MITOCHONDRIAL DNA SEQUENCES 5

III. SUMMARY 41

APPENDIX: SPECIMEN LOCALITIES 45

iv

ABSTRACT

Nucleotide sequences from the 2nd intron of the class 1 alcohol dehydrogenase

nuclear gene (Adh1-I2) were used to examine the boundaries of the genus Peromyscus

(sensu stricto), especially concerning the taxonomic affinities of Habromys,

Megadontomys, Osgoodomys, Podomys, and Neotomodon with regard to Peromyscus.

Relationships of the Peromyscus species groups also were tested. Adh1-I2 sequences

were analyzed using parsimony, maximum likelihood, and Bayesian methods and all

produced similar unresolved phylogenies regarding relationships between species groups.

Habromys, Megadontomys, Osgoodomys, Podomys, and Neotomodon were included in

clades containing members of the genus Peromyscus, however, support was lacking for

their exact placement. Genetic divergence values between the genus Peromyscus and the

5 genera in question ranged from 2.64% (Osgoodomys versus Peromyscus) to 4.61%

(Podomys versus Peromyscus). In addition, Adh1-I2 sequences were concatenated with

mitochondrial cytochrome b sequences and Bayesian methods were used to test the

boundaries of the genus. Support was strong for all species groups except the truei

group; however, an unresolved phylogeny was produced with respect to the relationships

of the species groups. Neotomodon, Habromys, and Osgoodomys were supported as

being included in the genus Peromyscus as traditionally defined, whereas Megadontomys

was placed in an unresolved clade with members of the genus Peromyscus and its

phylogenetic position with regard to Peromyscus (sensu stricto) remains uncertain.

v

LIST OF FIGURES

3.1 Strict consensus tree representing 132 equally parsimonious trees obtained from a parsimony analysis of unweighted DNA sequence characters from

Adh1-I2.

3.2 Adh1-I2 phylogeny generated using maximum likelihood with the HKY+G model of evolution.

3.3 Adh1-I2 phylogeny generated using Bayesian methods and the GTR+I+G model of evolution.

3.4 Adh1-I2---Cytb phylogeny generated using Bayesian methods and the GTR+I+G model of evolution.

1

CHAPTER I

INTRODUCTION

Peromyscus is a widely studied rodent genus and has been examined using

morphological, karyological, biochemical, and nucleotide sequence data. The genus has

undergone 3 major revisions beginning with Osgood (1909), followed by Hooper (1968)

and Carleton (1989). One of the most interesting controversies of these revisions

involves the phylogenetic and taxonomic status of the 5 subgenera (Osgoodomys,

Megadontomys, Isthmomys, Habromys, and Podomys) recognized by Osgood (1909) and

Hooper and Musser (1964) and later elevated to generic status by Carleton (1980; 1989)

based on morphological evidence.

Despite numerous studies involving various taxa of Peromyscus (Baker et al.

1983; Bradley and Schmidly 1987; Carleton 1979; Rennert and Kilpatrick 1986; Rogers

1983; Tiemann-Boege et al. 2000; Zimmerman et al. 1975), there has not been a

comprehensive phylogenetic study of the genus since Carleton (1980) and there has not

yet been an evaluation of the relationships among species groups. R. D. Bradley (pers

com) has developed an overview of the genus Peromyscus based on the mitochondrial

cytochrome b (Cytb) gene that would provide a maternally grounded phylogeny, but a

phylogenetic assessment utilizing a nuclear marker capable of detecting species level

differences is needed. One potential marker is the 2nd intron of the nuclear alcohol

dehydrogenase gene (Adh1-I2). Alcohol dehydrogenases, in mammalian systems, are

dimeric zinc-metalloenzymes that function to catalyze the oxidization of alcohols to

2

aldehydes or ketones (Cheung et al. 1999). Alcohol dehydrogenase has been shown to be

a highly polymorphic allozyme in rodents and capable of detecting species level

differences (Bradley et al. 1998). Amman et al. (In prep) has shown that intron 2 of the

class 1 alcohol dehydrogenase (Adh1-I2) is capable of detecting species level differences

in rodents.

This dissertation involves a phylogenetic treatment of the genus Peromyscus, as

defined by Carleton (1989), using nucleotide sequence data from the Adh1-I2 nuclear

marker. The primary objective of this study was to examine the classification of the

genus Peromyscus and allies by Carleton (1989) using nucleotide sequences from Adh1-

I2. A secondary goal was to use Adh1-I2 to examine the phylogenetic relationships of the

species groups within the genus Peromyscus.

The 2nd chapter of this dissertation represents the following manuscript being

prepared for publication and is formatted according to the specifications set forth by the

Journal of Mammalogy:

Amman, B. R. and R. D. Bradley. Molecular Systematics of

Peromyscus and Allies Based on Nuclear and Mitochondrial DNA

Sequences. In prep.

This chapter includes phylogenies produced by the analyses of the Adh1-I2 nuclear

marker via parsimony, maximum likelihood, and Bayesian methods. Also, Adh1-I2 and

mitochondrial Cytb nucleotide sequences were concatenated and analyzed using Bayesian

methods.

3

LITERATURE CITED

Baker R. J., L. W. Robbins, F. B. Stangl, Jr., and E. C. Birney. 1983. Chromosomal evidence for a major subdivision in Peromyscus leucopus. Journal of Mammalogy 64:356--359.

Bradley, R. D., and D. J. Schmidly. 1987. The glans penes and bacula in Latin American taxa of the Peromyscus boylii group. Journal of Mammalogy 68:595-- 616.

Bradley, R. D., R. M. Adkins, R. L. Honeycutt, and J. H. McDonald. 1998. Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys. Molecular Biology and Evolution 15:709--717.

Carleton, M. D. 1979. Taxonomic status and relationships of Peromyscus boylii from El Salvador. Journal of Mammalogy 60:280--296.

Carleton, M. D. 1980. Phylogenetic relationships in neotomine-peromyscine rodents (Muroidea) and a reappraisal of the dichotomy within New World Cricetinae. Miscellaneous Publications, Museum of Zoology, University of Michigan 157:1-- 146.

Carleton, M. D. 1989. Systematics and evolution. Pp. 7-141 in Advances in the study of Peromyscus (Rodentia) (G. L. Kirkland, Jr. and J. N. Layne, eds.). Texas Tech University Press, Lubbock, 366 pp.

Cheung, B., R. S. Holmes, S. Esteal, and I. R. Beacham. 1999. Evolution of class I alcohol dehydrogenase genes in catarrhine primates: gene conversion, substitution rates, and gene regulation. Molecular Biology and Evolution 16:23--36.

Hooper, E. T. 1968. Classification. Pp. 27-74 in Biology of Peromyscus (Rodentia) (J. A. King, ed.). Special Publication, The American Society of Mammalogists 2:1-- 593.

4

Hooper, E. T., and G. G. Musser. 1964. The glans penis in Neotropical cricetines (Family Muridae) with comments on classification of muroid rodents. Miscellaneous Publications, Museum of Zoology, University of Michigan 123:1-- 57.

Osgood, W. H. 1909. Revisions of the mice of the American genus Peromyscus. North American Fauna 28:1--285.

Rennert, P. D., and C. W. Kilpatrick. 1986. Biochemical systematics of populations of Peromyscus boylii I. Populations from east-central Mexico with low autosomal arm numbers. Journal of Mammalogy 67:481--488.

Rogers, D. S. 1983. Phylogenetic affinities of Peromyscus (Megadontomys) thomasi: Evidence from differentially stained chromosomes. Journal of Mammalogy 64:617--623.

Tiemann-Boege, I., C. W. Kilpatrick, D. J. Schmidly, and R. D. Bradley. 2000. Molecular phylogenetics of the Peromyscus boylii species group (Rodentia: Muridae) based on mitochondrial cytochrome b sequences. Molecular Phylogenetics and Evolution 16:366--378.

Zimmerman, E. G., C. W. Kilpatrick, and B. J. Hart. 1975. Biochemical genetics of the boylii and truei groups of the genus Peromyscus. Comparative Biochemical Physiology B: 52:541--545.

5

CHAPTER II

MOLECULAR SYSTEMATICS OF PEROMYSCUS AND ALLIES BASED ON NUCLEAR AND

MITOCHONDRIAL DNA SEQUENCES

The genus Peromyscus (Rodentia: Muridae), described by Gloger (1841), is 1 of

the most extensively studied groups of rodents. To date, several thousand papers have

been published concerning various aspects of Peromyscus natural history, ecology,

taxonomy, classification, and systematics. Consequently, Peromyscus has been referred

to as the Drosophila of North American Mammalogy (Wilson and Reeder 1993). The

genus has undergone 3 major revisions beginning with Osgood (1909), followed by

Hooper (1968) and Carleton (1989). One of the most interesting controversies of these

revisions involves the phylogenetic and taxonomic status of the 5 subgenera

(Osgoodomys, Megadontomys, Isthmomys, Habromys, and Podomys) recognized by

Osgood (1909) and Hooper and Musser (1964) and later elevated to generic status by

Carleton (1980; 1989) based on morphological evidence.

Osgoodomys was designated as a subgenus in recognition of its divergence from

Peromyscus largely based on phallic morphology (Hooper and Musser 1964a). Later

studies by Linzey and Layne (1969) and Carleton (1980) indicated a strong divergence of

Osgoodomys from Peromyscus. Karyological data presented by Rogers et al. (1984) and

Stangl and Baker (1984) did not resolve the status of Osgoodomys, but supported an early

origin as alluded to by Hooper (1968). The biochemical studies of Fuller et al. (1984)

6

and Schmidly et al. (1985) did not support the treatment of Osgoodomys as a distant

genus.

Megadontomys was 1st diagnosed by Merriam (1898) as a subgenus of

Peromyscus containing the species thomasi and nelsoni, the latter subsequently was

subsumed under thomasi as a subspecies (Musser 1964). Osgood (1909) retained

Megadontomys as a subgenus of Peromyscus; however, ensuing morphological (Carleton

1980; Hooper 1958; Hooper and Musser 1964a; Linzey and Layne 1969) and

biochemical (Werbitsky and Kilpatrick 1987) studies supported a distinction between

Megadontomys and Peromyscus. The divergence of Megadontomys relative to other

forms of Peromyscus was proposed to have occurred early in the origin of this genus

(Carleton 1980) and that it shared a common ancestry with Isthmomys; however, this

view was not supported by karyological evidence (Rogers 1983; Rogers et al. 1984;

Stangl and Baker 1984).

Although Bangs (1902) and Osgood (1909) allocated Megadontomys flavidus to

the subgenus Megadontomys, Hooper and Musser (1964a) suggested a dichotomy existed

in Megadontomys and proposed that M. flavidus, along with Peromyscus pirrensis, be

placed into a new subgenus (Isthmomys) to reflect differences in phallic morphology.

Carleton (1980) recommended that Isthmomys be elevated to generic rank and proposed a

relationship with Megadontomys based on a suite of morphological differences.

Habromys originally was placed in the mexicanus group by Osgood (1909), but

was elevated to subgeneric rank by Hooper and Musser (1964a) based on phallic

morphology. Later, morphological studies of Linzey and Layne (1974) and Carleton

(1980) corroborated the distinction of Habromys from Peromyscus. Karyological data

7

from Rogers et al. (1984) placed H. lepturus in an unresolved clade with many other

species of Peromyscus as well as Podomys and Neotomodon. Stangl and Baker (1984)

aligned H. lepturus with P. truei and attributed the relationship to a probable convergent

event, and suggested Habromys shared an early ancestor with Podomys and Neotomodon,

a view shared by Carleton (1980).

Osgood (1909) included Podomys as a subgenus of Peromyscus based on the

reduction in size and number of the plantar tubercles. Examinations of the male

reproductive tract also served to distinguish Podomys from Peromyscus (Carleton 1980;

Hooper 1958; Hooper and Musser 1964a; Linzey and Layne 1969). Carleton (1980)

reported consistent affinities between Podomys, Habromys, and Neotomodon based on

phallic morphology and accessory reproductive glands, although Osgoodomys and

Onychomys were sometimes included depending on the type of analysis and included

taxa. Karyotypic studies have been ambivalent concerning the hypothesized relationship

of Podomys to Habromys and Neotomodon (Carleton 1989). Yates et al. (1979) included

Podomys in an unresolved tetrachotomy with 3 members of the maniculatus group, P.

gossypinus, and Neotomodon. Robbins and Baker (1981) and Rogers (1983) also aligned

Podomys with Neotomodon and other species of Peromyscus. Biochemical studies also

alluded to the distinctness of Podomys (Avis et al. 1974) and its association with

Neotomodon (Patton et al. 1981).

Despite Carleton’s (1980) recognition as a separate genus, proposals have been

made to include Neotomodon (Stangl and Baker 1984; Yates et al. 1979) and possibly

Onychomys (Stangl and Baker 1984) in the genus Peromyscus, based on karyological

evidence. Bradley et al. (2004a) investigated the phylogenetic relationships of

8

neotomine/peromyscine rodents using mitochondrial cytochrome b (Cytb) sequence data

and found that Neotomodon was placed inside of the genus Peromyscus, whereas

Onychomys was not included.

In the most current classification, Carleton (1989) divided Peromyscus (sensu

stricto) into 2 subgenera, Haplomylomys and Peromyscus. Haplomylomys contains at

least 10 species divided into 2 species groups. Recently, Riddle et al. (2000) included the

addition of P. fraterculus to Haplomylomys based on mitochondrial DNA (mtDNA)

sequence data. Carleton (1989) placed 43 of his 53 recognized species within the

subgenus Peromyscus and further subdivided these into 11 species groups. Recent

studies added P. sagax (Bradley et al. 1996), P. schmidlyi (Bradley et al. 2004b), P. keeni

(Hogan et al. 1993), and P. beatae, P. levipes (Schmidly et al. 1988).

Despite numerous studies involving various taxa of Peromyscus, there has not

been a comprehensive phylogenetic study of the genus. Not since Carleton (1980) has

there been a treatment of the genus as a whole, and there has not yet been an evaluation

of the relationships between species groups. R. D. Bradley (pers com) has developed an

overview of the genus Peromyscus based on the mitochondrial Cytb gene providing a

maternally grounded phylogeny and, in effect, producing a need for a study utilizing a

nuclear marker to provide an alternative perspective.

Specifically, a nuclear marker is needed that evolves at a rate close to that of Cytb

and is capable of elucidating species level differences. One such marker is the 2nd intron

of the alcohol dehydrogenase gene. Alcohol dehydrogenases, in mammalian systems, are

dimeric zinc-metalloenzymes that function to catalyze the oxidization of alcohols to

aldehydes or ketones (Cheung et al. 1999). Alcohol dehydrogenase has been shown to be

9

a highly polymorphic allozyme in rodents and capable of detecting species level

differences (Bradley et al. 1998). Amman et al. (In prep) has shown that intron 2 of the

class 1 alcohol dehydrogenase (Adh1-I2) is capable of detecting species level differences

in rodents. The recommended nomenclature of vertebrate alcohol dehydrogenases

(Duester et al. 1999) will be used in this study and follows the use of Adh-1 (Enzyme

Commission No. 1.1.1.1) for mice.

The primary objective of this study was to examine the classification of the genus

Peromyscus and allies by Carleton (1989) using a nuclear molecular marker.

Specifically, Adh1-I2 was used to test the boundaries of the genus Peromyscus (i.e.,

should Habromys, Megadontomys, Osgoodomys, Podomys, and Neotomodon be

included?). Second, Adh1-I2 was used to examine the phylogenetic relationships of the

species groups within the genus Peromyscus. Third, use a combined nuclear and

mitochondrial data set to examine the genus and the species groups.

MATERIALS AND METHODS

Samples.---Tissues from individuals representing the genera Peromyscus,

Habromys (H. lepturus), Megadontomys (M. thomasi), Osgoodomys (O. banderanus),

Podomys (P. floridanus), and Neotomodon (N. alstoni) were obtained from naturally

occurring populations or museum loans. At least 2 representatives of each of the species

groups within the subgenus Peromyscus were examined except the crinitus (1 sample),

furvus (no sample), and hooperi (no sample) groups. One sample each from the

californicus and eremicus groups was used to represent the subgenus Haplomylomys and

at least 1 sample from Habromys, Megadontomys, Osgoodomys, Podomys, and

10

Neotomodon was examined. An attempt was made to obtain mitochondrial and nuclear

data from a single individual. When this was not possible, sequences from samples of the

same species and from close geographic proximity were used. If attainment of samples

from close geographic proximity was not possible, another representative from the same

genus was included (See Appendix for Specimens Examined.).

DNA Isolation and PCR.---DNA was isolated from liver samples (0.1g) using 2

methods. Mitochondrial DNA was extracted and purified using a Wizard Miniprep kit

(Promega, Madison, Wisconsin). Total genomic DNA was extracted from liver using

DNeasy tissue kits (Qiagen, Valencia, California) following the method of Smith and

Patton (1999).

The complete intron 2 of the Adh1 gene (598 bp) was amplified following the

methods of Amman et al. (In prep). Specifically, PCR parameters were: 25–30 cycles

94°C (1min) denaturation, ramped from 52°C to 46°C to 52°C at 0.5°C per second

annealing, 1°C per second to 73°C (1 min 30 sec) extension, and 1-cycle of 73°C (2 min)

extention. The primers utilized in PCR reactions were 2340-I, 2340-II, and Exon II F,

(Amman et al. In prep).

The complete cytochrome-b gene (1,143 bp) was amplified with the following

polymerase chain reaction (PCR) parameters modified from Saiki et al. (1988): 30 cycles

at 95°C (1 min) denaturation, 50°C (1 min) annealing, 72°C (2 min) extension, and a

final cycle of 72°C (7 min) extention. Primers utilized in PCR reactions (MVZ05 and

H15915) were those used by Smith and Patton (1993) and Irwin et al. (1991),

respectively.

11

Sequencing.---PCR products were purified using the QIAquick PCR purification

kit (Qiagen, Valencia, California) and PCR amplicons were sequenced using ABI Prism

Big Dye Terminator v3.1 ready reaction mix (PE Applied Biosystems, Foster City,

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

align and proof nucleotide sequences. Nucleotide sequences were analyzed on an ABI

3100 Avant automated sequencer (PE Applied Biosystems, Foster City, California)

Adh1-I2 amplicons were sequenced using the PCR primers in conjunction with 2

internal primers, Adh 350F and Adh 350R (Amman et al. In prep). Cytb amplicons were

sequenced using the following 6 cycle-sequencing primers: CWE-1 and 400F (Edwards

et al. 2001), 700L and WDRAT400R (Peppers and Bradley 2000), and PERO3' and 752R

(Tiemann-Boege et al. 2000). All DNA sequences were deposited in GenBank.

Data Analysis.---Nucleotide sequence data were analyzed using the software

package PAUP* (Swofford 2002). All nucleotide positions were treated as unordered,

discrete characters with 4 possible states, A, C, G, and T, for Cyt b, and 5 possible states:

A, C, G, T, and gaps (-) for Adh. Alignment of the Adh1-I2 sequences produced

hypothesized gaps that represent insertion or deletion events. Gaps were inserted based

on primary homology assessment. Graur and Li (2000) made a distinction between 2

types of gaps, terminal and internal, and recommend that terminal gaps (incomplete

sequence) should not be included in the analysis when alignment is with a complete

homologous gene or intron sequence; therefore incomplete Adh1-I2 sequences were

excluded from these analyses. For all analyses, Hodomys alleni, Reithrodontomys

fulvescens, and Baiomys taylori were used as outgroup taxa.

12

Parsimony analyses were performed on the Adh1-I2 data using equally-weighted

characters to avoid subjectivity inherent with a priori differential weighting. The

heuristic search and tree bisection-reconnection options were used to estimate the most

parsimonious tree(s). All phylogenetically uninformative characters were excluded from

these analyses. Robustness of topologies was evaluated using bootstrap (Felsenstein

1985) and Bremer (1994) support indices (Autodecay ver.3.03, Eriksson 1997).

The maximum likelihood analysis was conducted using the HKY+G model of

nucleotide substitution identified by MODELTEST (Posada and Crandall 1998) as the

most consistent model for the data. This model generated significantly better likelihood

scores than all other models and included the following parameters: base frequencies (A

= 0.3286, C = 0.1906, G = 0.1718, T = 0.3089) and gamma distribution (Γ = 1.1324).

A Bayesian analysis (Huelsenbeck and Ronquist 2001) was conducted to estimate

an independent phylogeny for comparison to that estimated by likelihood methodologies

and to generate values (posterior probability) for nodal support. Values ≥ 95% were

viewed as supportive following Alfaro et al. (2003), Douady et al. (2003), and

Hulsenbeck et al. (2002). The GTR+I+G model of evolution was employed with an

invariant gamma distribution, un-partitioned data, 4 Markov-chains, 2 million

generations, and sample frequency every 100th generation. The number of invariable

sites and gamma distribution were estimated from the data. After a visual inspection of

likelihood scores, the 1st 100 trees were discarded and the model was rerun using the

remaining stable likelihood values. A consensus tree (50% majority rule) was

constructed from the remaining trees.

13

The Kimura 2-parameter model of evolution (Kimura 1980) was used to estimate

genetic distances. This model of evolution was chosen in order to allow comparisons

between Adh1-I2 and values generated for Peromyscus using the Cytb data set. Mean

sequence divergences were used to examine levels of genetic divergence following

criteria outlined by Bradley and Baker (2001) and to assess levels of genetic differences

between Adh1-I2 and Cytb.

Adh1-I2 and Cytb sequence data were combined into a single analysis. An

incongruence length difference (ILD) test was not performed in light of recent studies

proclaiming the test was not a good predictor of data set compatibility (Yoder et al. 2001;

Dowton and Austin 2002). Huelsenbeck et al. (1996) also states that errors in the

statistical tests may incorrectly detect heterogeneity when conflicts among the data sets

actually do not occur. Complete sequences for both Adh1-I2 and the Cytb genes were

used in all but 2 samples (P. mexicanus missing 24 bases starting at 994 and P. nudipes

missing terminal 416 bp for Cytb). The overall data set was trimmed to include a single

representative of each taxon for both the Adh1-I2 and the Cytb sequences. A

mitochondrial Cytb nucleotide sequence for Podomys TK92502 was not available.

Hodomys alleni, R. fulvescens, and B. taylori were used as the outgroup taxa. This

resulted in a combined analysis of 28 taxa (1,740 bp). MODELTEST (Posada and

Crandall 1998) identified the GTR+I+G model of evolution as the most appropriate for

this data set and included the following parameters: base frequencies (A = 0.3148, C =

0.2979, G = 0.1459, T = 0.2414), rates of substitution (A-C = 1.5961, A-G = 4.2621, A-T

= 1.7426, C-G = 0.4237, C-T = 14.2589, G-T = 1.0), proportion of invariable sites (I =

0.2784) and gamma distribution (Γ = 0.4260). A Bayesian analysis was performed with a

14

site-specific gamma distribution with the following options: 4 Markov-chains, 2 million

generations, and sample frequency every 100th generation. After a visual inspection of

likelihood scores, the 100 trees were discarded and the model was rerun using the

remaining stable likelihood values. The Cytb data were partitioned according to codon

and the Adh1-I2 data were not partitioned. A consensus tree (50% majority rule) was

constructed from the remaining trees.

RESULTS

Adh1-I2.---A parsimony analysis was performed using a heuristic search based on

76 equally weighted informative characters. This analysis produced 132 equally most-

parsimonious trees with a tree length of 141, consistency index (CI) of 0.6312, and a

retention index (RI) of 0.8045. Bootstrap analysis (20,000 iterations with the rapid

option) provided moderate to good support for the species groups. A consensus of the

132 trees (Fig. 2.1) failed to resolve the relationships among species groups within the

genus; however, taxa forming species groups were well resolved. One moderately

supported (bootstrap/Bremer = 81/4) major clade was present containing all of the

ingroup taxa. Several subclades within the major clade also were present, representing

the species groups. One exception to this was the formation of a subclade containing

both the boylii and aztecus species groups.

The maximum likelihood analysis produced a topology (Fig. 2.2) with 7 major

clades (I--VII) and 4 subclades (A--D). Clades I--VI were unresolved with clade VII

being basal. Clade I contained Megadontomys and Habromys. Clade II was divided into

2 subclades (A and B). Subclade A contained Neotomodon, members of the melanophrys

species group (P. perfulvus and P. melanophrys), the megalops group (P. megalops), and

15

the truei group (P. gratus, P. difficilis, and P. attwateri). Subclade B contained members

of the boylii group (P. beatae, P. levipes, P. sp., P. boylii, and P. schmidlyi) and the

aztecus group (P. spicilegus and P. hylocetes). Clade III contained only P. pectoralis and

clade IV contained members of the mexicanus group (P. mexicanus and P. nudipes).

Clade V was divided into subclades C and D with subclade C containing the californicus

group (P. californicus) and the eremicus group (P. eremicus), both representing the

subgenus Haplomylomys. Subclade D contained members of the leucopus group (P.

leucopus) and the maniculatus group (P. maniculatus). Clade VI contained the crinitus

group (P. crinitus) and Podomys. Clade VII contained Osgoodomys.

The Bayesian analysis produced a resolved topology with 7 clades (I--VII) and 4

subclades (A--D) similar to that in the maximum likelihood analysis (Fig. 2.3). Clade I

contained Megadontomys and Habromys. Clade II was divided into 2 subclades, A and

B, with Neotomodon, members of the melanophrys species group, the megalops group,

and the truei group contained within subclade A. Subclade B contained members of the

boylii group and aztecus groups. Clade III contained only P. pectoralis and clade IV

contained members of the mexicanus group. Clade V contained Podomys and the crinitus

group. Clade VI was subdivided into subclades C and D with subclade C containing the

californicus and eremicus groups and subclade D containing the leucopus and

maniculatus groups. Clade VII contained Osgoodomys. Posterior probability values for

the species groups were supportive (posterior probability ≥ 95%) in most comparisons.

However, the subgenus Haplomylomys (clade VI C) was not supported (posterior

probability = 73%) and clade I containing M. thomasi and P. lepturus was not supported

16

(posterior probability = 25%). In addition, relationships among species groups and most

of the deeper nodes were not supported.

Combined Data Adh1-I2 and Cytb.---A Bayesian analysis performed on the

combined data (Adh1-I2 and Cytb) produced a topology (Fig. 2.4) with 7 major clades.

Clade I contained Megadontomys. Clade II contained 2 subclades (A and B). Subclade

A contained Neotomodon, the melanophrys group, the megalops group, and the

mexicanus group. Subclade B contained the truei group. Clade III is divided into 2

subclades (C and D) with subclade C containing members of the boylii group and

subclade D consisting of the aztecus group. Clade IV contained P. pectoralis and clade

V contained Habromys. Clade VI also was subdivided into 2 subclades (E and F) with

subclade E containing Osgoodomys and subclade F containing the subgenus

Haplomylomys, the leucopus group, and the maniculatus group. Clade VII contained the

crinitus group and was placed as the basal taxon. Nodal support was provided (posterior

probabilities ≥ 95) for subclade A, C, D, E, and F. Support also was provided for major

clade III, although major clade II was not supported (posterior probability of 91%). The

remaining clades were not supported (posterior probabilities < 95%).

Genetic Distances.---Genetic distance differences obtained (Table 2.1) ranged

from 0.0% between 2 samples of P. boylii to 10.96% between B. taylori and the genus

Peromyscus (sensu stricto). Pairwise comparisons among the genus Peromyscus,

outgroups, and the 5 genera in question ranged from 2.64% (Osgoodomys versus

Peromyscus) to 10.96% (B. taylori versus Peromyscus). Comparisons among R.

fulvescens and the 5 genera of interest ranged from 6.11% (Osgoodomys) to 8.35%

17

(Podomys). Comparisons between the 5 genera ranged from 1.99% (Megadontomys

versus Osgoodomys) to 4.18% (Neotomodon versus Podomys).

DISCUSSION

Adh1-I2.---Parsimony, maximum likelihood, and Bayesian analyses all produced

similar topologies that lacked support for mid-level nodes. In the parsimony analysis, all

mid-level clades collapsed forming 1 large clade (bootstrap/Bremer = 81%/4) with

several smaller subclades. Bayesian and maximum likelihood analyses produced similar

topologies and contained many unsupported nodes resulting in an unresolved topology.

The unresolved topologies are the result of too few informative characters associated with

the Adh1-I2 marker. The 76 informative characters provided sparse material for the

estimation of the topologies based solely on Adh1-I2.

Habromys and Megadontomys (clade I) were placed as sister taxa in both

maximum likelihood and Bayesian analyses; however, support values (posterior

probability = 25%) refute this association. Habromys and Megadontomys were

unresolved in the parsimony analysis, but both were contained within a large polytomy

with members of the genus Peromyscus.

Both maximum likelihood and Bayesian analyses placed members of the

melanophrys, megalops, and truei species groups and Neotomodon with members of the

boylii and aztecus groups into a supported clade (clade II, posterior probability = 100%)

that was subdivided into 2 subclades (A and B). Maximum likelihood and Bayesian

analyses associated members of the melanophrys, megalops, and truei species groups and

Neotomodon (clade II A), although lack of support refutes this association (posterior

probability = 83%). Support for the relationship between the melanophrys and megalops

18

species groups was present (posterior probability = 96%) in the Bayesian analysis, as was

the support for the truei species group (posterior probability = 96%). The parsimony

analysis grouped the melanophrys, megalops, and truei species groups, but excluded

Neotomodon. The relationship between the melanophrys and megalops species groups

received little support with the parsimony analysis (bootstrap/Bremer = 60%/1), but

support for the truei group was moderately high (bootstrap/Bremer = 86%/2).

Both maximum likelihood and Bayesian analyses placed members of the boylii

and aztecus species groups into a single clade (subclade II B, posterior probability =

100%). The Bayesian analysis separated the 2 groups but support for the topology was

insufficient and the clades collapsed. One sample of P. boylii was placed in the aztecus

group in the Bayesian analysis tree as sister to P. hylocetes, but without any support

(posterior probability = 7%). This may be indicative of a close relationship between the

boylii and aztecus species groups. Parsimony analysis also grouped the boylii and

aztecus species groups into 1 supported subclade (bootstrap/Bremer = 90%/2).

Peromyscus pectoralis (clade III) was depicted basal to the assemblage containing

the melanophrys, megalops, truei, boylii, and aztecus species groups in both maximum

likelihood and the Bayesian analysis. However, support for this arrangement was lacking

(posterior probability = 36%). In both instances, P. pectoralis grouped closer to the

boylii and aztecus assemblage, but the lack of support does not allow for any meaningful

inferences concerning the placement of P. pectoralis. Parsimony analysis placed P.

pectoralis between the truei and boylii groups, again in a clade by itself, further

elucidating the uniqueness of this species.

19

Both maximum likelihood and the Bayesian analysis placed the mexicanus group

(clade IV) basal to P. pectoralis, whereas the parsimony analysis placed it basal to the

boylii-aztecus assemblage. Due to the lack of support in the Bayesian analysis (posterior

probability = 53%), this relationship does not hold. Support for the species group

however, is present (posterior probability = 100%) as it is in the parsimony analysis

(bootstrap/Bremer = 100%/8).

P. crinitus and Podomys were depicted as sister in the Bayesian analysis (clade V)

but this arrangement was unsupported (posterior probability = 14%) negating the inferred

sister relationship. The subgenus Haplomylomys (californicus and eremicus species

groups) and members of the leucopus and maniculatus groups form 2 subclades (C and

D, respectively) in clade VI. The sister alignment between Haplomylomys and the

leucopus-maniculatus clade was supported (posterior probability = 100%) indicating a

close relationship, but the clade containing Haplomylomys was not supported (posterior

probability = 73%) and collapsed forming a polytomy with the supported leucopus-

maniculatus clade (posterior probability = 100%). Maximum likelihood produced the

same scenario, but with the clades reversed so that clade V contained Haplomylomys and

the leucopus and maniculatus species groups and clade VI contained P. crinitus and

Podomys aligned as sister taxa. The parsimony analysis also placed Haplomylomys with

the leucopus and maniculatus species groups (bootstrap/Bremer = 85%/2).

Osgoodomys was placed basal to the genus Peromyscus (clade VII) in both the

Bayesian and maximum likelihood analyses. In both instances, Osgoodomys was

positioned outside the genus Peromyscus, however, support for this arrangement in the

Bayesian analysis is poor (posterior probability = 57%), thus including Osgoodomys in

20

the clade with members of the genus Peromyscus. The parsimony analysis placed

Osgoodomys between Megadontomys and Neotomodon as separate genera and included

them in a clade containing members of the genus Peromyscus.

All of Carleton’s (1980) elevated genera (Habromys, Megadontomys,

Osgoodomys, Podomys, and Neotomodon) were included within clades containing

members of the genus Peromyscus based on evidence provided by Adh1-I2 data. All of

the accepted species groups remained intact and almost all were supported. The

exception to this was the boylii-aztecus assemblage where many of the internal nodes

collapsed forming the larger clade seen in all 3 topologies. Support for the remaining

species groups, however, may be an artifact of limited sample sizes.

Combined analysis.---Concatenating Adh1-I2 and Cytb data sets produced the

most resolved phylogeny when subjected to Bayesian analysis (Fig. 2.4). In this

combined analysis, support was provided for all of the recognized species groups with the

exception of the truei group. Again, this may be an artifact of limited sample sizes. As

in the maximum likelihood and Bayesian analyses of the Adh1-I2 data, the species groups

were monophyletic. Bayesian analysis of the combined data resulted in 7 clades (I--VII)

with clades II, III, and VI further divided into subclades. Support at the mid-level nodes

was improved by the addition of the Cytb data; however, the combined data still

produced nodes that collapsed due to insufficient support.

Similar to the analyses of Adh1-I2 data, Megadontomys was included in a clade

containing members of the genus Peromyscus. Initially, this genus was depicted as the

sole member of clade I using the combined data. This arrangement was not supported

however (posterior probability = 48%), and the clade collapsed forming the 1st of 4 major

21

groups. Maximum likelihood and Bayesian analysis showed Megadontomys sister to

Habromys using only Adh1-I2 data, but that relationship was not supported either

(posterior probability = 25%). Therefore, the only conclusion that could be extrapolated

from the analysis of the combined data, as well as the Adh1-I2 data, was that

Megadontomys was included in an unresolved polytomy with members of the genus

Peromyscus. Megadontomys traditionally has been aligned with Isthmomys (Carleton

1980) but due to the lack of an Isthmomys sample, Carleton’s (1980) alignment of

Megadontomys and Isthmomys could not be tested using Adh1-I2. Including

Megadontomys in the genus Peromyscus does, however, agree with the findings of

Rogers et al. (1983) who included Megadontomys in a clade with Neotomodon, Podomys,

and some members of the subgenus Peromyscus. Rogers et al. (1984) later included

Megadontomys in the genus Peromyscus based on karyological data.

Neotomodon was grouped with members of the melanophrys, megalops, and

mexicanus groups (clade II A) with support (posterior probability = 97%) indicating a

close relationship. These results differed from those of Carleton (1980), who reported

Neotomodon consistently uniting with Habromys and Podomys. Stangl and Baker (1984)

however, placed Neotomodon with the truei group, which agrees somewhat with the

topology produced by the combined data and with the topology produced using only

Adh1-I2 data. Yates et al. (1979) and Stangl and Baker (1984) proposed the inclusion of

Neotomodon in the genus Peromyscus and Bradley et al. (2004a) included Neotomodon

in Peromyscus based on mitochondrial Cytb sequence data. Both the Adh1-I2 and the

combined data in this study support the inclusion of Neotomodon in the genus

Peromyscus.

22

The relationships among the species groups in clade II A were supported except

for the sister relationship of the megalops group to the melanophrys group (posterior

probability = 90%). The addition of the Cytb data depicted the mexicanus group as sister

to the melanophrys and megalops groups (posterior probability = 97%) whereas it was

placed sister to pectoralis using only Adh1-I2 data and not supported (posterior

probability = 35%). This arrangement is indicative of a close relationship between these

species groups. Stangl and Baker (1984) placed the melanophrys group with 6 species of

the mexicanus group using karyological data, but their arrangement also included P.

pectoralis.

The relationship of the members of clade II A and members of the truei species

group (clade II B) was not supported (posterior probability = 91%). The sister

relationship between P. difficilis and P. attwateri was supported (posterior probability =

100%), but not their relationship to P. gratus (posterior probability = 88%) differing from

the analyses using only Adh1-I2 data, where the truei group was supported (posterior

probability = 96%, bootstrap/Bremer = 86%/2). This was the only species group not

supported and is suggestive of a division within the truei species group. This division was

in agreement with Tiemann-Boege et al. (2000) in their recognition of a difficilis

assemblage and a truei assemblage within the truei species group. Durish et al. (2004)

also identified a subdivision between a gratus and truei assemblage and a difficilis,

attwateri, and nasutus assemblage, and suggested that the latter assemblage include P.

pectoralis.

Members of the boylii group (clade III C) were again placed with members of

the aztecus group (clade III D). The sister relationship between the boylii and the aztecus

23

groups remained constant throughout all analyses and was supported in the combined

analysis (posterior probability = 100%). This suggests a close relationship between these

2 groups and clade probability values for each group (posterior probability = 100%)

support Carleton’s (1989) recognition of the aztecus species group as distinct from the

boylii species group. Tiemann-Boege et al. (2000) reported an average sequence

divergence for Cytb between the boylii and aztecus groups of 11.1%, whereas the

sequence divergence between boylii and truei was 13.2% and aztecus and truei was

13.6%, also indicating a close relationship between boylii and aztecus. The relationships

within these groups also were supported with the exception of the placement of P.

schmidlyi sister to a clade containing P. beatae and P. levipes (posterior probability =

88%).

Similar to the analysis of just the Adh1-I2 marker, the analysis of the combined

data places P. pectoralis (clade IV) outside of Neotomodon, the melanophrys, megalops,

mexicanus, truei, boylii, and aztecus species groups. Even though it was not supported

(posterior probability = 84%), a pattern has emerged throughout this investigation

suggesting pectoralis may not be as closely associated with the truei group as once

thought (Durish et al. 2004; Tiemann-Boege et al. 2000) and may be distinct enough to

justify the designation of its own species group. Support for such a designation has not

been provided to date but further investigation of this problematic genus is certainly

warranted. Because the support for clade IV was lacking, it collapsed into clade V, and

with clades II and III, would form the 2nd major group.

Habromys (clade V) was placed basal to P. pectoralis and the rest of the 2nd major

group with support (posterior probability = 98%). Bayesian and maximum likelihood

24

analyses of only Adh1-I2 data grouped Habromys with Megadontomys with no support

(posterior probability = 25%). Analyses of the combined data clearly placed Habromys

well within the boundaries of the genus Peromyscus. Carleton (1980) grouped Habromys

with Neotomodon as well as Podomys, and in a study by Rogers et al. (1984), Habromys

was placed sister to Neotomodon.

The combined analysis grouped Osgoodomys (clade VI E) with the californicus,

eremicus, leucopus, and maniculatus groups (clade VI F) with support (posterior

probability = 98%). Similar to Habromys, Osgoodomys was placed within the genus

Peromyscus based on the analysis of the combined data. Maximum likelihood and

Bayesian analyses of the Adh1-I2 data placed Osgoodomys basal to the genus

Peromyscus, whereas parsimony analysis of Adh1-I2 placed Osgoodomys between

Megadontomys and Neotomodon in a clade containing members of the genus

Peromyscus. These results disagree with the morphological data supporting the

distinctness of Osgoodomys (Carleton 1980; Hooper 1958; Hooper and Musser 1964;

Linzey and Layne 1969). Karyological data (Rogers et al. 1984; Stangl and Baker 1984)

do not support or refute Carleton’s (1980) elevation of Osgoodomys to generic status,

however, Schmidly et al. (1985) found that Osgoodomys clustered with the mexicanus

group based on biochemical evidence.

Another consistency among all of the analyses was the sister relationship between

the leucopus and maniculatus groups and their association with the californicus and

eremicus groups. Similar to the Adh1-I2 data, the combined Bayesian analysis showed

support for this arrangement (posterior probability = 100%). In addition, the clades

25

representing the subgenus Haplomylomys (posterior probability = 97%) and the leucopus

and maniculatus groups (posterior probability = 100%) were supported.

The crinitus group (clade VII) was placed basal to the genus, but support for this

arrangement was lacking (posterior probability = 81%) and inferences could not be made.

The crinitus group consistently was associated with Haplomylomys and the leucopus and

maniculatus groups in all analyses. It also has been grouped with Podomys, but a

nucleotide sequence for Cytb was unavailable preventing a test of this relationship with

the combined data. Osgood (1909) initially favored the allocation of crinitus to the

subgenus Haplomylomys but subsequent morphological studies retained it within the

subgenus Peromyscus (Hooper 1958; Linzey and Layne 1969). Zimmerman et al. (1978)

and Schmidly et al. (1985) aligned crinitus with members of the maniculatus group using

allozyme data. Greenbaum and Baker (1978) reported crinitus possessing chromosomal

banding identical to that of the ancestral condition. The placement of the crinitus group

in the combined analysis is in agreement with previous treatments of this species group

but the poor nodal support does not allow for any substantial inferences to be made at this

time. Because of the lack of support for the node forming clades VI and VII it collapsed

and formed of the 3rd major group containing Osgoodomys, Haplomylomys and the

leucopus and maniculatus groups, and the 4th major group containing only P. crinitus.

Concatenating Adh1-I2 and Cytb sequences clearly provided the best resolution

and support concerning the placement of Neotomodon, Habromys, and Osgoodomys.

Neotomodon was grouped with the melanophrys, megalops, and mexicanus complex and

was supported as being contained within the genus Peromyscus using the combined data.

Parsimony, maximum likelihood, and Bayesian analyses of Adh1-I2 also supported the

26

inclusion of Neotomodon in the genus Peromyscus. Habromys was supported as being

contained within the genus based on the evidence provided by the combined data. At the

very least, it was contained in a clade with other members of Peromyscus. Osgoodomys

was also contained within the boundaries of the genus Peromyscus based on the

combined data. Bayesian analysis of Adh1-I2 showed Podomys contained within the

genus Peromyscus, but the lack of informative characters would not allow for inferences

to be made concerning the placement of Podomys.

All 4 genera (Megadontomys, Habromys, Neotomodon, and Osgoodomys) were

included in a clade containing members of the genus Peromyscus with support (posterior

probability = 100%), however, support for the exact placement of Megadontomys within

the genus waned and inferences concerning relationships between this genus and other

members of the genus Peromyscus could not be made with any degree of certainty.

Genetic Distances.---Mean genetic divergences obtained from pairwise

comparisons of Adh1-I2 sequence divergence (Table 3.1) values indicate that

Megadontomys, Habromys, Neotomodon, Podomys, and Osgoodomys are more closely

associated with Peromyscus than Reithrodontomys, believed to be the closest relative of

the genus Peromyscus (Carleton 1980). Average divergence values ranged from 2.64%

(Osgoodomys versus Peromyscus) to 4.61% (Podomys versus Peromyscus), whereas

Reithrodontomys versus Peromyscus was 7.85% divergent. Comparisons with Cytb also

indicate a closer relationship between the 5 genera and Peromyscus than

Reithrodontomys and Peromyscus. Values for Cytb ranged from 13.26% (Neotomodon

versus Peromyscus) to 14.96% (Osgoodomys versus Peromyscus) compared to 17.23%

between Reithrodontomys and Peromyscus. Comparisons of Adh1-I2 sequence

27

divergence values were made between Megadontomys, Habromys, Neotomodon,

Podomys, and Osgoodomys and selected species within the genus Peromyscus. The

average sequence divergence ranged from 1.61% (Osgoodomys versus P. boylii) to

7.10% (Podomys versus P. maniculatus). Comparable values for Cytb ranged from

13.02% (Megadontomys versus P. boylii) to 15.10% (Osgoodomys versus P.

maniculatus). Comparisons of average sequence divergences for Adh1-I2 between

Reithrodontomys, P. boylii, and P. maniculatus were 7.10% and 9.30%, respectively.

Similar comparisons for Cytb were 16.87% and 16.56%, respectively. Based on average

genetic distance values, Megadontomys, Habromys, Neotomodon, Podomys, and

Osgoodomys are clearly more similar to Peromyscus than Reithrodontomys is to

Peromyscus.

Taxonomic Conclusions.---Despite the morphological evidence presented by

Carleton (1980), retaining the generic status of Neotomodon, Habromys, and

Osgoodomys, would require the elevation of several more species or species groups to

generic status. For example, if Neotomodon were to retain its generic rank, evidence

provided by the combined data analysis would necessitate the distinction of the

melanophrys, megalops, mexicanus, and truei groups as separate genera. In addition, the

boylii and aztecus groups would also constitute a separate genus. This also is true for

Habromys. Because of its basal position in the clade containing Neotomodon, and the

melanophrys, megalops, mexicanus, truei, boylii, and aztecus groups, everything outside

of that arrangement would constitute a separate genus including the leucopus,

maniculatus, and crinitus groups. The retention of the generic rank of Osgoodomys

would necessitate the elevation to generic rank of members of Haplomylomys, the

28

leucopus, maniculatus, and crinitus groups. Megadontomys and Podomys were not

supported as being contained within the genus Peromyscus, however, the average

sequence divergence between these 2 genera and the genus Peromyscus suggest they are

more similar to species groups within Peromyscus. Therefore, based on the molecular

evidence presented here, it seems best to treat Megadontomys, Habromys, Neotomodon,

Podomys, and Osgoodomys as subgenera within the genus Peromyscus.

Although Adh1-I2 provided some indication of the nature of the relationships

between the 5 genera in question and the genus Peromyscus, there were too few

characters to provide resolution at the mid-level nodes, thus a clear representation of the

relationships between those individuals contained within the boundaries of the genus was

not obtained. The addition of the Cytb data bolstered the Adh1-I2 data and provided

support for the inclusion of Neotomodon, Habromys, and Osgoodomys in the genus

Peromyscus. However, the combined data were still unable to provide a fine scale

resolution and therefore, unable to provide definitive information concerning

relationships within the boundaries of the genus.

29

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Table 2.1. Average Kimura 2-parameter genetic distances for selected comparisons of taxa examined in this study. Genetic distances are based on sequences from the 2nd intron of the nuclear alcohol dehydrogenase (Adh1-I2) and mitochondrial cytochrome b (Cytb) genes. ------------------------------------------------------------------------------------------------------------ Taxon Adh1-I2 Cytb ------------------------------------------------------------------------------------------------------------ R. fulvescens versus Megadontomys 0.07165 0.16707 R. fulvescens versus Osgoodomys 0.06119 0.17209 R. fulvescens versus Neotomodon 0.08308 0.16406 R. fulvescens versus Podomys 0.08353 0.18386 R. fulvescens versus Habromys 0.07496 0.16707 Megadontomys versus Osgoodomys 0.01999 0.15152 Megadontomys versus Neotomodon 0.03843 0.14319 Megadontomys versus Podomys 0.03871 ---------- Megadontomys versus Habromys 0.0255 0.16002 Osgoodomys versus Neotomodon 0.03069 0.15386 Osgoodomys versus Podomys 0.03276 ---------- Osgoodomys versus Habromys 0.02158 0.14984 Neotomodon versus Podomys 0.04183 ---------- Neotomodon versus Habromys 0.03249 0.12877 Podomys versus Habromys 0.04025 ---------- B. taylori versus Peromyscus 0.10958 0.17197 H. alleni versus Peromyscus 0.08399 0.20712 R. fulvescens versus Peromyscus 0.07847 0.17232 Megadontomys versus Peromyscus 0.03341 0.13721 Neotomodon versus Peromyscus 0.03396 0.13264 Podomys versus Peromyscus 0.04606 ---------- Osgoodomys versus Peromyscus 0.02636 0.14956 Habromys versus Peromyscus 0.03687 0.13871 Megadontomys versus P. boylii 0.02362 0.13025 Megadontomys versus P. maniculatus 0.06146 0.13935 Osgoodomys versus P. boylii 0.01612 0.13948 Osgoodomys versus P. maniculatus 0.05714 0.15094 Neotomodon versus P. gratus 0.02330 0.12588 Neotomodon versus P. maniculatus 0.06277 0.14224 Podomys versus P. boylii 0.03839 ---------- Podomys versus P. maniculatus 0.07091 ---------- Habromys versus P. boylii 0.02341 0.13336 Habromys versus P. maniculatus 0.06097 0.14689 Reithrodontomys versus P. boylii 0.07102 0.16870 Reithrodontomys versus P. maniculatus 0.09290 0.16558

37

Figure 2.1. Strict consensus tree representing 132 equally parsimonious trees obtained from a parsimony analysis of unweighted DNA sequence characters from

Adh1-I2. Numbers above branches indicate bootstrap values and numbers below branches refer to Bremer support values.

Baiomys taylori

Hodomys alleni

Reithrodontomys fulvescens

Megadontomys thomasi

Osgoodomys banderanus


Neotomodon alstoni

P. californicus

P. eremicus

P. leucopus

P. leucopus

P. maniculatus

P. crinitus

Podomys floridanus

P. perfulvus

P. melanophrys

P. megalops

P. gratus

P. difficilis

P. attwateri

P. pectoralis

P. beatae

P. levipes

P. sp

P. beatae

P. boylii

P. boylii

P. boylii

P. schmidlyi

P. schmidlyi

P. spicilegus

P. spicilegus

P. spicilegus

P. hylocetes

P. mexicanus

P. nudipes

P. nudipes

Habromys lepturus

57

81

93

85

68

96100

60 94

86 86

90

87

63

6363

100mexicanus group

aztecus group

boylii group

truei group

megalops group

melanophrys group

crinitus group maniculatus group

leucopus group

Haplomylomys

8

11

12

2

4

22

1 3

2

1

2

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Figure 2.2. Adh1-I2 phylogeny generated using maximum likelihood with the HKY+G model of evolution. Roman numerals refer to major clades and letters refer to subclades as identified in the text.

Baiomys

Hodomys

Reithrodontomys

Megadontomys

Habromys

Neotomodon

P. perfulvus

P. melanophrys

P. megalops

P. gratus

P. difficilis

P. attwateri

P. beatae

P. levipes

P. sp.

P. beatae P. boylii

P. boylii

P. boylii

P. schmidlyi

P. schmidlyi

P. spicilegus

P. spicilegus

P. spicilegus

P. hylocetes

P. pectoralis

P. mexicanus

P. nudipes

P. nudipes

P. californicus

P. eremicus

P. leucopus

P. leucopus

P. maniculatus

P. crinitus

Podomys floridanus



0.005 substitutions/site

I

II

III

IV

V

VI

VII

A

B

C

D

melanophrys group

truei group

boylii group

mexicanus group

megalops group

aztecus group

Haplomylomys

leucopus group

maniculatus group

crinitus group

39

Figure 2.3. Adh1-I2 phylogeny generated using Bayesian methods and the GTR+I+G model of evolution. Roman numerals indicate major clades and letters indicate subclades as discussed in the text. Clade probability values are shown above branches.

Baiomys taylori

Hodomys alleni


P. californicus

P. eremicus

P. leucopus

P. leucopus


Osgoodomys banderanus 5 changes

45

25

6

melanophrys group

megalops group

36

53

57


Habromys lepturus

P. beatae

P. pectoralis

P. nudipes

Neotomodon alstoni

P. perfulvus

P. melanophrys

P. megalops

P. gratus

P. difficilis

P. attwateri

96

truei group 39

83

100

96

100

100 P. beatae P. sp

P. levipes

P. boylii

P. hylocetes

P. boylii

P. boylii

P. schmidlyi

P. schmidlyi

P. spicilegus

P. spicilegus

P. spicilegus

P. crinitus

Podomys floridanus

11

P. mexicanus

P. nudipes

100

100

100

100 100

73

14

100 34

7

9

96

93

97

8

1

3

10

100

boylii group

mexicanus group

crinitus group

Haplomylomys

P. maniculatus

leucopus group

maniculatus group

I

II

A

B

III

IV

V

VI C

D

VII

aztecus group

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Figure 2.4. Adh1-I2---Cytb phylogeny generated using Bayesian methods and the GTR+I+G model of evolution. Roman numerals indicate major clades and letters indicate subclades as discussed in the text. Clade probability values are shown above branches.

Baiomys taylori

Hodomys alleni



Neotomodon alstoni

P. perfulvus

P. melanophrys

P. megalops

P. mexicanus

P. nudipes

P. gratus

P. difficilis

P. attwateri

P. beatae

P. levipes

P. schmidlyi

P. boylii

P. boylii

P. spicilegus

P. hylocetes

P. pectoralis

Habromys lepturus


P. californicus

P. eremicus

P. leucopus

P. maniculatus

P. crinitus 50 changes

86

100

48

98

84

96

91

97

97

90

100

100

88

100

100 88

100

100 100

100

81

98

100

97

100

melanophrys group

megalops group

mexicanus group

truei group

boylii group

aztecus group

Haplomylomys

leucopus group

maniculatus group

crinitus group

I

II

A

B

E

D

C

F

III

IV

V

VI

VII

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CHAPTER III

CONCLUSIONS

The genus Peromyscus has undergone 3 major revisions beginning with Osgood

(1909), followed by Hooper (1968) and Carleton (1989). One of the most interesting

controversies of these revisions involves the phylogenetic and taxonomic status of

Megadontomys, Habromys, Neotomodon, Podomys, and Osgoodomys with regard to

Peromyscus. Once designated as subgenera within the genus Peromyscus (Osgood 1909;

Hooper and Musser 1964), these 5 genera were elevated to generic status by Carleton

(1980, 1989). In this his dissertation, I described the utilization of the 2nd intron of the

nuclear alcohol dehydrogenase gene (Adh1-I2) to examine the boundaries of the genus

Peromyscus as defined by Carleton (1989), and to evaluate the taxonomic placement of

Megadontomys, Habromys, Neotomodon, Podomys, and Osgoodomys with regards to

Peromyscus (sensu stricto). The Adh1-I2 marker also was used to examine the

relationships between the species groups contained within Peromyscus.

Three phylogenies were estimated using parsimony, maximum likelihood, and

Bayesian analyses (Huelsenbeck and Ronquist 2001). Due to the relatively small number

of informative characters, 76 of 598 available bases, mid-level nodes collapsed and none

of the aforementioned analyses were able to produce a completely resolved phylogeny.

Thus, relationships between the species groups were not elucidated. All 5 of the genera

in question were consistently contained in large, unresolved clades with members of the

genus Peromyscus suggesting more affinity with Peromyscus than not based on Adh1-I2

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sequence data. The lack of support associated with the Bayesian analysis, however,

would not allow for any specific inferences concerning the taxonomic placement of

Megadontomys, Habromys, Neotomodon, Podomys, and Osgoodomys based solely on the

analysis of Adh1-I2 sequence data.

Concatenating Adh1-I2 and mitochondrial cytochrome b (Cytb) sequence data

also was unable to provide fine-scale resolution; information concerning the relationships

of species groups within Peromyscus was not obtained. Combining the data did,

however, provide support for the inclusion of Habromys, Neotomodon, and Osgoodomys

in the genus Peromyscus when analyzed using Bayesian methods (Huelsenbeck and

Ronquist 2001). Again, Megadontomys was contained within an unresolved clade with

members of the genus Peromyscus but insufficient support would not allow any specific

inference concerning its taxonomic placement. A Cytb sequence was unavailable for

Podomys preventing the examination of its taxonomic placement with regard to

Peromyscus based on Adh1-I2--- Cytb sequence data.

As a result of this study, it has become clear that although Adh1-I2 provided some

indication of the nature of the relationships between the 5 genera in question and the

genus Peromyscus, there were too few characters to provide resolution at the mid-level

nodes. Therefore, a clear representation of the relationships between those individuals

contained within the boundaries of the genus was not obtained. For future studies,

inclusion of 1 or more additional Adh1 introns may add to the number of informative

characters and provide the necessary resolution required to make inferences concerning

relationships between species groups. Also, utilizing other nuclear markers with rates of

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evolution approaching half of that found in Cytb may prove informative when combined

with Adh1-I2 and Cytb.

The addition of the Cytb data bolstered the Adh1-I2 data and provided support for

the inclusion of Neotomodon, Habromys, and Osgoodomys in the genus Peromyscus.

However, the combined data were still unable to provide a fine scale resolution and

therefore, unable to provide definitive information concerning relationships within the

boundaries of the genus. Again, the inclusion of additional Adh1 introns or other nuclear

markers may increase the resolution at the deeper nodes.

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LITERATURE CITED


Carleton, M. D. 1989. Systematics and evolution. Pp. 7-141 in Advances in the study of Peromyscus (Rodentia) (G. L. Kirkland, Jr. and J. N. Layne, eds.). Texas Tech

University Press, Lubbock, 366 pp.



Huelsenbeck, J. P., and F. Ronquist. 2001. Mr. Bayes: Bayesian inference of phylogeny. Bioinformatics 17:754--755.


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APPENDIX

LOCALITIES OF SPECIMENS EXAMINED; PEROMYSCUS ARE LISTED BY

SPECIES GROUP.

The following is a list of localities for samples examined in this study. Specimens

examined are listed by species group and type of sequence (Adh1-I2 or Cytb). Species

names are followed by locality. Museum or collector number (Duke S. Rogers—DSR,

Ira F. Greenbaum—GK, and Texas Tech University tissue number—TK) and the

GenBank accession number are provided in parentheses. Published sequences used in

this study do not include a locality and are followed by their citation.

Adh1-I2

Baiomys taylori.— MEXICO: Jalisco; 2 Km NW Mesconcitos, (TK 93084, AY994205).

Hodomys alleni.— (TK45042, AY817627) (Longhofer and Bradley In prep).

Reithrodontomys fulvescens.—USA:Oklahoma; MiCintosh Co, 4.9 km E Dustin,

(TK23469, AY994207). Megadontomys thomasi.— MEXICO: Guerrero; 6.4 km SSW

Filo de Caballo (TK93388, AY994208). Habromys lepturus.— MEXICO: Oaxaca; 2.25

km N Llano de Las Flores (TK93160, AY994239). Neotomadon alstoni.— MEXICO:

Michoacán; Ladera W Cerro del Burro (TK45302, AY994210). Podomys floridanus.—

USA: Florida; Columbia Co, O’leno State Park (TK92502, AY994214). Osgoodomys

banderanus.— MEXICO: Michoacán; Coalcoman, Tehuanepec, 17.5 km WSW

Coalcoman (Sample 1, TK 45952, AF 994209). MEXICO: Michoacán; Villa Victoria,

6.3 km SSW Villa Victoria (Sample 2 TK45964, AY994231). Peromyscus pectoralis.—

MEXICO: Jalisco; 30 km W Huejuquilla el Alto (TK48645, AY994221). melanophrys

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group.— Peromyscus perfulvus, MEXICO: Michoacán; Tunel de Riego, 2 km E Cerro

Colorado (TK47926, AY994215); P. melanophrys, MEXICO: Durango; 2.2 km S, 2.5

km E Vicente Guerrero (TK448848, AY994216). megalops group.— Peromyscus

megalops, MEXICO: Guerrero; 6.4 km SSW Filo de Caballo (TK93375, AY994217).

truei group.— Peromyscus gratus, MEXICO: Michoacán; Aguililla, Zona Arqueologica

Tres Cerritos, 4 km E Cuitzeo (TK46354, AY994218); Peromyscus difficilis, MEXICO:

Tlaxcala; 2 km NE Tepetitla (TK93120, AY994219); Peromyscus attwateri, USA:

Oklahoma; McIntosh Co, 4.9 km E Dustin (TK23396, AY994220). boylii group.—

Peromyscus beatae, MEXICO: Veracruz; Xometla (Sample 1, GK3954, AY994222);

MEXICO: Chiapas; Yalentay (Sample 2, TK93279, AY994223); Peromyscus levipes,

MEXICO: Michoacán; Las Minas, 3km SW Tuxpan (TK47819, AY994224);

Peromyscus species, MEXICO: Michoacán; km 81 Carr Ario de Rosales-la Huacana

(TK47890, AY994230); Peromyscus boylii, USA: California: San Diego Co, Heise

County Park (Sample 1, TK90233, AY994225); USA: Utah; Washington Co, Bever Dam

Wash (Sample 2, TK24389, AY994226); MEXICO: Jalisco; 2 km NW Mesconcitos

(Sample 3, TK93089, AY994227); Peromyscus schmidlyi, MEXICO: Durango; 6.1 km

W Coyotes, Hacienda Coyotes, (Sample 1, TK72443, AY994228), MEXICO: Durango;

San Juan de Camarones (Sample 2, TK70812, AY994229). aztecus group.—

Peromyscus spicilegus, MEXICO: Michoacán; km 81 Carr Ario de Rosales-la Huacana

(Sample 1, TK47888, AY994232), MEXICO: Michoacán; Aguililla, 5 km E Dos Aguas

(Sample 2, TK45255, AY994234), MEXICO: Michoacán; Aguililla, Dos Aguas (Sample

3, TK45262, AY994233), Peromyscus hylocetes, MEXICO: Michoacán; Estacion

Microodnas, Cerro Burro (TK45309, AY994235). mexicanus group.— Peromyscus

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mexicanus, MEXICO : Chiapas; 14.48 km N. Ocozocoaulta (TK93314, AY994236);

Peromyscus nudipes, NICARAGUA: Selva Negra; Cody Trail (Sample 1 TK113541,

AY994237); NICARAGUA, Los Mongos (Sample 2 TK93600, AY994238).

californicus group.— Peromyscus californicus, USA: California; Los Angeles Co,

Chatsworth Reservoir Park (TK93600, AY994211). eremicus group. — Peromyscus

eremicus, USA: California; Los Angeles Co, Kanan Dome Rd. (TK90458, AY994212).

leucopus group.— Peromyscus leucopus, USA: Texas; Presidio Co, Las Palomas WMA

(Sample 1 TK54008, AY994240); USA: Texas; La Salle Co, Chaparral Wildlife

Management Area (Sample 2 TK112167, AY994241). maniculatus group.—

Peromyscus maniculatus, USA: Arkansas; Mississippi Co, Dillahunty Pecan Orchard

(TK90458, AY994242). crinitus group.— Peromyscus crinitus, USA: Utah; Vintah Co,

Cottonwood Canyon (DSR6171, AY994213).

Cytb

Baiomys taylori.—(TK 93084, AF548471) (Amman and Bradley 2004). Hodomys

alleni.—(TK45042, AF186801) (Edwards and Bradley 2001). Reithrodontomys

fulvecens.—(TK23469, AF176257) (Bell et al. 2001). Megadontomys thomasi.—

MEXICO: Guerrero; 6.4 km SSW Filo de Caballo (TK93388, AY195795). Habromys

lepturus.— MEXICO: Oaxaca; 2.25 km N Llano de Las Flores (TK93160, DQ000482).

Neotomadon alstoni.— (TK45302, AY195796) (Bradley et al. 2004). Osgoodomys

banderanus. — MEXICO: Michoacán; Coalcoman, Tehuanepec, 17.5 km WSW

Coalcoman (Sample 1, TK 45952, DQ000473). Peromyscus pectoralis.— MEXICO:

Jalisco; 30 km W Huejuquilla el Alto (TK48644, DQ000476). melanophrys group.—

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Peromyscus perfulvus, MEXICO: Michoacán; Tunel de Riego, 2 km E Cerro Colorado

(TK47926, DQ000474); Peromyscus melanophrys, MEXICO: Durango; 2.2 km S, 2.5

km E Vicente Guerrero (TK448848, AY322510). megalops group.— Peromyscus

megalops, MEXICO: Guerrero; 6.4 km SSW Filo de Caballo (TK93375, DQ000475).

truei group.— Peromyscus gratus, (TK46354, AY376421) (Durish et al. 2004);

Peromyscus difficilis, (TK93120, AY376416); (Durish et al. 2004); Peromyscus

attwateri, (TK23396, AF155384) (Durish et al. 2004). boylii group.— Peromyscus

beatae, MEXICO: Veracruz; Xometla (Sample 1, GK3954, AF131921); Peromyscus

levipes, MEXICO: Michoacán; Las Minas, 3km SW Tuxpan (TK47819, DQ000477);

Peromyscus boylii, USA: Utah; Washington Co, Bever Dam Wash (Sample 2, TK24389,

DQ000478); (Sample 3, TK93089, AY322504) (Bradley et al. 2004b); Peromyscus

schmidlyi, (Sample 1, TK72443, AY370610) (Bradley et al. 2004b). aztecus group.—

Peromyscus spicilegus, MEXICO: Michoacán; km 81 Carr Ario de Rosales-la Huacana

(Sample 1, TK47888, DQ000480); Peromyscus hylocetes, MEXICO: Michoacán;

Estacion Microodnas, Cerro Burro (TK45309, DQ000481); mexicanus group.—

Peromyscus mexicanus, (TK93314, AY376425) (Durish et al. 2004); Peromyscus

nudipes, (Sample 1 GenBank no. AY041200) (Rinehart,T.A., Grahn,R.A. and

Wichman,H.A. unpublished data). californicus group.—Peromyscus californicus, (TK

83632, AF155393) (Tiemann-Boege et al. 2000). eremicus group.—Peromyscus

eremicus, (TK91184, AY322503) (Durish et al. 2004). leucopus group.—Peromyscus

leucopus,USA: Texas; La Salle Co, Chaparral Wildlife Management Area (Sample 2

TK112168, DQ000483). maniculatus group.—Peromyscus maniculatus, USA AR:

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Mississippi Co; Dillahunty Pecan Orchard (TK11695, DQ000484). crinitus group.—

Peromyscus crinitus, (DSR6171, AY376413) (Durish et al. 2004).

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