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1997 Relative Rates of Molecular Evolution in Rodents and Their yS mbionts. Theresa Ann Spradling Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Spradling, Theresa Ann, "Relative Rates of Molecular Evolution in Rodents and Their yS mbionts." (1997). LSU Historical Dissertations and Theses. 6527. https://digitalcommons.lsu.edu/gradschool_disstheses/6527

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RELATIVE RATES OF MOLECULAR EVOLUTION IN RODENTS AND THEIR SYMBIONTS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Zoology and Physiology

ly Theresa Ann Spradling B.S., Emporia State University, 1988 M.S., University of North Texas, 1991 August 1997

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Acknowledgments

Many people have contributed to my graduate studies through their extreme generosity with their time and knowledge. I thank my major professor, Dr. Mark Hafner, for his patience and hard work in teaching me more about the process of science. As a graduate student of Dr. Hafner's, I never felt like a second-tier priority. My past and present committee members (Drs. C. F. Bryan, D. A. Good, D. Prowell, J. V. Remsen, F. H. Sheldon, R. M. Zink) also deserve special thanks. Dr. P. D. Sudman was instrumental in helping me get these research projects started; his advice and tutoring are greatly appreciated. Dr. D. W. Foltz also contributed to this research through discussions of population genetics and rates of evolution. Dr. X. Xia modified the Codrates program for use with mitochondrial DNA and assisted with data analysis. Dr. D. A. Good made available the MacSequence program and a program for calculating probabilities based on the binomial equation. People in the Museum of Natural Science and in the Systematics and Evolution group have helped in ways too numerous to mention. I especially thank B. S. Arbogast, A. L. Bass, J. M. Bates, P. O. Dunn, S. J. Hackett, C. A. Lehn, S. Meiers, D. A. McClellan, D. L. Reed, and L. A. Whittingham. Dr. J. W. Demastes was helpful in nearly every aspect of this project from beginning to end, assisting in specimen collection, providing advice on data analyses, and providing helpful criticisms of drafts of this manuscript. Several people at other institutions helped to make the research presented here possible. C. T. Beagley, It Okimoto, and D. R.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wolstenholme made available PCR primers for the amplification of mitochondrial DNA of both pocket gophers and chewing lice. B. J. Verts and L. N. Carraway took time to teach me how to catch the "big" gophers. Paul Sherman provided tissues of naked mole-rats from his collection, and the Museum of Southwestern Biology at the University of New Mexico provided tuco-tuco tissues. Drs. I. F. Greenbaum and E. Nevo made available tissues from the blind mole-rat. Finally, Dr. Roger Price was extremely helpful in confirming the identifications of many of the chewing lice included in this research. I appreciate financial support from the American Society of Mammalogists, the National Science Foundation Doctoral Dissertation Im provem ent Program (DEB-9321395), Sigma Xi, the NSF-Louisiana Education Quality Support Fund (ADP-02), the Theodore Roosevelt Memorial Fund of the American Museum of Natural History, and the Louisiana Board of Regents. Finally, I thank my parents, Cloyce and Bernadette Spradling, for their support. They fostered my earliest interest in rodents and genetics during my hamster-ranching days.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table of Contents

Acknowledgments ...... ii

List of Tables...... v i

List of Figures...... v ii

A bstract...... ix

C hapter 1 Introduction to the Study of Rates of Molecular E v olution ...... 1 TYPES OF DNA SUBSTITUTIONS...... 3 POSSIBLE CAUSES OF EVOLUTIONARY RATE HETEROGENEITY...... 4 RELATIVE RATES OF EVOLUTION Tests Based on the Fossil Record ...... 7 Tests Based on Cospedating Taxa ...... 9 Analyses Based on Relative-Rate Tests ...... 11 FOCUS OF THIS DISSERTATION...... 16

2 Phylogeny and Relative Rates of Evolution of R odents ...... 18 MATERIALS AND METHODS...... 22 RESULTS AND DISCUSSION Phylogenetic Utility of Cytochrome b ...... 26 Implications for the Evolutionary History of R odents ...... 34 Relative Evolutionary Rates Among Rodents 38 Selectionist and Neutralist Explanations of Rate Heterogeneity ...... 49 Absolute Rates of Evolution in Rodents ...... 61 CONCLUSIONS...... 63

3 Historical Assodations of Pocket Gophers and Chewing Lice...... 68 POCKET GOPHERS AND CHEWING LICE...... 71 SPECIFIC AIMS OF THIS STUDY...... 73 MATERIALS AND METHODS Specimens Examined ...... 74 DNA Isolation, Cloning, and Sequencing ...... 76

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Data Analysis ...... 77 RESULTS AND DISCUSSION Phylogenetic Relationships of Chewing Lice ...... 80 Phylogenetic Relationships of Pocket Gophers 86 Historical Associations Between Hosts and Parasites ...... 90 Relative Rates of Evolution Within Each G roup ...... 98 Do Chewing Lice Have a Higher Rate of E volution? ...... 99 CONCLUSIONS...... 108

Literature Cited ...... 110

Appendixes A ...... 127

B...... 128

C...... 148

D ...... 155

E...... 156

F ...... 160

G ...... 167

V ita...... 174

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2.1 Specimens for which cytochrome b sequences were examined. 20

2.2 Results of relative-rate tests comparing four pocket gopher genera to Mus and Rattus, w ith Oryctolagus as the outgroup 39

2.3 Results of relative-rate tests based on the full maximum- likelihood model ...... 40

2.4 Pairwise comparisons of relative rate of evolution based on the maximum-likelihood model ...... 41

2.5 Relative rates of evolution of nonsynonymous (above diagonal) and synonymous (below diagonal) substitutions ...... 44

2.6 Estimated basal metabolic rate, body mass, and rate of nonsynonymous substitution from cluster analysis ...... 52

2.7 Estimated average FST of rodents for which cytochrome b sequences were examined ...... 57

2.8 Estimated average generation time of females and frequency of reproduction ...... 60

2.9 Sequence divergence and rate of evolution per million years (my) for rodents for which divergence time can be estimated from fossil data ...... 62

3.1 Specimens examined of pocket gophers ( Thomomys) and chewing lice (Geomydoecus and Thomomydoecus) ...... 75

3.2 Branch lengths from maximum-likelihood trees based on COI nucleotide sequences for pocket gophers and chewing lice ...... 101

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. List of Figures

2.1 Strict consensus of six most-parsimonious trees (629 steps) based on cytochrome b amino-add sequences ...... 27

2.2 Results of maximum-likelihood analysis of nudeotide sequences when a 1.3:1.0 transitionrtransversion ratio is assum ed ...... 29

2.3 Results of parsimony analysis of nudeotide sequences based on an equal weighting of transitions and transversions ...... 31

2.4 Results of parsimony analysis ...... 32

2.5 UPGMA analysis of rate of nonsynonymous substitution ...... 47

3.1 Maximum-likelihood estimate of relationships among two genera of chewing lice, Geomydoecus and Thomomydoecus ...... 82

3.2 Results of parsimony analysis of COI sequences of two genera of chewing lice, Geomydoecus and Thomomydoecus ...... 83

3.3 Areas of disagreement between COI maximum-likelihood (a) and parsim ony (b) trees ...... 85

3.4 Results of maximum-likelihood (a) and parsimony (b) analysis of COI data from seven spedes of pocket gophers ( Thomomys ) ...88

3.5 Relationships of Thomomys and Geomydoecus indicated by maximum-likelihood analysis of COI sequence data ...... 93

3.6 Comparison of relationships of Thomomys (left) and Thomomydoecus (right) indicated by maximum-likelihood analysis of COI sequence data ...... 94

3.7 Comparison of relationships of Thomomys (left) and Thomomydoecus (right) indicated by parsimony analysis of gopher COI sequences and by parsimony and maximum- likelihood analysis of the louse COI sequences ...... 95

3.8 Reduced major-axis regression (through the origin) of pocket gopher and chewing louse maximum-likelihood branch lengths (data from Hafner et al., 1994) ...... 102

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.9 Plots of maximum-likelihood branch lengths (from Table 3.2) for pocket gophers and chewing lice ...... 104

3.10 Comparison of maximum-likelihood branch lengths (considering all substitutions; Table 3.2) of sister taxa of pocket gophers and chwing lice...... 106

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abstract

The focus of this dissertation is the relative tempo of molecular evolution among organisms of varying levels of relatedness. Because this is a rapidly expanding area of research, Chapter 1 is devoted to a review of the current state of knowledge on rates of molecular evolution. In Chapter 2,1 present an analysis of relative rates of evolution among several distantly related rodents. The heterogeneity in rate of evolution of the cytochrome b gene observed here cannot be explained by metabolic rate, body size, generation time, or nudeotide- generation time. Likewise, there is no evidence that these differences in rate of evolution are due to differential selection. The strongest potential correlate of rate of evolution in these data appears to be population subdivision, a factor long thought to influence heterozygosity of nuclear genes through genetic drift. However, a large number of variables, and complex interactions among variables, may influence rate of evolution of any particular gene; the relative importance of these variables probably differs from gene to gene, even within a species. Therefore, although differences in rate of molecular evolution exist, it likely will remain difficult to predict which species and which genes will show foster (or slower) rates because of the complex interactions among the numerous variables that influence rate of evolution. Cospedating taxa offer a powerful means to compare rates of evolution between groups of organisms. Congruence in the phylogenies of two symbiotic lineages likely indicates contemporaneous dadogenic events. In Chapter 3, the phylogenies of three symbiotic groups (pocket gophers and two genera of chewing lice) are compared to evaluate their

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. level of phylogenetic congruence, or cospedation. Although statistical tests indicate a history of widespread cospedation between these hosts and parasites, analysis of relative rates of evolution in these groups does not indicate the disparity in rates of evolution reported by Hafner et al. (1994).

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Introduction to the Study of Rates of M olecular Evolution

To gain a dear understanding of the process of evolution, one must understand the major factors that influence genetic change, induding positive natural selection (adaptation), purifying selection (removal of deleterious mutants from a population), genetic drift, and mutation. Each factor can play a role in the genetic and phenotypic differentiation that we observe among populations and spedes. These factors are represented in various general models of molecular evolution, including Kimura's neutral model of evolution (1968), Ohta's "nearly neutral" or "effectively neutral" model (Kimura and Ohta, 1974; Ohta, 1973,1974,1976), and several selection-based models (Gillespie, 1991, 1994). Traditionally, natural selection has been thought to be the primary evolutionary force molding small genetic changes (caused by mutation and recombination) into observed evolutionary phenomena. In contrast, the neutral theory of molecular evolution (Kimura, 1968) suggests that genetic drift is among the most powerful forces for evolutionary change. Debate concerning the relative influence of natural selection and neutral factors on phenotypic and molecular evolution is widespread in the literature (e. g., Gillespie, 1991,1994; Kimura, 1986). Arguments concerning the relative influence of different evolutionary forces frequently depend on presumed trends in the mode and tempo of molecular change (e. g., Gillespie, 1991; Kimura, 1987). For

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. example, over 30 years ago, observations on the extent of genetic divergence among taxa (Margoliash, 1963; Zuckerkandl and Pauling, 1965) led to the formulation of the molecular clock hypothesis, which suggests that orthologous molecules in different taxa evolve at a similar rate. This hypothesis is under almost constant scrutiny as more and more molecular data become available. Although molecular evolution often appears to proceed in a dock-like fashion, examples to the contrary are increasing in number. Understanding the biological cause or causes of these deviations is important in providing a more accurate view of the evolutionary role of genetic variation. Ideally, further comparative study of molecular data will allow a greater understanding of the causes and consequences of the changes that we observe in nudeotide and protein sequences. In addition, the validity of the molecular dock hypothesis has practical implications for the likelihood of recovering correct phylogenetic relationships from molecular data and for the accuracy of estimates of timing of biogeographical events based on levels of genetic divergence. Several approaches have been used to compare rates of molecular evolution within and among taxa. These approaches rely on comparing genetic data with some estimate of time. Estimates of time may be based on direct fossil evidence or on indirect, comparative estimates of time since divergence based on either cospedation studies or relative-rate tests. Each approach offers unique benefits and is constrained by unique limitations that will be outlined later in this chapter along with the insights to molecular evolution provided by studies that use each approach.

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TYPES OF DNA SUBSTITUTIONS Many studies described in the following sections were designed to treat separately the relative rate of synonymous, or silent, substitutions (nucleotide replacements that leave amino add sequence unaltered) and of nonsynonymous, or replacement, substitutions (those replacements that result in a change in amino add sequence). The implication of differences between rates in these two categories is not completely dear. Often, synonymous substitutions are considered to be selectively neutral, affected predominantly by mutation and drift, whereas nonsynonymous substitutions are considered to be exposed to natural selection (Gillespie, 1991; Ohta, 1995). Many nonsynonymous sites are probably under greater selective constraint than are most synonymous sites. However, many synonymous substitutions may not be completely free from selection (nudeotide composition at many of these sites appears not to be independent of nudeotide composition at neighboring, nonsynonymous sites; Myers et al., 1995), and many nonsynonymous substitutions may have little or no effect on relative fitness. Therefore, the two categories may represent a continuum of levels of selection. Because synonymous substitutions occur more frequently than nonsynonymous substitutions, these two categories may be useful for investigating evolutionary patterns among taxa with a range of divergence times. For example, among dosely related taxa, synonymous substitutions may have accumulated to a level that allows statistical comparison, whereas nonsynonymous substitutions may be so rare that statistical tests can provide no resolution of differences among taxa. On the other hand, synonymous sites are likely to become "saturated" after

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long periods of time, obliterating the history of substitutions as multiple changes occur at the same nucleotide position (Muse and Gaut, 1994). Saturation, then, would leave an appearance of rate homogeneity among taxa at synonymous sites that may have appeared as rate heterogeneity earlier in time. Therefore, heterogeneity in evolutionary rates may be most observable at synonymous sites in recently diverged taxa and at nonsynonymous sites in taxa that diverged long ago (Mindell et al., 1996; Muse and Gaut, 1994). POSSIBLE CAUSES OF EVOLUTIONARY RATE HETEROGENEITY A greater understanding of the selective advantage, disadvantage, or neutrality of genetic differentiation may be gained by taking a more detailed look at factors associated with observed differences in rate of evolution. For example, many studies seem to indicate that generation time is associated with rate of evolution, such that more rapidly reproducing organisms show a higher rate of evolution (Laird et al., 1969). This hypothesis suggests that rate of evolution is driven primarily by mutation rate, with the majority of mutations accumulating during DNA replications in the germ line (Li et al., 1987). Because purifying natural selection should eliminate most deleterious substitutions, it has been suggested that the "generation-time effect" may be more apparent in synonymous substitutions than in nonsynonymous substitutions (Li et al., 1987; Ohta, 1995). A more elaborate form of the generation-time hypothesis is the "nucleotide generation-time hypothesis" (Martin and Palumbi, 1993a), which implicates generation time, mass-specific resting metabolic rate, and body size as interrelated factors correlated with rate of silent

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. substitution. This explanation also invokes selective neutrality, in that each of these factors is thought to be correlated with the average amount of time that elapses between DNA replications and, therefore, the number of opportunities for mutations to occur in a given amount of time. Additionally, higher metabolic rate may contribute to a greater amount of oxidative damage to DNA (Adelman et al., 1988), thereby further increasing the rate of mutation in organisms with high metabolic rates. Differences in rate of silent substitution of sharks and mammals may be explained by differences in nucleotide generation time. Furthermore, these differences in silent substitution rate seem to be sufficient to explain observed differences in rate of amino add evolution in these taxa (Martin and Palumbi, 1993b). Therefore, it appears that, at least in some cases, differences in mutation rate may account for differences in rate of protein evolution without invoking differences in selective constraint (Martin and Palumbi, 1993b). "Nearly-neutral" theory (Ohta, 1973,1974) predicts that effective population size and generation time both influence rate of molecular evolution. This theory relies on the inverse relationship between these two factors; i.e., organisms that have long generation times generally have small effective population sizes (Chao and Carr, 1993). As noted above, shorter generation time seems to be assodated with a faster rate of evolution of synonymous substitutions. When effective population size is large, the power of purifying selection to remove slightly deleterious mutations is relatively stronger than it would be in a small population. Thus, mutations that are slightly deleterious, and that would be selected against in a large population, might be effectively

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neutral in a small population. These slightly deleterious mutations can then reach fixation within the population by genetic drift (Kimura, 1983; Ohta, 1973,1974,1976,1993), meaning that smaller average population size tends to promote a higher rate of evolution in a species. Proponents of nearly-neutral theory suggest that effective population size and generation time are factors that oppose each other, such that rate of evolution of nonsynonymous substitutions (those that are most likely to be exposed to selection) is constant on a per-year basis among taxa of different generation times (Qiao and Carr, 1993; Ohta, 1993,1995). Therefore, synonymous substitutions may be influenced by generation time to a greater degree than are nonsynonymous substitutions because natural selection has less influence on synonymous substitutions. In many instances, however, generation time and absolute time both seem to be poor predictors of rate of evolution. In some cases, dramatic exposure to mutagens seems to be enough to increase rate of evolution measurably (Baker et al., 1996). Perhaps exposure to natural environmental mutagens on a normal, but geographically variable, scale commonly influences rate of evolution? Other explanations of differing rates of evolution include the possibility of variation in the effectiveness of DNA replication and repair systems (Britten, 1986). Finally, substitutions at both synonymous and nonsynonymous sites may occur at different rates in different taxa due to different selective constraints imposed by different life-history or environmental factors (Gillespie, 1986). Relaxed selective constraints on the proteins of warm­ blooded vertebrates (because the proteins operate under constant environmental temperature) has been proposed as a possible

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. explanation for the higher rate of evolution of birds and mammals relative to fishes and amphibians (Adachi et al., 1993). RELATIVE RATES OF EVOLUTION Tests Based on the Fossil Record Direct estimation of evolutionary rates based on fossil evidence has the unique advantage of being the only way in which rates of evolution can be calibrated in absolute time. Unfortunately, however, this method is not available for most organisms because of a fragmentary or nonexistent fossil record of their history. Furthermore, even in taxa with relatively complete fossil records, a large margin of error is associated with attempts to determine the time since divergence between two taxa. Literal interpretation of the fossil record (i.e., the assumption that the observed first occurrence of a taxon in the fossil record represents its actual evolutionary origin) leads to a systematic underestimation of the time of origin of the taxon, artificially inflating calculated rates of evolution (Springer, 1995). Moreover, different levels of incompleteness of the fossil records of different organisms can lead to inappropriate conclusions about evolutionary rate heterogeneity in these groups. Hillis and Moritz (1990) have suggested that the statistical error in estimating time of divergence based on the fossil record is generally so large that it severely limits the utility of subsequent estimates of rate of evolution. Finally, when comparing rates of evolution in different organisms, it is important that the divergence times between pairs of taxa selected from each group are similar, thus decreasing the chance that multiple nucleotide substitutions per site will result in greater levels of saturation in older comparisons, artificially

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producing results that indicate a slower rate of evolution in these taxa (B. S. Axbogast, in review). Despite the many limitations of the fossil record, estimates of rates of molecular evolution calibrated by use of fossil evidence represent a large proportion of the rate studies published to date. For example, Brown et al. (1979,1982) used fossil data to calculate that mitochondrial-DNA divergence among primates has occurred at a rate of about 2% per million years (my). Shields and Wilson (1987) calculated a similar rate estimate for mitochondrial-DNA divergence in birds. The rate estimate for birds was based on a single divergence time, estimated from the fossil record, between two genera of geese (Anser and Branta). Although a rate estimate based on such limited data should be viewed with caution, Wilson et al. (1985) considered the bird example to be indicative of a growing body of evidence pointing to a rate of mitochondrial DNA evolution that is uniform in animals, with only rare exceptions. The nucleotide generation-time hypothesis developed by Martin and Palumbi (1993a) also was based on divergence times estimated from the fossil record. Similarly, Bowen et al. (1993) suggested, again based on fossil data, a slower rate of evolution of the mitochondrial cytochrome b gene in marine turtles than in ungulate mammals and dolphins. This slower rate of evolution was observed in comparisons of transitions, transversions, and overall sequence differences. Bowen et al. (1993) observed that their results fit well with the nucleotide generation-time explanation of rates of evolution, as marine turtles have exceptionally long lives and low metabolic rates.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tests Based on Cosperiating Taxa Relative rates of molecular evolution can be calculated for taxa with parallel evolutionary histories (cospedating taxa). Groups of organisms may share evolutionary histories because they were exposed to the same biogeographic factors (vicariance and dispersal) or because they are coevolving due to physiological or ecological interdependence (strict coevolution; Futuyma and Slatkin, 1983). Although the ultimate cause of common phylogenetic histories is interesting, it is not important for studies of rates of evolution. The only important consideration is that corresponding pairs of organisms in different groups diverged at approximately the same time, thereby allowing for comparison of relative amounts of change along each corresponding lineage in the two groups. Although this method yields estimates of relative (not absolute) rates of change, it is a relatively straightforward procedure that avoids problems assodated with using fossil data. Therefore, comparison of amounts of molecular change in organisms with common biogeographic histories may have great utility as an index of relative rates of evolution in distantly related organisms. In few cases is the strength of association between two organisms greater than in symbionts such as hosts and parasites. Because this assodation is so strong, congruence in the phylogenies of the two lineages is espedally likely to indicate contemporaneous dadogenic events. Therefore, if host and parasite phylogenies are congruent, then it is appropriate to assume that corresponding pairs of hosts and pairs of parasites diverged at approximately the same time. When this is the case, we have the opportunity to compare rates of evolution in distantly

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related organisms that have vastly different life histories. Unfortunately, appropriate groups of organisms seem to be relatively rare, and studies including rigorous tests of congruence between host and parasite phylogenies based on genetic data are still few in number. Some chewing louse species have been shown to have relationships that mirror the relationships of their pocket gopher hosts to a greater degree than would be expected due to chance or factors other than cospedation (Demastes and Hafner, 1993; Hafner and N adler, 1988; Hafner et al., 1994). Hafner et al. (1994) compared rates of evolution in these cospedating taxa and found a significantly higher rate of evolution in the mitochondrial cytochrome c oxidase subunit I gene of chewing lice than in that of pocket gophers. This difference in rate of evolution was observed in analyses of all substitutions and of substitutions at only the silent, fourfold degenerate sites. This rate difference could be the result of differences in the hosts' and parasites' generation times, body sizes, metabolic rates, or in numerous other factors. In contrast, divergence in nudear DNA (allozymes) in pocket gophers and chewing lice seems to proceed at approximately equal rates (Hafner and Nadler, 1988). These results suggest that different factors may influence evolutionary rates in the mitochondrial and nudear genes studied, or that rate differences are more easily resolved in the more rapidly evolving mitochondrial genome. Aphids and their endosymbiotic bacteria also appear to have cospeciated throughout much of their evolutionary history. Moran et al. (1995) showed evidence of a higher rate of evolution of the ribosomal DNA in the endosymbiotic bacteria than of the orthologous gene in the

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aphids. This difference in rate of evolution is consistent with the generation-time hypothesis, as the organisms with the higher rate of evolution also have a shorter generation time. Clearly, many other factors also might explain this difference in rate of evolution. Analyses Based on Relative-Rate Tests Relative-rate tests provide a method for studying rates of evolution without the need for fossil data and without the special requirement for organisms with congruent evolutionary histories. Relative rates of evolution of two taxa can be compared and tested for departure from equal rates. This method simply requires that something is known about the phylogenetic relationships of the taxa being compared and that an appropriate outgroup taxon is available for study. Unfortunately, an appropriate outgroup taxon is not always available because of extinction events or uncertain phylogenetic relationships. Additionally, levels of genetic divergence between available outgroup and ingroup taxa may be so high that rate heterogeneity among the ingroup taxa is masked by saturation (i.e., differences between the outgroup and ingroup taxa are so great that it is not clear which differences between ingroup taxa are ancestral and which are derived, leading to the false conclusion of rate homogeneity; Springer, 1995). Often, a lack of observable rate heterogeneity is interpreted as evidence for dock-like evolution (e.g., Esteal, 1990,1991; O'hUigin and Li, 1992). This condusion may be correct in many instances. However, Scherer (1989) pointed out that "passing the relative rate test does not guarantee a uniform rate of molecular evolution." Therefore, conclusions of rate homogeneity always should be viewed with caution.

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Certain molecular evidence has suggested that rate of molecular change has slowed in the hominoid lineage (for review, see Li et al., 1996). This controversial issue is best addressed by conducting relative- rate tests, rather than depending on fragmentary paleontological evidence. Using a New World monkey as a reference, Li et al. (1996) compared the rate of evolution of a pseudogene, several introns, and flanking and untranslated gene regions in an Old World monkey and human. For each comparison, the human showed equal or significantly lower rates of nucleotide substitution than did the Old World monkey. This result is in contrast to Esteal's (1991) analysis of similar data, which indicated little rate heterogeneity between humans and other primates. The source of this discrepancy is not clear, but the use of more closely related outgroup taxa by Li et al. may account for the different results. Li et al. (1996) also summarized relative-rate data for Homo versus rodent protein-coding DNA sequences and found a significantly higher rate of amino add substitution in the rodent lineage than in Homo. Furthermore, their re-analysis of data presented by Esteal and Collet (1994) also indicated a slightly higher rate of synonymous substitution in rodents than in Homo, despite apparently high levels of saturation in these substitutions. Conversely, Esteal and Collet (1994) interpreted their data as evidence for a constant rate of evolution at synonymous sites in rodents and Homo. These interpretations led to fundamentally different conclusions about the nature of molecular evolution in rodents and Homo. For example, Li et al. (1996) suggested that these taxa differ in rate of substitution at synonymous sites because of differences in the underlying mutation rate, which also could account for at least

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some of the difference in rate of evolution of proteins. Therefore, Li et al. (1996) interpreted the results of the Old World monkey/human and rodent/human comparisons described above as evidence for the effect of generation time on rate of evolution. In contrast, Esteal and Collet (1994) suggested that rodents and Homo do not differ in rate of evolution of synonymous sites and, therefore, underlying mutation rate does not differ. Instead, Esteal and Collet suggested that differences in rate of evolution of proteins are caused by differences in rate of fixation of non-neutral mutations, which could be the result of differences in effective population size. However, numerous factors other than generation time and population size differ between these mammals and also may account for these differences in rate of evolution. Relative-rate tests also have provided support for the idea that birds generally have a slower rate of molecular evolution than do mammals. These results are in contrast to the conclusions reached by Shields and Wilson (1987; discussed above). For example, Adachi et al. (1993) found significantly fewer mitochondrial amino add substitutions in the chicken than in Homo (using a frog as an outgroup). Likewise, Mindell et al. (1996) found evidence for a generally lower rate of evolution in the chicken than in four mammal species (human, cow, and two rodents) in both mitochondrial and nudear protein-coding genes. Also, ribosomal DNAs (rDNAs—16S and 12S) were examined by Mindell et al. (1996), but did not show a trend toward lower rates of evolution in birds; the authors attributed this to a postulated higher level of selective constraint on the rDNAs than on the other gene sequences studied. Among the protein-coding genes analyzed, mitochondrial genes showed the greatest

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rate heterogeneity at first- and second-base positions of codons and in transversional substitutions and nonsynonymous changes. Nuclear genes showed the greatest rate heterogeneity in third-base positions of codons, in transitional substitutions, and in synonymous changes. These trends seem to suggest that the more slowly evolving nuclear genes have accumulated few transversional or nonsynonymous changes since the divergence between birds and mammals, whereas the more rapidly evolving mitochondrial genes have accumulated many such changes. Furthermore, the mitochondrial sequences might be saturated in terms of synonymous changes, accounting for the lack of observable differences at these sites. Mindell et al. (1996) suggested that the difference in distribution of rate heterogeneity in mitochondrial versus nuclear genes indicates that the slower rate of evolution in birds cannot be attributed solely to selective forces because the trend includes both synonymous and nonsynonymous changes. However, neutral explanations involving metabolic rate are not consistent with these data, especially when one considers that birds and mammals have similar metabolic rates despite their different rates of molecular evolution (M indell et al., 1996). Relative-rate tests among groups of birds based on DNA-DNA hybridization data (Mooers and Harvey, 1994) showed a significant correlation between generation time and rate of evolution. However, the same study produced no evidence of a correlation between mass- specific metabolic rate (or body size) and rate of evolution, which is predicted by the nucleotide generation-time hypothesis of Martin and Palumbi (1993a). A similar study of mammals (Bromham et al., 1996)

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revealed a correlation between mutation rate, as measured by substitutions at fourfold degenerate sites, and both generation time and body size, but not metabolic rate. Crozier et al. (1989) used relative-rate tests to show that the honeybee (Apis mellifera) has had a higher rate of amino add substitution in the mitochondrial cytochrome c oxidase subunits I and H than has the fruit fly ( Drosophila yakuba). Although these two lineages diverged approximately 280 million years ago (mya) and probably have undergone many changes in life-history parameters since that time, m odem Apis have longer generation times than do Drosophila (Crozier et al., 1989). This difference in rate of evolution, therefore, also opposes the generation-time hypothesis. However, Crozier et al. (1989) suggested that several other factors may explain this observed difference in rate of evolution, including a smaller effective population size in Apis versus Drosophila. Studies documenting significant heterogeneity in evolutionary rate generally involve organisms that are distantly related, hence biologically dissimilar. However, Zhang and Ryder, 1995 showed significant heterogeneity in the overall rate of evolution of the mitochondrial genome within a single spedes of mammal, Kirk's dik-dik ( Madoqua kirkii). Demonstrable rate heterogeneity among dosely related lineages, although rare, suggests that factors other than effective population size, body size, generation time, metabolic rate, body temperature, rate of cell division, differential selection pressure, DNA repair efficiency, and nucleotide composition can influence rates of molecular evolution.

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FOCUS OF THIS DISSERTATION Many studies described above seem to hint at factors that either directly influence or are correlated with rate of molecular evolution. However, no mechanism suggested to account for differential rates of evolution (effective population size, body size, generation time, metabolic rate, body temperature, efficiency of DNA repair, differential selection pressure, and rate of cell division) is supported consistently by the available evidence. Therefore, perhaps none of these factors acts as the sole determinant of rate of molecular evolution in all groups of organism s. General trends in rate of molecular evolution among nuclear genes may differ from trends among mitochondrial genes (Vawter and Brown, % 1986). Even within a single genome, studies of rate heterogeneity that include multiple genes usually reveal varying levels of rate heterogeneity among those genes (e.g., Gu and Li, 1992; Li et al., 1987; Li et al., 1996). Differences in the life histories of different taxa (or in the environments they occupy) may influence rates of molecular evolution through a complex interaction of neutral and non-neutral forces. Certain of these forces may be important under certain circumstances and not others, and some of these forces may influence only certain classes of DNA substitutions and not others. Clearly, far more data are needed before a general synthesis of factors influencing rate of molecular evolution can be reached. The principal focus of this dissertation is the comparative mode and relative tempo of molecular evolution among organisms of varying levels of relatedness. In Chapter 2 ,1 present an analysis of relative rates

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of evolution among several distantly related rodents. The initial observation that pocket gophers show a higher overall rate of mitochondrial cytochrome b evolution than do certain other species of rodent (DeWalt et al., 1993) prompted this more detailed comparison of relative rates of evolution among rodents. Rate heterogeneity within the Rodentia was unexpected, given the relative rarity of reported rate differences among closely related taxa. Thus, my earlier study (DeWalt et al., 1993) indicated a need for a more thorough examination of rates of evolution among a wider variety of rodent species. Similarly, cospedating taxa provide an opportunity to compare rates of evolution between groups of organisms. Generally, however, cospedating groups (e.g., vertebrate hosts and their invertebrate parasites) are more distantly related to each other and show more dramatic disparities in life histories than do organisms amenable to relative-rate tests. Therefore, comparisons involving cospedating taxa allow assessment of the extent to which heterogeneity in rate of molecular evolution may occur among organisms with the most dramatic of life history differences. Hafner et al. (1994) reported that pocket gophers and their ectoparasitic chewing lice show observable differences in rate of molecular evolution. Chapter 3 indudes a comparison of molecular evolutionary patterns within another genus of pocket gopher (Thomomys) and within each of two genera of chewing lice (Ceomydoecus and Thomomydoecus) that occur on these gophers.

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Phytogeny and Relative Rates of Evolution of Rodents

Rodents appear to have generally higher rates of molecular evolution than do other mammals; this trend has been observed in a number of studies with a variety of techniques (e.g., Britten, 1986; Catzeflis et al., 1987; Esteal and Collett, 1994; Gu and Li, 1992; Li et al., 1996; but, also see Irwin and Amason, 1994; Janke et al., 1994). Additional evidence suggests that pocket gophers may have even higher rates of evolution than do the other rodent species previously examined (DeWalt et al., 1993). A clear understanding of the biological factors that influence rate of molecular evolution can be gained only by study of organisms that differ in rate of molecular evolution. Although heterogeneity in rate of molecular evolution is often observed among distantly related organisms (e.g., among orders of mammals or between mammals and sharks), among closely related organisms there are fewer such examples (Fieldhouse et al., 1997, Zhang and Ryder, 1995). Even though comparisons of distantly related organisms have provided some clues as to potential causes of rate heterogeneity, study of more closely related organisms that exhibit different rates of molecular evolution should provide even greater insight to the factors influencing rate of molecular evolution, because closely related organisms differ less dramatically in life-history traits, physiology, and nucleotide composition. Therefore, among closely related organisms, it may be more feasible to determine which factors are most important in influencing rate of molecular evolution.

18

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Relative-rate tests have documented significantly more nucleotide substitutions in the cytochrome b gene of two pocket gophers, Cratogeomys castanops and Pappogeomys bulleri, than in that of two muroid rodents, Mus musculus and Ratfus noroegicus (DeWalt et al., 1993). This difference in rates is unexpected, given that pocket gophers belong to the same suborder as do the muroid rodents (Wilson and Reeder, 1993) and, thus, might be expected to show similar evolutionary rates. Pocket gophers also have similar, or slightly longer, generation times than do M. musculus and R. noroegicus (Nowak, 1991), which, according to the generation-time hypothesis (Laird et al., 1969), would lead to the expectation of equal or slightly slower rates of evolution in pocket gophers relative to these muroid rodents. Furthermore, metabolic rates of fossorial rodents, such as pocket gophers, tend to be lower than expected for their body sizes compared to metabolic rates of other rodents (Contreras and McNab, 1990; McNab, 1988). Therefore, a number of factors, including phytogeny, generation time, and metabolic rate, all suggest that pocket gophers should have an equivalent or slower rate of DNA substitution relative to the muroid rodents studied if rate of molecular evolution in pocket gophers is influenced by the same factors implicated in other studies (Martin and Palumbi, 1993a). Thus, the ultimate cause of the higher rate of cytochrome b evolution of pocket gophers compared to the muroid rodents studied remains unknown. To address this issue, tests of relative rates of evolution were conducted using Cratogeomys, Pappogeomys, Mus, Rattus, and 17 additional rodent species (Table 2.1; see Appendix A for common names). Among these, two other genera of pocket gophers and a member of the

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Table 2.1—Specimens for which cytochrome b sequences were examined.4 Sequence Accession Taxonb reference0 number^ Order Lagomorpha Oryctolagus cunniculus 5 U07566 Order Rodentia Suborder Sciurognathi Family Sciuridae Sciurus carolinensis 8 none Marmota flaviventris 8 none Spermophilus tridecemlineatus 8 none Family Geomyidae Cratogeomys castanops 3 L11902 Pappogeomys bulleri 3 L11900 Geomys bursarius 3 L11901 Thomomys bulbivorus 1 LSUMZ 31308 Family Heteromyidae Chaetodipus penicillatus 1 LSUMZ 34420 Family Muridae Mus musculus 2 J01420 Rattus noroegicus 4 X14848 Spalax ehrenbergi 1 M 6094 Suborder Hystricognathi Family Bathyergidae Heterocephalus glaber 1 LSUMZ 35916 Family Hystriddae Hystrix africaeaustralis 7 X70674 Family Erethizontidae Coendou bicolor 6 U34852 Family Caviidae Cavia porcellus 7 none Family Dasyproctidae - Myoprocta pratti 6 U34850 Family Ctenomyidae Ctenomys boliviensis 1 NK15907 Family Echimyidae Echimys chrysurus 6 L23341 Euryzygomatomys spinosus 6 U34858 Proechimys simonsi 6 U35414 Thrichomys apereoides 6 U34854

recognized here as separate genera and Spalax is not referred to here as Nannospalax. b See Appendix A for common names. c Sequence references: 1) This study; 2) Bibb et al., 1981; 3) DeWalt et aL, 1993; 4) Gadaleta et aL, 1989; 5) Irw in and Amason, 1994; 6) Lara et aL, 1996; 7) Ma et aL, 1993; 8) Thomas and M artin, 1993. d Genbank accession numbers (where available). "LSUMZ” and "M" indicate accession numbers for Louisiana State University Museum of Natural Science; "NK" indicates accession number for University of New Mexico Museum of Southwestern Biology.

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closely related family Heteromyidae ( Chaetodipus) were included to test the phylogenetic limits of the increased rate of cytochrome b evolution. Three other fossorial rodents more distantly related to pocket gophers (Spalax, Heterocephalus, and Ctenomys) also were included to determine if some aspect of life in the fossorial niche has lead to the increased rate of evolution in pocket gophers. Finally, sampling of non-fossorial rodents was expanded to include several representatives of each of the two rodent suborders Sciurognathi and Hystricognathi. The greater breadth of taxonomic sampling of rodents in this study allows examination of relative rates of molecular evolution among rodents with a wider variety of life-history traits. In addition, the compilation of these DNA sequences allows an investigation of the utility of cytochrome b in recovering phylogenetic relationships among rodents. Because heterogeneity in rate of molecular evolution among taxa may influence the outcome of phylogenetic analyses (Honeycutt et al., 1995; Philippe and Douzery, 1994), it is useful to determine to what extent relatively well-established phylogenetic relationships among rodents can be recovered using cytochrome b sequences. Relative-rate tests were performed on synonymous and nonsynonymous substitutions separately. Most nonsynonymous substitutions are likely to be subject to greater purifying selection than are most synonymous substitutions. In addition, nonsynonymous substitutions accumulate at a much slower rate than do synonymous substitutions. Therefore, this approach allows the comparison of rates of molecular evolution among taxa to be made independently in groups of

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substitutions that differ both in their exposure to natural selection and in their propensity toward saturation. MATERIALS AND METHODS Previously published cytochrome b sequences were used in conjunction with several cytochrome b sequences generated specifically for this study (Table 2.1). Genomic DNA was isolated from rodent liver or kidney tissues following the technique outlined by Hillis et al. (1990). The cytochrome b gene was amplified using the polymerase chain reaction (PCR) with Thermus aquaticus DNA polymerase (Promega, Madison, WI; Saiki et al., 1986,1988). Double- and single-stranded amplifications and sequencing reactions were performed using various combinations of the following primers, depending on the rodent species being amplified: L14724 (5'-CGAAGCTTGATATGAAAAACCATCGTTG- 3'), L15049 (5'-GCCTGTACATCCACATCGGACGAGG-3'), L15171 (5'-CCAT GAGGACAAATATCATTCTGAGG-3'), L15408 (5'CAGATTGCGGCAAA GTACCATTCCA-3'), L15513 (5'-CTAGGAGACCCTGACAACTA-3'), H15149 (5,-AAACTGCAGCCCCTCAGAATGATAnTGTCCTCA-3/), H15154 (5'-GCAGCCCCTCAGAATGATATTTGTCCTC-3')/ H15579 (5 -CCT AGTTTATTTGGAATGGATCGTAG-3 ), H15906 (S'-CATTTCCGGTTTAC AAGACCAGTGTAAT-3), and H15915 (5-AACTGCAGTCATCTCCGGT TTACAAGAC-3’). Primer names indicate the DNA strand (H=heavy or L=light) and the position of the 3' end of the oligonucleotide sequence relative to the human sequence (Anderson et al., 1981). Primer sequences for L14724, L15513, H15149, and H15915 were taken from tw in et al. (1991); the sequence for L15408 is a modification of that reported by Irw in

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et al. (1991). The sequence for H15154 is that of "MVZ04" in Smith and Patton (1993). Double-stranded PCR amplifications were performed in 50 pi reaction volumes, usually using primers L14724 w ith H15149 or H15154, L15049 with H15579, H15906 or H15915, and L15513 with H15906. Each reaction included 3 |jl of each primer (10 pM), 2 fil of MgClj (10 niM), 2 pi of

deoxynudeoside-triphosphate mixture (dATP, dGTP, dCTP, and dTTP, each 1 mM), 5 |xl of 10X Taq buffer, and 0.25 |il of Taq DNA polymerase. Between 34 and 38 PCR cycles were performed with the following thermal-cycling parameters: 1 min. denaturation at 95°C, 1 min. annealing at 50-57°C, and 1-1.5 min. extension at 72°C. Often, the first four PCR thermal-cycles were performed with lower annealing temperatures, ranging from 38-45°C. Reaction success was assessed by electrophoresing 5 jil of the PCR product on a 1% agarose gel, staining with ethidium bromide, and viewing under UV light. For double-stranded PCR products that were difficult to sequence, a 5- pl sample of the double-stranded product was then used to generate single-stranded DNA. Asymmetric PCR reactions using only one primer were performed under the same conditions as the symmetric reactions described above. Approximately 35 thermal cycles were performed under the same regime as that used in the last cycles of the symmetric reactions. Prior to sequencing, the double- or single-stranded PCR product was purified using PCR Select ID spin columns (5 prime, 3 prime, Inc., Boulder, CO). Sequencing reactions were performed on 7 pi of the DNA template using T7 DNA polymerase (Sequenase version 2.0, United States Biochemical, Cleveland, OH) following standard dideoxy chain-

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termination protocols (Sanger et al., 1977) and modifications recommended by the manufacturer. Presumed amino-acid sequences based on the nucleotide sequence data, number of potentially phylogenetically informative nucleotides, and pairwise numbers of synonymous and nonsynonymous substitutions were assessed using the MacSequence program (version 4.2, D. A. Good, unpublished). This program uses amino-acid sequences to determine silent versus replacement changes and provides matrices of changes by codon position and type (transitions versus transversions). All phylogenetic analyses were conducted using Oryctolagus (a rabbit) to root the rodent tree. Maximum-likelihood estimates of relationships among these rodents and levels of support for relationships, as determined by bootstrapping, were estimated using the UNIX-based versions of the fastDNAml (Felsenstein, 1981; Olsen et al., 1994) and PHYLIP (Phytogeny Inference Package; Felsenstein, 1993) programs. To insure that the best maximum-likelihood tree was found, a variety of transition:transversion ratios were incorporated into the model. In addition, the input order of taxa was jumbled, and the global rearrangement option was used in both programs until a tree with the best tog-likelihood score was found a minimum of five times. Maximum-likelihood analysis of amino-acid sequences was conducted employing the mtREV-22 model of amino-acid sequence evolution (Adachi and Hasegawa, 1996) and a quartet-puzzling algorithm (PUZZLE 2.5; Strimmer and von Haeseler, 1996). Parsimony analysis was performed on the amino add and nucleotide sequence data using the computer program PAUP (Phylogenetic Analysis Using Parsimony, version 3.1.1; Swofford, 1993). Skewness of tree-length

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. distribution as a measure of phylogenetic signal (Hillis and Hulsenbeck, 1992) was estimated using PAUP to calculate g i statistics based on 10,000 randomly generated trees. Relative-rate tests were performed using the Codrates program of Muse and Gaut (1994), which was modified for mitochondrial DNA (mtDNA). This approach uses the codon as the unit of evolution and treats synonymous and nonsynonymous substitutions separately. The tests, as used here, involve building a maximum-likelihood tree for two ingroup taxa and an outgroup taxon ( Oryctolagus) and then comparing this tree to additional trees in which either synonymous or nonsynonymous substitutions (or all substitutions simultaneously) are constrained to be equal in both ingroup taxa. If the constrained tree is judged significantly less likely than an unconstrained tree by a likelihood- ratio test (which approximates a distribution; Muse and Gaut, 1994), then the null hypothesis of equal rates of substitution among the ingroup taxa is rejected. To represent the data graphically, UPGMA (unweighted pair-group method using arithmetic averages) was used to cluster the values (R Package program; Legendre and Vaudor, 1991). Rates of evolution among these rodents were further examined using relative-rate tests (Li and Graur, 1991) on numbers of synonymous and nonsynonymous substitutions observed in pairwise comparisons of taxa. The binomial test (Mindell and Honeycutt, 1990) was used to determine if differences were statistically significant based on a two-tailed test (Allard and Honeycutt, 1992; DeWalt et al., 1993).

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RESULTS AND DISCUSSION Phylogenetic Utility of Cytochrome b Sequence data for the first 1140 base pairs of the cytochrome b gene of 21 rodents and the rabbit (Table 2.1 and Appendix B), yielded 621 variable characters, 533 of which were potentially phylogenetically informative. Within the ingroup taxa (i.e., excluding the rabbit sequence), there were 615 variable nucleotide positions, 529 of which were potentially phylogenetically informative. Of the 380 characters in the presumed amino-acid sequence (Appendix C), 129 were variable and 115 were potentially phylogenetically informative. Both parsimony and maximum-likelihood analyses of amino add sequences recover generally accepted evolutionary relationships (Fig. 2.1), which are reflected in the current taxonomy of these rodents (Table 2.1; Wilson and Reeder, 1993). For example, each family represented in this study (Sduridae, Geomyidae, Muridae, and Echimyidae) is shown to be monophyletic based on analysis of cytochrome b amino-add sequences. These data also are consistent with monophyly of the geomyid- heteromyid superfamily Geomyoidea, monophyly of the ctenomyid- echimyid superfamily Octodontoidea, and monophyly of the suborder Hystricognathi. Analysis of the amino-add sequences does not suggest monophyly of the suborder Sciurognathi; only four of the six most- parsimonious trees place the Geomyoidea with the Sduridae. Also, the sdurognath family Muridae is not dustered with the other sciurognath families (Sduridae, Geomyidae, and Heteromyidae) in any of the six most-parsimonious trees or in the maximum-likelihood tree.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cratogeomys Pappogeomys 92 (90) Geomys 32. (75) Thomomys Chaetodipus Mus 1001— p 3 ) | ____Rattus (71) Spalax Heterocephalus Hystrix Coendou (64) (61) c Myoprocta Ctenomys

SSL Echimys (96) (99) Proechimys (62) (98) Euryzygomatomys Thrichomys Cavia Sciurus 98 j Marmota m m i — (96)1------Spermophilus Oryctolagus

Figure 2.1—Strict consensus of six most-parsimonious trees (629 steps) based on cytochrome b amino-add sequences. Bootstrap values are shown in bold font above each branch that was supported in more than 50% of 1,000 replicates. Maximum-likelihood analysis of amino-add sequences produced a tree that was less resolved, but entirely congruent (support from quartet puzzling is shown in parentheses below branches).

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Maximum-likelihood analysis of DNA sequences yields a slightly different tree (Fig. 2.2). The transitionrtransversion ratio of 1.3:1.0 produced the best log-likelihood score (-15309.61), although ratios of l.Orl.O, l.lrl.O,1.2:1.0,1.4.T.0, and 15:1.0 produced the same tree topology. Higher ratios (1.6-2.0:1.0 and 7.0:1.0) produced a tree with only minor topological differences (i.e., the positions of Spalax and the Mus/Rattus clade are reversed from those shown in Fig. 2.2). Maximum-likelihood analysis of 100 bootstrap replicates indicated differing levels of support for the various nodes (Fig. 2.2). For example, nodes indicating monophyly of the Geomyidae, the Geomyoidea, and the Sduridae are reasonably well supported. However, the Hystricognathi and the Echimyidae each appear as monophyletic groups in only 50-60% of the bootstrap replicates. This level of support is similar to that provided by analysis of amino-add sequences (Fig. 2.1). As in other analyses of the cytochrome b data, monophyly of the Sdurognathi is not supported; murids, sdurids, and geomyoids formed a monophyletic group in less than 1% of maximum- likelihood bootstrap replicates. Unlike the parsimony and maximum- likelihood analyses of amino-add sequences, maximum-likelihood analysis of nudeotides fails to recover the presumed monophyletic relationship of the Muridae ( Rattus, Mus, and Spalax form ed a monophyletic group in only 7% of the bootstrap replicates of nudeotide sequences). Parsimony analysis of nudeotide sequences was conducted using several weighting schemes to investigate the effect of transition bias and of indusion or exdusion of rapidly evolving sites on the outcome of the analysis. Equal weighting of transitions and transversions and of each

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7 4 — Cratogeomys Pappogeomys 76 Geomys 87 Thomomys Chaetodipus 70 Spalax Rattus 97 42 cMus Heterocephalus 25 3 1 1 Hystrix 50 c Coendou 54 Cavia 34 c Myoprocta 27 Ctenomys

4 3 1-----— Echimys 98 381 I___ Thrichomys 60 Euryzygomatomys Proechimys Sciurus 94 Marmota lio^l— Spermophilus Oryctolagus

Fig. 2.2—Results of maximum-likelihood analysis of nucleotide sequences w hen a 1.3:1.0 transition:transversion ratio is assumed. Bootstrap values (based on 100 replicates) are shown for each node.

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nucleotide position within a codon yields one most-parsimonious tree (Fig. 2.3). A few nodes in this tree are well supported, as indicated by bootstrap analysis (Fig. 2.3). However, many other nodes are supported in less than 50% of the 1,000 bootstrap replicates. Among these, the grouping ofHeterocephalus with Spalax is not seen in any other analysis and is in conflict with the presumed monophyly of both the Muridae and the Hystricognathi (Table 2.1). Attempts to reduce the effect of homoplasy on parsimony analysis of nucleotide sequences by use of various weighting schemes produced trees with slightly different topologies (Fig. 2.4); differences include the placement of Coendou and Hystrix and rearrangements within the Echimyidae. No single method of phylogenetic analysis recovers all of the generally accepted relationships indicated by current taxonomy (Table 2.1). Analyses that place little or no emphasis on silent changes (Fig. 2.1, 2.4c, and 2.4d) depict the family Muridae as monophyletic (i.e., including Spalax). This result suggests that analyses that incorporate silent substitutions may be confounded by different levels of saturation of silent changes in Spalax or in the Mus/Rattus lineage. As discussed by Lara et al. (1996), cytochrome b nucleotide sequences provide little resolution of relationships within the Echimyidae; however, monophyly of the Echimyidae and of the echimyid-ctenomyid superfamily, Octodontoidea, are indicated by all analyses. Likewise, monophyly of the Geomyidae and of the geomyid-heteromyid superfamily Geomyoidea are recovered by all analyses, as is monophyly of the Sduridae. Monophyly of the Cavoidea (Cavia and Myoprocta) is indicated in all analyses except those based on amino-add sequences. All analyses of amino-add sequences and

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6 6(— Cratogeomys ' 89 I I----- Pappogeomys 87 Geomys 73 Thomomys Chaetodipus

91 Rattus c Mus Heterocephalus 53 c Spalax Hystrix

Cavia Myoprocta Coendou Ctenomys Echimys 96 Thrichomys Euryzygomatomys Proechimys Sciurus 87 l i o o p - Marmota SpermophUus Oryctolagus

Fig. 2.3—Results of parsimony analysis of nucleotide sequences based on an equal weighting of transitions and transversions. Values above branches indicate level of support (%) for those nodes that were found in more than 50% of 1000 bootstrap replicates.

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7* f Pappogeomys Pappogeomys 97 ^ — Cratogeomys Cratogeomys Geomys Geomys Thomomys Thomomys Chaetodipus Chaetodipus 97 mm Mus Mus fc— Rattus Rattus Spalax Spalax C Heterocephalus C Heterocephalus Coettdou Hystrix Cavia Coendou Myoprocta Cavia Hystrix Myoprocta Ctenomys Ctenomys Echimys Proechimys Thrichomys Thrichomys Euryzygomatomys Euryzygomatomys Proechimys Echimys 901 Scrums Sciurus Marmota Marmota iool— Spermophilus E Spermophilus > Oryctoiagus Oryctoiagus 67-— Pappogeomys Pappogeomys 97 ■■—> Cratogeomys Cratogeomys Geomys Geomys 86 Thomomys ■ Thomomys Chaetodipus i ■ ■ ■ ■ Chaetodipus 99^» Mus 9£|—•M us > Rattus I L— Rattus < Spalax I. ■ — Spalax 55 ■ Heterocephalus ■ - - — Heterocephalus >Hystrix ■ ■ ■ — Hystrix • Coendou ■ ■ Coendou Cavia '65^— Cavia Myoprocta Myoprocta Ctenomys Ctenomys 95 Proechimys Echimys Thrichomys Thrichomys Euryzygomatomys Euryzygomatomys Echimys 1 Proechimys 931 Sciurus Sciurus Marmota Marmota ilnl— Spermophilus lO O k — Spermophilus . Oryctoiagus Oryctoiagus

Fig. 2.4—Results of parsimony analysis. Analyses include giving third position transitions 1/2 (a) and 1/7 (b) the weight of other changes, eliminating third position transitions (c; strict consensus of two trees), and eliminating all transitions (d; strict consensus of three trees). Level of support (%) is indicated for nodes found in more than 50% of 1000 bootstrap replicates.

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maximum-likelihood analysis of nudeotide sequences indicate a monophyletic Hystricognathi; parsimony analysis of nudeotide sequences does not. Monophyly of the Sdurognathi is not supported by any analysis of the cytochrome b data; however, four of the six most-parsimonious trees based on the amino-add data indicate that the Geomyoidea and the Sduridae are sister groups, as do some of the trees based on nudeotide sequences with transitions either partially or completely eliminated from the analysis. It is dear that this data set contains homoplasy, probably in the form of substitutional saturation at freely evolving sites, which makes it difficult to ascertain relationships among certain taxa. In addition, accumulation of changes at less-variable sites may be too slow to allow recovery of certain phylogenetic relationships. Nevertheless, the data set contains a significant amount of phylogenetic signal, whether analyzed as amino- add or nudeotide sequences (gi statistics for 10,000 random trees are -0.70 and -0.68, respectively; p<0.01; Hillis and Hulsenbeck, 1992). Additionally, both the amino-add and nudeotide sequence data sets retain significant phylogenetic signal when they are reduced to indude only one member of each family (gi statistics range from -0.48 to 0.87, p<0.01; Hillis and Hulsenbeck, 1992). This result suggests that the data sets contain meaningful information about higher-level relationships, despite generally lower bootstrap values at the basal regions of the trees. It is likely, therefore, that other factors, such as rapid phyletic radiations and heterogeneity in rate of evolution, account for much of the lack of resolution in certain regions of the phylogenetic trees.

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Implications for the Evolutionary History of Rodents The relationships recovered here in the analyses of cytochrome b nucleotide and amino-add sequences are those that are most strongly supported by morphological data (e.g., monophyly of the Geomyidae, Geomyoidea, and Hystricognathi; Luckett and Hartenberger, 1985). These relationships also have been supported in other molecular analyses (Nedbal, 1996). Some ambiguous relationships in this study also have been difficult to resolve based either on morphology (e.g., the relationship of Spalax to other rodents and monophyly of the Sdurognathi; Luckett and Hartenberger, 1985) or with molecular and morphological data combined (e.g., relationships within the Hystricognathi; Nedbal et al., 1994). Lara et al. (1996) proposed that relationships within the Echimyidae are ambiguous based on cytochrome b data because the diversity present within extant echimyids is the result of a relatively old (late Miocene) and rapid radiation. In fact, many evolutionary relationships may be impossible to recover based on any amount of molecular data if the time since divergence of taxa is too long relative to the time of common ancestry (de Jong, 1985; Lanyon, 1988). Most evidence suggests that Spalax belongs to a lineage that diverged from other murids early in the history of the group (Luckett and Hartenberger, 1985). However, the postulated sister relationship of Spalax to the Mus/Rattus dade is not recovered consistently by analyses of cytochrome b data (Figs. 2.2-2.4). Furthermore, bootstrap values uniting Mus, Rattus, and Spalax in parsimony analyses of nudeotide sequences are low (19-36%), and support for alternative hypotheses based on these data (e.g., that Spalax is more dosely related to Heterocephalus) also is

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weak. Therefore, divergence between the early hystricognaths and the muroids and between the two muroid lineages (Spalax and Mus/Rattus) may have occurred rapidly, thus confounding subsequent morphological and molecular analyses. The apparent paraphyly of the Sciurognathi indicated by these data is not surprising given that the Sciurognathi is often considered a "wastebasket group" (Hartenberger, 1985:29). W hether the Geomyidae and Sduridae are actually sister groups relative to the other taxa examined here or whether the lineage leading to extant sdurids was derived earlier than the split between geomyids and other rodents is undear based on these data. Taxonomic affinities of the Geomyoidea also are not dear based on morphology; geomyoids have been recognized as sduromorphs or as myomorphs, depending on which characters are emphasized (Falbusch, 1985). Relationships among most of the sdurognath families studied here are poorly resolved (Figs. 2.1-2.4) and analysis of 12S rRNA data, induding a more thorough representation of outgroup taxa, also fails to provide robust support for any of the alternative relationships among these particular families (Nedbal et al., 1996). As in the analysis of muroid taxa, this lack of resolution, whether based on morphological or molecular data, may indicate a rapid radiation of the ancestors of these rodents, the history of which is difficult to resolve regardless of the data set analyzed. This problem would be further compounded by potential differences in rate of evolution among sdurognath lineages. As mentioned earlier, the cytochrome b data provide support for the monophyly of the Hystricognathi, although bootstrap values are not high

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(Fig. 2.1-2.2). Furthermore, the data suggest that the Hystricognathi shared a common ancestor with members of the Muridae (Figs. 2.1-2.2). Parsimony analyses tend to place one of the hystricognaths (Heterocephalus) with members of the Muridae (Figs. 2.3-2.4). Alternatively, Nedbal et al. (1996) suggested weak support for a closer relationship between the Hystricognathi and the Sduridae based on 12S rRNA data. The dose relationship between the Muridae and the Hystricognathi suggested by the cytochrome b data is interesting given that the Muroidea appear to have been hystricomorphous primitively, leading to earlier suggestions of a link between muroids and hystricognaths (Flynn et al., 1985; Luckett and Hartenberger, 1985; Vianey- Liaud, 1985). The relationships among rodents suggested by these data contradict relationships proposed by Graur et al. (1991) and Li et al. (1992) based on protein sequences. These two studies suggested that caviomorph rodents may have diverged before the divergence of myomorph rodents and primates and that the Hystricognathi should, therefore, be elevated in taxonomic rank and recognized as an order separate from the Rodentia. However, both studies indicating a polyphyletic Rodentia (Graur et al., 1991; Li et al., 1992) involved building a phylogenetic tree for each gene analyzed using only four ingroup taxa, including only two of the small number of rodent species examined and two distantly related mammals (primates and artiodactyls). Given the limited numbers of rodents sampled in the studies, the problems with phylogenetic analysis of small numbers of taxa (Philippe and Douzery, 1994), and the distantly related outgroups used (marsupials, birds, and amphibians), results conflicting

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with traditional taxonomy should be viewed with caution. More recently, DErchia et al. (1996) analyzed the complete mitochondrial genomes of 14 mammals and suggested that murid rodents arose before the Hystricognathi diverged from primates, ungulates, cetaceans, and lagomorphs. This is not the same scenario suggested by Graur et al. (1991) and Li et al. (1992), but it also implies a polyphyletic origin of rodents. However, despite a large number of sequence characters, the mtDNA analysis of DErchia et al. (1996) suffered from limited taxon sampling, including representatives of only two of 29 extant families (three of 426 genera) of rodents (Nowak, 1991). Poor sampling of taxa can result in phylogenetic trees that appear robust, but are misinformative (Philippe and Douzery, 1994). Therefore, it seems appropriate to rely on the larger body of morphological, paleontological, developmental, and genetic data that suggests that rodents are a monophyletic group (Frye and Hedges, 1995; Honeycutt and Adkins, 1993; Luckett and H artenberger, 1993; Nedbal et al., 1996; Novacek, 1992) and that the Lagomorpha is most likely the sister order to the Rodentia (Honeycutt and Adkins, 1993; Luckett and Hartenberger, 1993). This controversy concerning rodent monophyly serves to highlight the potential importance of assumptions about evolutionary rate constancy. G raur et al. (1991) suggested that, instead of indicating a polyphyletic Rodentia, the protein data they examined may instead indicate a faster rate of nucleotide substitution in guinea pig and myomorph genes than in human genes and that this rate heterogeneity may be great enough to influence the results of parsimony analysis. Given what is now known about rates of evolution in rodents and humans (e.g., Esteal and Collet,

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1994; Gu and Li, 1992; Li et al., 1996), this explanation is likely. Furthermore, if the Rodentia are monophyletic, the protein sequences examined suggest a higher rate of evolution in many guinea pig proteins than in the myomorphs examined (Li et al., 1991). A dramatically higher rate of evolution in a large number of proteins may seem unlikely (Li et al., 1991), but Frye and Hedges (1995) have shown that the rapid evolution of many of these proteins may be due to compensatory changes as a result of altered action of a single gene. When all such biochemically related genes are removed horn consideration, rodent monophyly is supported strongly (Frye and Hedges, 1995). Relative Evolutionary Rates Among Rodents Comparison of relative rates of cytochrome b evolution of the pocket gophers, Cratogeomys and Pappogeomys, w ith Mus and Rattus using the codon-based maximum-likelihood approach (Table 2.2) confirms earlier observations that the gophers show evidence of a higher relative rate of evolution than do the muroid rodents (DeWalt et al., 1993), particularly in nonsynonymous substitutions. In only one comparison, that between Pappogeomys and Mus, was there a significant difference in number of synonymous substitutions. The other pocket gopher genera, Geomys and Thomomys, also exhibited evidence of higher relative rates of nonsynonymous substitution. Comparisons of relative rates of cytochrome b evolution for all rodents included in this study yielded many significant differences in overall rate of substitution (Table 2.3). Many of these differences are attributable to differences in the rate of nonsynonymous substitution (Table 2.4, above diagonal). Analysis of numbers of substitutions estimated from direct

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VO u> with 0.05; Rattus, (* = P < = (* 1.54 1.54 ns 0.00 ns 1.26 1.26 ns 0.51 0.51 ns 3.80 ns 2.41 2.41 ns and 5.84 5.84 * 0.12 ns Synonymous Mus 14 ♦ * * * <4 14 <4 14 ♦ 14 * * * 14 * 7.60 5.26 7.01 5.27 Nucleotide Substitutions 6.68 * 6.65 5.86 ns 9.54 " 6.69 * 6.94 * 6.75 9.88 ** 7.13 11.48 11.48 ** 6.73 13.59 13.59 ** Rattus Mus Mus as the outgroup. In every comparison, the pocket gopher showed a higher rate of 0.01; ns = not significant). The criticalX2 value for comparison of all substitutions is 5.99; for Thomomys Rattus Pappogeomys Rattus Geomys Cratogeomys Rattus Cratogeomys Geomys Thomomys Mus Taxa comparedPappogeomys Mus All Nonsynonymous nonsynonymous and synonymous comparisons, the criticalX2 value is 3.84. Oryctolagus ** = P P < = ** evolution. Values for each comparison represent the valueX2 for the relative-rate test Table 2.2--Results of relative-rate tests comparing four pocket gopher genera to

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Table 2.3-Results of relative-rate tests based on the full maximum-likelihood model. X2 values are shown, with statistically significant comparisons underlined (critical value = 5.99, P = 0.05). Taxon abbreviations are the first four letters of generic names given in Table 2.1. «C i a Cl o 6*|un o ci i y a Al u a « O 6 o s R boo^UKZncomzssuusauua.^ aaooiu(D«4«hay6>oi£itOh «hO£fi«3auaa>ioojrHOiaoMi veHeei*)inci«9N(*iOHoono«H > veHeei*)inci«9N(*iOHoono«H B>uoe>u o>u e b v u v a b f l e oo>u o « i j o o - * o t > o > i j ® > o o £ u i o u i H H n c t i n H i A q c j ^ v j v q i n ^ p j i t e ^ O} O n « m « « H ^ ( i A O 0 H i l 4 A « 9 i A j H n A O < H n O c t l O f l f I I VO las |r ^Hmoo> t l^iH^mnovo<>« IctnHOciftonninctHON i IctnHOciftonninctHON |«CtSAtktOC(NOO«CBNHt HqdmeoHo ia:-i qd \q « nqvj i o O O H o o jn v q in H* Knonni tnHfjm H rtin N M in n n « H<*) o n (K ov mvo HMn< m aof^ <*tovoetaD*p 4 r® t or*® h o

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pairwise comparisons of sequences also shows that most rate differences are attributable to different rates of nonsynonymous substitutions (Table 2.5, above diagonal). Several significant differences in synonymous substitution rates also were observed based on the maximum-likelihood test (Table 2.4, below diagonal), particularly in comparisons involving Ctenomys, Euryzygomatomys, Geomys, Pappogeomys, and Proechimys. However, direct comparison of uncorrected numbers of synonymous substitutions (Table 2.5, below diagonal) indicates that rates of synonymous substitution in these taxa generally are not significantly different. Considering the lengthy time since divergence of many of these taxa (considerably more than 35 million years for some comparisons; Hartenberger, 1985), the measures of sequence divergence at synonymous positions (Tables 2.4-2.5) probably include a considerable amount of substitutional saturation. Honeycutt et al. (1995) also found that synonymous sites were saturated in the cytochrome c oxidase subunit H (COII) gene of rodents. Under these conditions, the maximum-likelihood test probably has greater resolving power (Muse and Gaut, 1994). However, lack of correspondence between the two tests (Tables 2.4 and 2.5) is disturbing and indicates either that those results indicating differences in rates of synonymous substitution are spurious or that some (possibly many) results indicating similar rates among taxa are misleading because of multiple substitutions at many of the more freely evolving nucleotide positions. Therefore, although there seems to be some evidence of heterogeneity in rate of synonymous substitution among rodents, saturation at synonymous sites may have rendered the data

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unreliable for determining which taxa have accumulated the greatest number of changes over time. Cluster analysis of multiple pairwise comparisons of rates of nonsynonymous substitution indicates two major groups of taxa (Fig. 2.5). These two groups represent clusters of taxa with similar rates of nonsynonymous substitution, as determined by values from relative- rate tests (Table 2.4). Of the rodents included here, those examined by DeWalt et al. (1993) seem to represent both extremes of a range of rates of cytochrome b evolution; pocket gophers are among the most rapidly evolving rodents, and Mus and Rattus are among the slowest evolving rodents (Table 2.4, Fig. 2.5). In addition to Mus and Rattus, the slowest evolving group (group 0 in Fig. 2.5) includes the three sdurid rodents and Hystrix. The more rapidly evolving taxa are further divided by cluster analysis into three subgroups (Fig. 2.5). Two of these groups (groups 1 and 2 in Fig. 2.5) show evidence of rates of nonsynonymous substitution intermediate between those of the slowly evolving group (group 0 in Fig. 2.5) and the rapidly evolving group (group 3 in Fig. 2.5). Although some of these taxa with an intermediate rate of evolution (e.g., Cavia and Coendou) show few, if any, significant differences in rate of nonsynonymous substitution from any of the other rodent species examined, maximum-likelihood branch lengths (Table 2.4) reveal a consistent pattern in these rodents of fewer substitutions than are found in the most rapidly evolving species (group 3 in Fig. 2.5) and more substitutions than are found in the most slowly evolving species (group

0).

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Pappogeomys Geomys Thomomys | Cratogeomys Heterocephalus Thrichomys | Chaetodipus Echimys Proechimys Myoprocta Coendou HCtenomys Spalax H Euryzygomatomys Cavia Rattus Mus Hystrix Marmota I Sciurus I Spermophilus

i IT i i i i 5.0 4.0* 3.0 2.0 1.0 0.0 Fig. 2.5—UPGMA analysis of rate of nonsynonymous substitution. Pairwise comparisons of likelihood-ratio scores (X^ values from Table 2.4) are clustered into groups of taxa with similar rates of nonsynonymous substitution. A value of 3.84 (*) is a statistically significant difference in rate of evolution in this analysis. Groups of taxa with similar rates of evolution are numbered from slowest (0) to fastest (3) based on relative numbers of substitutions in Table 2.4.

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The pocket mouse (Chaetodipus penicillatus), the closest relative of the pocket gophers included here, appears to have a rate of nonsynonymous substitution that is similar to that of pocket gophers. In the absence of other data, this finding would suggest the importance of a phylogenetic, or lineage, effect on rates of evolution. However, many of the more distantly related rodents also show high rates of evolution. Among these, the three other fossorial rodents ( Spalax, Heterocephalus, and Ctenomys) appear to have elevated rates of substitution, which are most dramatic in Heterocephalus and Ctenomys. Nevertheless, many of the non-fossorial rodents also have relatively high rates of evolution, and rate of nonsynonymous substitution in the terrestrial Thrichomys is approximately equal to rates measured in pocket gophers. Therefore, a fossorial existence seems to have little direct effect on rate of substitution at nonsynonymous sites of the cytochrome b gene. Rate of nonsynonymous substitution appears to correspond little with phylogeny. For example, the most slowly evolving group includes members of the suborders Hystricognathi and Sciurognathi (i.e., Hystrix and members of the families Sduridae and Muridae), as does the most rapidly evolving group (i.e., Heterocephalus, Thrichomys, and members of the Geomyidae). Therefore, if there is any nonrandom, biological component to rate of cytochrome b evolution, then it is associated with factors that are not lineage specific. The possibility remains, then, that life-history parameters common to multiple lineages are the key to understanding these differences in rate of evolution.

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Selectionist and Neutralist Explanations of Rate Heterogeneity An increase in rate of molecular evolution may result from increased positive selection, weaker purifying selection, or simply a higher substitution rate at the nucleotide level. Of course, the converse of all of these explanations would lead to a decreased rate of evolution. But for sake of clarity, higher rates of evolution (relative to pocket gophers) will be discussed here. Mutations beneficial to an organism should increase in frequency in a population (positive Darwinian selection). If distantly related organisms occupying similar niches have similar demands on their cytochrome b gene products, then there may be enough selection pressure to produce evolutionary convergence. Under these conditions, positive selection could alter the rate of fixation of nonsynonymous substitutions, but it should have little affect on synonymous substitutions, which affect protein function less directly, if at all. Because of substitutional saturation, potential differences in rate of synonymous substitutions are difficult to examine in this data set. Nevertheless, nonsynonymous substitutions are the most important subset of nucleotide changes to consider when assessing the importance of positive selection on rates of molecular evolution. If positive selection has influenced rate of cytochrome b evolution through convergence, then there should be some demonstrable, characteristic way of interacting with the environment that is shared by most or all members of each rate group (0-3 in Fig. 2.5). The most striking ecological attribute of the rapidly evolving pocket gophers (group 3) is their fossorial lifestyle. Although all other fossorial rodents show rates of

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evolution that are higher than those of the slowly evolving group (group 0), several nonfossorial rodents also show elevated rates of evolution (i.e., Chaetodipus, Coendou, Myoprocta, Thrichomys , and others). Additionally, the fossorial rodents are dispersed among three rate groups (1-3, Fig. 2.5), and the highly fossorial blind mole-rat ( Spalax) seems to be evolving more slowly than pocket gophers, Ctenomys , or Heterocephalus. Mo obvious distinguishing ecological or morphological characteristics are shared only by the rapidly evolving rodents, leaving absent a plausible explanation for evolutionary convergence through positive selection. Furthermore, positive selection of advantageous mutants must be rare in nature relative to selection against deleterious mutants (purifying selection) or fixation of neutral or slightly deleterious mutants (Kimura, 1983; Kimura and Ohta, 1974) simply because most mutations, if they affect protein function at all, are more likely to lead to physiological deficiencies rather than to selective benefits for the organism. It seems logical that this would be especially true for critically important metabolic genes, such as cytochrome b. It also seems unlikely that there would be a sufficient number of sites along the cytochrome b molecule subject to positive selection pressure to generate measurably higher rates of evolution in distantly related rodent species. Weaker purifying selection also is invoked often as an explanation for higher rates of evolution. It is possible that the cytochrome b genes of the rapidly evolving rodents are, or have been, under less selective constraint, allowing a higher proportion of mutations to become fixed in these populations. However, it is not clear why the rapidly evolving rodents would be under weaker purifying selection because, again, there

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are no apparent ecological, morphological, or physiological characteristics that are shared by all rapidly evolving rodents that are not also found in certain slowly evolving species. One proposed mechanism for weaker purifying selection and higher rate of evolution in the cytochrome b gene focuses on this gene's important role in the animal's metabolic pathway (Irwin and Amason, 1994). The central premise of this argument is that organisms with lower metabolic rates have less stringent demands on their metabolic machinery than do organisms with higher metabolic rates. Irwin and Amason (1994) suggested this hypothesis based on their observation of a higher rate of evolution of first- and second-position nucleotides in humans and elephants (large mammals with low metabolic rates) relative to Mus (a small mammal with a high metabolic rate). If this hypothesis is correct, then the slowly evolving rodents examined in this study should have higher metabolic rates than do the rapidly evolving rodents. The data (Table 2.6) do not fit this prediction. Instead, the metabolic rates of the rodents in the slowly evolving group (group 0) span the entire range of metabolic rates reported. For example, a member of the genus Marmota has the lowest estimated metabolic rate of all rodents studied here. The other two sdurids examined have metabolic rates that are slightly below the mean rate, and Mus and Rattus have the highest metabolic rates of the rodents examined. No significant association is apparent between metabolic rate and the four nonsynonymous substitution rate classes (Kruskal-Wallis analysis of variance, P = 0.58). Furthermore, no association is observed between metabolic rate and "slow substitution rate" (class 0) and "fast substitution rate" (classes 1, 2,

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Table 2.6—Estimated basal metabolic rate, body mass, and rate of nonsynonymous substitution from duster analysis (Fig. 2.5; 0 = most slowly evolving group; 3 = most rapidly evolving group) of rodent spedes for which cytochrome b sequences were examined. Where basal metabolic rates are unavailable, data for a similarly sized congeneric taxon are induded (designated by parentheses).

Metabolic rate* Body Rate

Taxon ______(cm Q2 /g*hr) mass (g) dass Suborder Sciurognathi Family Sduridae Sciurus carolinensis 4 (0.69) (624) 0 Marmota flaviventris 4 (0.25) (2650) 0 Spermophilus tridecemlineatus 4 0.57 182 0 Family Geomyidae Geomys bursarius 2 0.70 197 3 Thomomys bulbivorus 2 (0.76) (249) 3 Family Heteromyidae Chaetodipus penicillatus 4 (1.07-1.30) (15) 2 Family Muridae Mus musculus 5 (1.7) (17) 0 Rattus noroegicus 4 (1.27) (132) 0 Spalax ehrenbergi 2 0.62-1.03 121-104 1 Suborder Hystricognathi Family Bathyergidae Heterocephalus' glaber 2 0.66 35 3 Family Erethizontidae Coendou bicolor 1 (0.28) (3280) 2 Family Caviidae Cavia porcellus 1 0.55 629 1 Family Dasyproctidae Myoprocta pratti 1 (0.55) (914) 2 Family Ctenomyidae Ctenomys boliviensis 2 (0.45) (490) 2 Family Echimyidae Proechimys simonsi 3 (0.63) (498) 2 Thrichomys apereoides 1 0.64 323 3 a Metabolic rate data from: i Arends, 1985; 2 Contreras and McNab, 1990; 3 McNab, 1982; 4 McNab, 1988; 5 Pearson, 1947

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and 3 combined; Mann-Whitney U test, P = 0.53). Therefore, if different levels of selective constraint act on the cytochrome b genes of different rodents, thereby producing evolutionary rate heterogeneity, then the differences in selective constraint are not the result of differences in any obvious life-history parameter (such as fossoriality) or differences in metabolic rate. The heterogeneity in rate of evolution observed in the cytochrome b gene appears to be paralleled by other mitochondrial genes, casting further doubt on the possibility that different levels of positive or purifying selection account for the observed differences in rate of evolution. For example, Honeycutt et al. (1995) reported a significantly greater overall number of substitutions in the COII gene of Cratogeomys and Geomys than in that of Mus and Rattus. As in the cytochrome b data, there appeared to be no significant difference in rate of synonymous substitution (measured by third position transversions) in the COII gene, but synonymous sites clearly were saturated. When only nonsynonymous substitutions were examined, no significant difference in rate of evolution was evident between Geomys and Mus (Adkins et al., 1996), providing some evidence that differences in rate of evolution among taxa may not be as great in the COII gene as they are in the cytochrome b gene. Nedbal et al. (1996) provided evidence for a higher rate of evolution of the mitochondrial 12S rRNA gene in Perognathus. Chaetodipus, a close relative of Perognathus, has a relatively high rate of cytochrome b evolution. Therefore, high rate of evolution in the cytochrome b gene may be caused by factors that also affect rate of evolution in COII and 12S rRNA genes. It is unlikely that different levels

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of either positive or purifying selection would affect rate of evolution of these three genes in a similar manner. Even if different levels of selective constraint do not seem to account fully for the observed differences in rate of evolution of the cytochrome b gene, purifying selection may affect the rate of fixation of nonsynonymous substitutions through differences in average population size of different species of rodents. Nearly-neutral theory holds that, when effective population size is large, the power of purifying selection to remove slightly deleterious mutations is relatively stronger than it would be in a small population. Thus, slightly deleterious mutations that would be selected against in a large population might be effectively neutral in a small population, reaching fixation in the population through genetic drift (Kimura, 1983; Ohta, 1973,1974,1976,1993). Therefore, smaller average population size may tend to promote a higher rate of evolution in a species. For example, Drosophila populations provide empirical evidence for a rapid rate of evolution in the mtDNA of small populations with repeated founder events (DeSalle and Templeton, 1988). Although little is known about the population dynamics of most of the species studied in this analysis, qualitative examination of population structure and rate of evolution among rodents is consistent with the possibility that population size may affect rate of evolution of the cytochrome b gene in rodents. Among the most rapidly evolving rodents (rate group 3, Fig. 2.5), pocket gophersCratogeomys, 0 Geomys, Pappogeomys, and Thomomys ) are characterized by small effective population sizes and high levels of population subdivision, with genetic diversification among populations as much as two to five times greater

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than that of other small mammals (Patton, 1990). Heterocephalus glaber also occurs in disjunct, genetically differentiated populations, each of which consists of only a very small number of breeding individuals (Jarvis and Bennett, 1990). Genetic data suggest frequent population bottlenecks in H. glaber (Honeycutt et al., 1991). Another fossorial rodent, the tuco-tuco (Ctenomys , rate group 2) also is characterized by a patchy distribution, population dynamics that favor differentiation (Reig et al., 1990), and high observed levels of genetic subdivision among populations (Gallardo et al., 1994). Little is known about the population dynamics of the other rodents with relatively high rates of evolution (groups 1 and 2, Fig. 2.5). Effective population.size and degree of population subdivision are not known for Chaetodipus species (Patton and Rogers, 1993). However, population size in Chaetodipus, an inhabitant of arid regions, appears to fluctuate dramatically over time (up to 100 fold) in accord with unpredictable climatic cycles (Brown and Hamey, 1993), potentially contributing to the higher rate of evolution observed here. Spalax, which has an intermediate rate of evolution (group 1 in Fig. 2.5), also shows evidence of population subdivision, although in some parts of its range, the degree of subdivision appears lower than that seen in other fossorial rodents (Nevo, 1979; Savic and Nevo, 1990). Among the most slowly evolving rodents (rate group 0, Fig. 2.5), Sciurus carolinensis shows little population subdivision (Moncrief, 1987), as does Mus musculus, at least when measured over large areas (Wright, 1978). Another of the most slowly evolving rodents, Marmota flaviventris, despite having a patchy geographic distribution, shows little population subdivision as a result of high levels of male dispersal (Schwartz and Armitage, 1980). Finally,

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Hystrix africaeaustralis shows no evidence of either small population size or patchy distribution, as dispersal of young individuals seems to be retarded by high population densities in the absence of culling (van Aarde, 1987). The foregoing accounts suggest differences in the population size of rodents with different rates of cytochrome b evolution. This observation is supported by quantitative estimates of population subdivision, as m easured by Fsx (Wright, 1951). Although available data are few, Fgx values are associated significantly with rate of nonsynonymous substitution (Table 2.7; Mann-Whitney U test, P = 0.0495). This result suggests a strong possibility that population size is a factor in determining rate of evolution of the cytochrome b gene in rodents. Nevertheless, final resolution of this issue will require analysis of additional genetic data on the population structure of a larger number of rodent species. In attempting to explain the apparently dock-like behavior of nonsynonymous substitutions observed in certain studies, proponents of nearly-neutral theory have suggested that effective population size and generation time are factors that oppose one another, such that rate of evolution of nonsynonymous substitutions (those most likely to be exposed to selection) is constant on a per-year basis among taxa with different generation times (Chao and Carr, 1993; Ohta, 1993,1995). However, rates of nonsynonymous substitution are not always equal among taxa in a study (e.g., Adachi et al., 1993; Crozier et al., 1989; Li et al., 1996; Mindell et al., 1996; this study). If, in fact, rate of evolution results from a dynamic balance between population size and generation time,

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Table 2.7—Estimated average F$x of rodents for which cytochrome b sequences were examined (values in parentheses represent data from congeners) and rate of nonsynonymous substitution from cluster analysis (see Fig. 2.5; 0 = most slowly evolving group; 3 = most rapidly evolving group). There is a significant association between F$x and rate of substitution when rodents are divided into groups of rapidly (groups 1, 2, and 3) and slowly (group 0) evolving taxa (Mann-Whitney U test, P = 0.0495).

Taxon fst Rate class Referencea Suborder Sciurognathi Family Sduridae Sciurus carolinensis 0.056 0 3 Marmota flaviventris 0.070 0 5 Family Geomyidae Geomys bursarius 0.276 3 1 Thomomys bulbivorus (0.258) 3 4 Family Muridae Mus musculus 0.017 0 6 Suborder Hystricognathi Family Ctenomyidae Ctenomys boliviensis (0.248) 2 2 a Data from: 1) Cothran and Zimmerman, 1985; 2) Gallardo et al., 1995; 3) Moncrief, 1987; 4) Patton and Smith, 1990; 5) Schwartz and Armitage, 1980; 6) Wright, 1978

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then evolutionary rate heterogeneity may be the result of significant deviations from the normal equilibrium between these two factors in certain species. If so, a higher relative rate of nonsynonymous substitution may result either from a long-term reduction in population size, or from frequent population bottlenecks not compensated for by increased length of generation time. This hypothesis will be exceedingly difficult to test given the paucity of information on effective population size and generation time for most species and the almost complete absence of information on variance in effective population size through time within a single species. Therefore, population size, whether operating in conjunction with generation time or not, remains a viable, but weakly tested, explanation for the observed differences in rate of cytochrome b evolution among rodents. Higher rate of mutation (substitution at the DNA level) also should contribute to a higher overall rate of evolution. Variation in mutation rate generally is implicated in studies that reveal heterogeneity in rate of synonymous substitution. In the present study, there is no clear pattern in rate of synonymous substitution. Given the long time since divergence among many of these rodent species (and between rodents and the outgroup), this lack of resolution could be the result of multiple substitutions at synonymous sites, such that the synonymous substitution data are saturated. Saturation would cause apparent rate homogeneity among taxa that actually showed rate heterogeneity earlier in time. It follows that heterogeneity in rate of DNA substitution at synonymous sites may be most observable between recently diverged taxa and at nonsynonymous sites between more anciently diverged taxa (Mindell et

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al., 1996; Muse and Gaut, 1994). Although many nonsynonymous substitutions are likely to be detrimental to the organism, those substitutions that persist are likely to be selectively neutral or nearly neutral. As such, the possibility that differences in rate of nonsynonymous substitution are the result of heterogeneity in mutation rate deserves further consideration. Short generation time has long been thought to potentially increase mutation rate (Laird et al., 1969). The range of generation times shown by the rodents included in this study provides an outstanding opportunity to test the generation-time hypothesis. Generation times range from slightly over two months in Mus to approximately two years in Coendou, and new litters can be produced nearly every month in certain taxa (Mus and Rattus) or as rarely as once a year in others (Table 2.8). Analysis of these data reveals no significant association between the four nonsynonymous rate classes and either generation time or frequency of reproduction (Kruskal-Wallis analysis of variance, P = 0.68 and 0.98, respectively). When nonsynonymous substitution rates are divided into two groups (group 0 versus groups 1,2, and 3), there also is no significant association with either generation time or frequency of reproduction (Mann-Whitney U test, P = 0.81 and 0.88, respectively). Therefore, generation time, alone, seems to be a poor predictor of relative rate of evolution among rodents. Other potential correlates of mutation rate are body size and metabolic rate, such that smaller organisms, which generally have higher metabolic rates, may show higher rates of nucleotide substitution (Martin and Palumbi, 1993a). As discussed previously, metabolic rate is not associated

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Table 2.8—Estimated average generation time of females and frequency of reproduction (Nowak, 1991) for rodent species for which cytochrome b sequences were examined, compared with rate of nonsynonymous substitution from cluster analysis (see Fig. 2.5; 0 = most slowly evolving group; 3 = most rapidly evolving group). Where generation time estimates are unavailable, data from a similarly sized congeneric taxon are shown (designated by parentheses).

Frequency of Generation reproduction Rate Taxon time (months)a (m onths) _____ class Suborder Sciurognathi Family Sduridae Sciurus carolinensis 12 6 0 Marmota flaviventris 24 12 0 Spermophilus tridecemlineatus 12 12 0 Family Geomyidae Cratogeomys castanops 9 6 3 Geomys bursarius 7-12 6-12 3 Thomomys bulbivorus (12) (12) 3 Family Heteromyidae Chaetodipus penicillatus (6) (6) Family Muridae Mus musculus 2.25 1.8 0 Rattus noroegicus 3-4 1-12 0 Spalax ehrertbergi 12 1 Suborder Hystricognathi Family Bathyergidae Heterocephalus glaber 12 3 Family Hystriddae Hystrix africaeaustralis 12-19 6-12 0 Family Erethizontidae Coendou bicolor 25 10 2 Family Caviidae Cavia porcellus 2.5 1 Family Dasyproctidae Myoprocta pratti 11-15 2 Family Ctenomyidae Ctenomys boliviensis 12 2 Family Echimyidae Proechimys simonsi (8-9) (2.6) 2 Thrichomys apereoides ______10-12 4-6 3 a Gestation period plus time to first reproduction

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significantly with rate of nonsynonymous substitution in these rodents. Likewise, body size, alone, is not a good predictor of rate of evolution in this group; i.e., body mass shows no significant association with substitution rate (Table 2.6) either for the four nonsynonymous substitution rate groups (Kruskal-Wallis analysis of variance; P = 0.71) or for the fast (groups 1, 2, and 3) versus slow (class 0) nonsynonymous rate classes (Mann-Whitney U test; P = 0.78). Therefore, none of the factors previously considered to influence mutation rate in other organisms (generation time, body size, and metabolic rate; Martin and Palumbi, 1993a) seems to be associated with the observed heterogeneity in rate of cytochrome b evolution in rodents. The likelihood that these differences in rate of evolution result solely from differences in mutation rate appears small, but cannot be discounted. Absolute Rates of Evolution in Rodents Fossil data were used to estimate time of divergence between taxa to calculate an absolute rate of evolution for substitution-rate classes. Comparison of absolute rate of evolution between two taxa known to show different rates of evolution would not be meaningful; thus, the number of possible comparisons is limited to taxon pairs that lack significant rate differences and for which fossil data are available (Table 2.9). Analysis of absolute rates of evolution (Table 2.9) indicates a roughly five- to six-fold difference in estimated rate of evolution between the most rapidly evolving and most slowly evolving taxa. hi contrast, relative-rate tests indicate less than a two-fold difference in rate of nonsynonymous substitution between the most rapidly and most slowly evolving groups of taxa (determined both by comparison of branch

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N> ON 1.8 1.7 6.2 2.4 ____ 49.5 37.4 31.8 33.7 8.4 represent non- 1.1 1.1 37.4 6.2 0.27 1.35 Sciurus 6.3 0.21 52.1 7.4 0.25 6.6 5.4 6.4 and 2nd positions 3rd positions 0.7 0.7 2.8 3.7 21.6 16.8 2.8 14.8 and for the two comparisons of Russell, 1968a; 3 Thomas and Martin, 1993 2 5-7 5-7 16.8 3-5 13.5 13.1 1.0 3.7 29-30 29-30 21.4 Geomys Estimated Uncorrected percentage sequence divergence Sciurus—Spermophilus3 Spermophilus—Marmota3 Sciurus—Marmot3 Pappogeomys—Cratogeomys^ Geomys—Cratogeomys2 Geomys—Pappogeomys2 3 3 0 3 0 0 a Divergence times from: * Russell, 1968b; time can be estimated from fossil data. Taxa are grouped by overall rate of evolution (using the groups in Fig. independent estimates of divergence time. Substitutional saturation undoubtedly has masked many of the Rate divergence All positions 1st 2.5). 2.5). Data for the two comparisons of more dramatic than it actually is in this comparison. Table 2.9-Sequence divergence and rate of evolution per million years (my) for rodents for which divergence sequence changes between taxa with older divergence times, making evolutionary rate heterogeneity appear

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lengths from maximum-likelihood relative-rate tests in Table 2.4 and by absolute numbers of nonsynonymous substitutions in Table 2.5). The disparity in absolute rates of evolution between rapidly and slowly evolving rodents (Table 2.9) probably is artificially inflated, because when estimated divergence times differ considerably between groups being compared, levels of substitutional saturation also differ, confounding comparisons of evolutionary rate (B. S. Arbogast, in review). For example, the rapidly evolving pocket gophers diverged much more recently than did the slowly evolving taxa compared in Table 2.9. Because of this, the degree of saturation of DNA substitutions should be far lower in the pocket gopher comparison than in the older comparisons. Thus, much of the apparent difference in absolute rates of evolution in Table 2.9 may simply be an artifact of increased substitutional saturation in the taxa with older divergence times. To control for this possibility, one must compare pairs of slowly evolving and rapidly evolving taxa with similar divergence times. Unfortunately, such comparisons are not possible at present. CONCLUSIONS Phylogenetic analysis of sequence data from the cytochrome b gene of 21 species of rodents supports several of the well-established relationships among rodents. Relationships that have been difficult to determine based on other data also are not well-resolved based on these limited molecular data. This lack of resolution is not surprising, given the lengthy time since divergence of many of these taxa relative to the number of phylogenetically informative characters available from the analysis of a single gene. In addition, heterogeneity among taxa in rate of evolution

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may further complicate phylogenetic analysis of mtDNA sequence data of rodents (Honeycutt et al., 1995; Philippe and Douzery, 1994). However, that certain relationships difficult to resolve using molecular data also have been difficult to determine based on morphological and paleontological data suggests that lack of resolution may result from several periods of rapid diversification in the evolution of the Rodentia, rather than horn inherent weaknesses of the data themselves. The heterogeneity in rate of evolution of the cytochrome b gene observed here cannot be explained by any of the conventionally dted sources for increased rate of mutation, including metabolic rate, body size, generation time, or nucleotide generation time. Likewise, no evidence suggests that these differences in rate of evolution are the result of either differential positive selection or purifying selection. The strongest correlate of rate of evolution in this data set appears to be population subdivision, a factor that has long been thought to influence allele frequency and heterozygosity of nuclear genes through genetic drift (Wright, 1949, 1951). Because the mitochondrial genome is maternally inherited in a clonal fashion, the effective population size of mtDNA is even smaller than that of nuclear genes, thereby leading to an even higher rate of fixation through drift in mitochondrial genes than in nuclear genes (Birkey et al., 1983). Effective population size has been implicated as a possible factor influencing rate of evolution in other animals (Crozier et al., 1989; DeSalle and Templeton, 1988; Esteal and Collet, 1994). If high levels of population subdivision, small effective population size, or frequent population bottlenecks can be shown to influence rate of nonsynonymous substitution in genes such as

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cytochrome b, then it follows that these nonsynonymous substitutions must belong to the category of "effectively neutral" substitutions discussed by Kimura (1983) and Ohta (1973,1974,1976,1993). For nearly every idea advanced to explain molecular rate heterogeneity, at least one counter example exists in the literature. Thus, drawing general conclusions about patterns or sources of differences in rate of evolution based on any single data set is risky. Numerous studies have dealt with taxa that exhibit different rates of evolution; however, most studies amount to "point samples" rather than comprehensive analyses including large numbers of taxa. Adding to the confusion, some studies have dealt with nuclear genes and others with mitochondrial genes; the relative importance of factors that influence rate of evolution of mitochondrial genes may differ for nuclear genes. For example, studies comparing rate of evolution in rodents and primates indicate a higher rate of evolution in rodents when based on nuclear data (Catzeflis et al., 1987; Esteal and Collet, 1994; Li et al., 1996) and a higher rate in humans when mitochondrial data are compared (Irwin and Amason, 1994; Janke et al., 1994). It is not yet clear whether this discrepancy between studies based on mitochondrial versus nuclear genes is real or artifactual. Further adding to the confusion is the relationship between increased time since divergence and increased substitutional saturation. Arbogast (in review) has shown that many rate differences attributed to body size, generation time, and metabolic rate disappear when time since divergence is factored into the analysis. For example, many of the large taxa studied by Martin and Palumbi (1993a) diverged relatively long ago, subjecting their DNA sequences to greater amounts of saturation, which

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would artificially reduce the apparent rate of divergence between these taxa. An increasing number of studies report no significant relationship between rate of evolution and metabolic rate (this study; Bromham et al., 1996; Mindell et al., 1996; Mooers and Harvey, 1994) or generation time (this study; Crozier et al., 1989; Zhang and Ryder, 1995). Finally, heterogeneity in rate of evolution may be distributed differently among codon positions of protein-coding regions. Certain studies have provided evidence of heterogeneity in rate of nonsynonymous substitutions, others only in rate of synonymous substitutions, and still others have considered overall amount of change without regard for substitution type. Often, a selective value is assumed for nonsynonymous substitutions and selective neutrality is assumed for synonymous substitutions. However, this distinction may not be so simple; for example, it is possible that neutral forces, such as mutation rate, can influence rate of nonsynonymous substitution as well as rate of synonymous substitution (Mindell et al., 1996). Clearly, there is a need for large-scale, comprehensive analyses of rate of evolution before any general conclusions concerning sources of rate heterogeneity can be reached. Currently, it is impossible to predict which taxa will have similar rates of evolution and which will have different rates, although more distantly related organisms seem far more likely to exhibit significantly different rates. Perhaps, when more data are available, we will learn which factors in an organism's environment, physiology, population biology, or life history are important in determining the rate at which changes will accumulate in its DNA.

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Alternatively, it seems more likely that a large number of variables, and complex interactions among those variables, that can influence rate of evolution of any particular gene. Furthermore, the importance of each variable that influences rate of evolution probably varies over time and across genes, even within a species. Additional evidence suggests that patterns of substitution are not independent among genes, as research in complexity theory has demonstrated an apparent correlation between nucleotide composition at sites located from 100 to 10,000 or more nucleotide positions apart (Amato, 1992; Peng et al., 1992). Therefore, although differences in rate of evolution are known and observable, we may never be able to predict exactly where those differences will exist or what the boundaries of those differences will be simply because there is no way to account for interactions among all of these variables. This suggests that application of complexity theory ("chaos" theory) to studies of evolutionary rates may be informative. The limited number of examples of rate heterogeneity available at present have hinted at some of the factors that may influence rate of evolution (e.g., metabolic rate, generation time, and population size), but as examples of rate heterogeneity increase in number, these initial explanations may be shown to be gross oversimplifications of an extremely complex process.

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Historical Associations of Pocket Gophers and Chewing Lice

In few cases is the strength of association between two organisms greater than in symbionts, such as vertebrate hosts and their invertebrate parasites. Because this association is so strong, congruence in the phylogenies of the two lineages is especially likely to indicate contemporaneous cladogenic events. Comparative studies of host and parasite phylogenies are valuable because they provide for a better understanding of the history of a host-parasite association. Potentially of greater significance, however, is the perspective on molecular evolution that can be gained from studying two or more groups of organisms with common evolutionary histories but vastly different life histories. Congruence in the phylogenies of hosts and their parasites is a pattern commonly acknowledged in parasitology. This phenomenon is described by Fahrenholz’s Rule ("the natural classification of some groups of parasites corresponds with that of their hosts;” Eichler, 1948:588). In some cases, this trend may be the result of strict coevolution ("reciprocal adaptive responses between ecologically interacting species;" Brooks and McLennan, 1991:190). However, in many host-parasite associations, congruence in phylogenies is likely the result of a shared biogeographical history that includes responses to the same fragmentation of ranges through vicariance and host dispersal. Therefore, congruent phylogenies may result either because of strict host-spedficity (classical coevolution) or because the hosts and parasites

68

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have responded in tandem to the same extrinsic factors, such as vicariant events. Regardless of the ultimate cause of the association, this relationship has been referred to as parallel dadogenesis by Futuyma and Slatkin (1983:9). Alternatively, when host and parasite phylogenies are not congruent, it is presumed either that the parasites colonized their respective hosts independently (host switching) or that factors such as sampling error, extinction events, or multiple lineages of parasites cospedating on a single lineage of hosts have led to apparent incongruence in the phylogenies (Page, 1993b). Page (1996:165) described why, even in hosts and parasites that have a strong historical assodation and have experienced little or no host switching, it is unlikely that phylogenies of hosts and parasites will be perfectly congruent. Higher rates of spedation and extinction in parasites than in their hosts or failure of parasites to colonize both descendants of a host spedation event will almost certainly result in differences between the host and parasite phylogenies. Parallel dadogenesis, as reflected by Fahrenholz's Rule, is often thought of as common in parasitological relationships. However, in only a small number of cases has the assumption of parallel dadogenesis been tested rigorously. Crudal to revolutionary studies is the independence of proposed host and parasite phylogenies (Hafner and Nadler, 1990; Hafner and Page, 1995). Frequently, widespread acceptance of Fahrenholz's Rule has lead to the classification of parasites based on host phylogenies (Brooks, 1977; Brooks and Overstreet, 1978) or to inferences of host relationships based on parasite phylogenies (Brooks and Glen, 1982; Hopkins, 1949; Timm, 1983; Wenzel et al., 1966).

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However, it is critical in studies of parallel dadogenesis that host and parasite phylogenies are derived independently, as relationships of one group may have been influential in determining the taxonomy of the other group, thereby introducing circularity into the argument for a shared history (Lyal, 1986). Studies based on independently derived phylogenies for two distantly related groups are still relatively rare, but are increasing rapidly in number (e.g., Baverstock et al., 1985; Chenuil and McKey, 1996; Demastes and Hafner, 1993; Hafner and N adler, 1988; Hafner et al., 1994; Moran et al., 1995; Page et al., in review). Independent colonization provides a suitable null hypothesis of host- parasite relationships that can be tested statistically in coevolutionary studies (Hafner and Nadler, 1988,1990). Although patterns of phylogenesis are rarely identical between hosts and their parasites, associations may show evidence of widespread congruence in which phylogenetic similarity is not absolute, but is statistically more abundant than expected by random association or association due to factors other than common descent. If it can be shown that the phylogenies of hosts and their parasites are significantly more similar than would be expected by chance, then the alternative hypothesis of a shared evolutionary history is supported (Page, 1990). Only associations typified by a statistically significant level of phylogenetic congruence allow the inference that pairs of hosts diverged at approximately the same time as their parasites, thereby permitting examination of relative rates of evolution in the two groups of organisms (Hafner and Nadler, 1990; Hafner and Page, 1995; Page, 1996).

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POCKET GOPHERS AND CHEWING LICE A shared evolutionary history has been demonstrated for several species of pocket gophers and their chewing lice based on the congruence of host and parasite phylogenies as assessed by both allozyme differentiation (Demastes and Hafner, 1993; H afner and Nadler, 1988) and mitochondrial DNA sequences (Hafner et al., 1994). Pocket gophers (Geomyidae) are rodents that lead an almost entirely subterranean existence. Their underground burrows are typically plugged with soil at all entrances. Pocket gophers are asocial animals, only known to purposefully encounter other individuals during brief mating encounters and during the maternal care of offspring. Consequently, the asocial, fossorial nature of pocket gophers, coupled with the non­ overlapping geographic distribution of gopher species, makes widespread transfer of parasites between species of gophers unlikely. Chewing lice of pocket gophers are wingless insects of the order Phthiraptera (Trichodectidae) that feed on skin detritus (Marshall, 1981) and possibly on other arthropods found on the host (Oniki and Butler, 1989). The entire life cycle of chewing lice (egg, nymph, and adult) takes place on the host. Because chewing lice are wingless, obligate ectoparasites, transfer of chewing lice between gophers is thought to occur only during physical contact between hosts (Timm, 1983). Thus, the natural histories of both the gophers and lice result in few opportunities for host switching. Therefore, much of the phylogenetic congruence observed in this host-parasite assemblage may result from the asocial nature and allopatric distribution of the pocket gopher hosts, which could produce islands of chewing louse habitat that correspond

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directly to the geographic distribution of pocket gopher taxa. Data from transfer experiments indicate that some spedes of chewing lice are capable of living on pocket gophers on which they do not naturally occur and that lack of opportunity to colonize new host taxa may be an important factor driving cospedation between gophers and lice (Reed and Hafner, 1997). The patchy distribution of pocket gopher individuals, populations, and spedes seems to be of primary importance in limiting these colonization opportunities (Demastes, 1996). Regardless of the cause of cospedation between pocket gophers and chewing lice, their shared history has allowed comparison of genetic divergence in the orthologous DNA sequences of radically different organisms over common periods of time (Hafner et al., 1994; Page, 1996). These analyses have suggested that chewing lice have a higher rate of evolution of the mitochondrial cytochrome c oxidase subunit I gene (COI) than do pocket gophers, espedally when one considers changes in the third positions of codons (primarily four-fold degenerate sites in which changes in nudeotide composition do not alter amino add composition). However, this trend deserves further examination. By using different models of molecular evolution and varying hypotheses about which taxa have cospedated, resulting hypotheses of rate differences between hosts and parasites are altered (Hafner and Page, 1995; Page, 1996). Analysis of other groups of gophers and lice should provide a broader foundation for the study of patterns of cospedation and rates of evolution among cospedating taxa.

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SPECIFIC AIMS OF THIS STUDY This study focuses on the comparison of nucleotide sequence differentiation, phylogenetic relationships, and relative rates of evolution of a portion of the COI gene in two lineages of symbiotic organisms, pocket gophers and chewing lice. Because a mitochondrial gene is examined, the relationships discussed here represent mitochondrial gene trees, rather than species trees. It has already been demonstrated that mitochondrial gene trees do not always reflect biological species boundaries (Patton and Smith, 1994), and a variety of processes in the evolution of any single gene may lead to incongruence between divergence in this gene and divergence among populations (Doyle, 1992; Pamilo and Nei, 1988; Patton and Smith, 1994). Nevertheless, previous work has shown that gopher and louse mtDNA phylogenies are generally consistent with phylogenies based on nuclear- encoded characters (i.e., allozymes, morphological characters, chromosomal characters; Hafner and Nadler, 1988; Hafner et al., 1994; Page et al., 1995). Future studies of cospedation between pocket gophers and chewing lice based on nudear DNA will provide additional information concerning patterns of cospedation and relative rates of evolution in other genes; however, this study will be restricted to determining if the mitochondrial DNA of pocket gophers and their chewing lice provides evidence of cospedation among these organisms, and if so, how the relative rates of evolution compare among these organism s. The hosts studied here are pocket gophers of the genus Thomomys, a genus induded in previous cospedation studies only as an outgroup.

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The genus Thomomys consists of seven species that range over the western United States, southwestern Canada, and much of Mexico (Hall, 1981). Species of Thomomys are generally allopatric in distribution, and the few zones of contact between congeneric species are usually narrow zones of parapatry. The parasites studied here include many species of chewing lice that have not been examined previously using molecular data. These lice belong to two genera, Geomydoecus and Thomomydoecus, both restricted to pocket gophers; together they include 122 recognized species and subspecies (Hellenthal and Price, 1994; Page et al., 1995). Many individual gophers of the genus Thomomys host both genera of lice. However, it is unusual for a pocket gopher population to host more than one species of louse from each genus; i.e., congeners rarely are sympatric (Hellenthal and Price, 1984a). Because the two genera of chewing lice appear to be reciprocally monophyletic (Hellenthal and Price, 1984b; Nadler and Hafner, 1993), they offer two independent lineages of parasites that can be compared with Thomomys. MATERIALS AND METHODS Specimens Examined Representatives of all seven species of Thomomys were examined, including specimens from 20 localities (Table 3.1, Appendix D). Lice of the genus Geomydoecus were collected from each of these 20 localities, whereas representatives of the genus Thomomydoecus were present at only nine localities (Table 3.1). Louse specimens were identified to species level based on morphological features (Hellenthal and Price, 1994).

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Table 3.1—Spedmens examined of pocket gophers ( Thomomys ) and chewing lice {Geomydoecus and Thomomydoecus ). Locality numbers refer to sites listed in Appendix D.

Hockfit Gophers______Chewing Lice Locality Thomomys Geomydoecus Thomomydoecus number

subgenus subgenus subgenus Thomomys T h a e le riu s Jamespattonius T. mazama couchi G. thaeleri 1 T. mazama mazama G. thaeleriP 2 T. monticola G. c r a ig i 3 T. talpoides £ossor G. thomomyus T. a r le n a e 4 T. talpoides immunisa G. thaeleri^ 5 T. talpoides levis G. craigi T. zacatecae^ 6 T. talpoides nebulosus G. thomomyus T. barbarae 7

subgenus subgenus subgenus Megascapheus Geomydoecus Thomomydoecus T. bottae connectens G. aurei T. minor 8 T. bottae leucodon G. shastensis 9 T. bottae opulentus G. centralis T. minor 10 T. bulbivorus G. oregonus 11 T. townsendii bachmani G. idcihoensis 12 T. townsendii townsendii G. idahoensis 13 T. umbrinus atrovarius G. m u scu li 14 T. umbrinus chihuahuae G. chihuahuae T. asymmetricus15 T. umbrinus chihuahuae G. wannanaec T. asymmetricus 16 T. umbrinus goldmani G. w e l l e r i c 17 T. umbrinus intermedius G. crovelloi T. bimeyi 18 T. umbrinus pullus G. welleric 19 T. umbrinus tolucae G. welleric T. neocopei 20 a Specimens from this locality have been reported as T. talpoides previously; however, these specimens are not morphologically distinguishable from T. mazama. b Louse population densities were very low at these localities; only females were collected. Because females of this species are indistinguishable from G. betleyae, identifications listed are not definitive. c Identification is inferred from specimen collection locality (Price and Hellenthal, 1981a, 1981b). d Thomomydoecus zacatecae is a member of the subgenus Thomomydoecus. Most other populations of Thomomys talpoides host chewing lice of the subgenus Jamespattonius (Hellenthal and Price, 1991).

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DNA Isolation. Cloning, and Sequencing Genomic DNA was isolated from rodent liver or kidney tissues following the technique outlined by Hillis et al. (1990). Louse DNA was extracted by grinding a single individual in a solution of 100 mM EDTA (pH 8.0), 10 mM Tris (pH 8.0), 10 mM NaCl, and 2 mg/ml proteinase K. The mixture was incubated at 55°C for at least six hours (up to overnight) and followed by one phenol-chloroform extraction. DNA was precipitated overnight, on ice, in 2.5 volumes of 95% ethanol with 10 pg of yeast transfer RNA, and then dried under vacuum. DNA from a single louse was resuspended in 50 pi of water. An orthologous 379 base-pair region of the COI gene was amplified from the DNA of pocket gophers and chewing lice using the polymerase chain reaction (PCR; Saiki et al., 1986,1988). Amplifications were performed using the following degenerate primers: L6625 (5- CCGGATCCTTYTGRTmTYGGNCAYCC-3') and H7005 (5- CCGGATCCACNACRTARTANGTRTCRTG-3’). Primer names indicate the DNA strand (H=heavy or L=light) and the position of the 3' end of the oligonucleotide sequence of humans (Anderson et al., 1981). One microliter of genomic DNA was used in a 50 pi PCR reaction, which included 3 pi of each primer (10 pM), 3 fil of deoxynucleoside- triphosphate mixture (dATP, dGTP, dCTP, and dTTP, each 1 mM), 3 pi of MgCl2 (25 mM), and 1-2 units of Thermus aquaticus DNA polymerase (Promega Corporation, Madison, WI). The reactions were carried out in a solution of 50 mM KC1,10 mM Tris-HCl (pH 9.0), and 0.1% Triton X- 100. Four thermal-cycles were performed, each with a 1 min. denaturation at 95°C and a 1 min. annealing period at 45°C, followed by

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a 1 min. extension period at 72°C. Twenty-five to 30 additional cycles were performed with the same parameters, except that denaturation was carried out at 94°C and annealing at 60°C. Each PCR reaction was completed with a 10 min. extension period at 72°C. Fresh PCR products were cloned using the TA cloning kit of Invitrogen (San Diego, CA) or the pT7Blue T-Vector cloning kit of Novagen (Madison, WI). Plasmid DNA containing the COI insert was isolated from 5 ml bacterial cultures using the Spin-Bind Miniprep kit sold by Farmada (Rockland, ME). Sequencing reactions were performed on the denatured plasmid DNA using T7 DNA polymerase (Sequenase version 2.0, United States Biochemical, Cleveland, OH) and following standard dideoxy chain-termination protocols (Sanger, 1977). Sequencing primers complementary to the plasmid were used. Generating sequence data from doned DNA allowed use of sequencing primers that matched the vector perfectly and, therefore, provided more reliable sequences than could be produced using the degenerate PCR primers in sequencing reactions. Also, this approach was important in determining COI sequences of chewing lice, because it prevented mistakes that could result from amplification of residual pocket gopher DNA from the gut of the louse. To guard against errors in nudeotide-sequence determination resulting from errors in PCR, sequence data were collected from dones derived from at least two different PCR reactions. Data. Analysis The MacSequence program (version 4.2, D. A. Good, unpublished) was used to determine the number of potentially phylogenetically

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informative nucleotides and pairwise numbers of substitutions between taxa in each data set. Maximum-likelihood estimates of relationships were determined using the UNIX-based fastDNAml program (Felsenstein, 1981; Olsen et al., 1994). To increase the probability of finding the best maximum-likelihood tree, a variety of transition:transversion ratios were incorporated into the model, hi addition, the input order of taxa was jumbled, and the global rearrangement option was used in both programs until a tree with the best log-Iikelihood score was found a minimum of four times. Maximum-likelihood bootstrap analysis also was conducted using the fastDNAml program. Parsimony analysis was performed using the computer program PAUP (Phylogenetic Analysis Using Parsimony, version 3.1.1; Swofford, 1990). Heuristic searches of tree topology (varying the input order for 20-40 replicates) and bootstrap analyses were conducted with third position transitions and transversions weighted by the optimal ratio obtained horn maximum-likelihood analyses. Pairwise estimates of sequence divergence among taxa were generated also using this optimal ratio with the maximum-likelihood option of PHYLIP (Phylogeny Inference Package; Felsenstein, 1993). Lengths of alternative trees were compared using the MacClade program, with all character-state changes weighted equally (Maddison and Maddison, 1992). To determine if parallel dadogenesis predominates the history of this host-parasite assodation, host and parasite phylogenies were compared to determine if their topological similarity was greater than expected by chance. The TreeMap program (Page, 1994b) was used to reconcile all

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host and parasite trees to estimate the number of potential cospedation events between the two groups. Tests of significance were conducted by randomizing the parasite trees 1,000 times; the fit between these randomly generated parasite trees and the actual host tree was compared to the fit between the actual host and parasite trees. The Fit command of the COMPONENT program (Page, 1993a) also was used to reconcile the host and parasite dadograms, thereby estimating the minimum number of events that must have occurred ("number of leaves added" and "number of losses;" Page, 1994a) to produce the observed level of incongruence in the host and parasite trees if no host switching has occurred. Tests of significance were conducted by comparing the measure of fit between the actual host and parasite trees to the fit between the parasite tree and 1,000 randomly generated host trees using the Random and Map onto all trees commands. Host and parasite genetic data were further analyzed for significant assodation by comparing maximum-likelihood sequence-divergence matrices (Mantel, 1967) through the R-Package program (Legendre and Vaudor, 1991). The null hypothesis of this test is that there is no linear relationship between the two matrices (Legendre and Vaudor, 1991). If this test indicates a greater assodation between distance matrices than would be expected by chance, then host-parasite assodations are considered to be a result of common evolutionary histories (Demastes and Hafner, 1993). Clock-like behavior of DNA substitutions within the pocket gopher and chewing-louse lineages was tested using relative-rate tests (Allard and Honeycutt, 1992; Mindell and Honeycutt, 1990). Relative rates of

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evolution of gophers and lice were estimated through comparisons of branch lengths of gopher and louse maximum-likelihood trees generated using the optimal maximum-likelihood transition: transversion ratio. Orthologous host and parasite branches were compared and model I and model II regression was used to find the slope of the best-fit line using the SYSTAT program (version 5.2, SYSTAT, Inc., Evanston, IL). RESULTS AND DISCUSSION Phylogenetic .Relationships .of Chewing Lice Sequence data were collected for multiple louse individuals at four localities (14,15,19, and 20; Appendix D). Level of sequence divergence am ong Geomydoecus chewing lice from the same host individual, measured using maximum-likelihood with a transitionrtransversion ratio of 3:1, ranged from 0.3% to 0.5%. Sequence divergence among chewing lice from different host individuals collected at the same locality ranged from 1.4% to 2.4%. Measured using allozyme data, variance in allele frequencies of chewing lice collected from different hosts at the same locality averaged 9.2% (Nadler et al., 1990). The COI sequence data collected for Thomomydoecus and Geomydoecus (Appendices E and F) included 71 and 144 potentially phylogenetically informative positions, respectively. Of 127 third positions in codons, 98 (in Thomomydoecus ) and 125 (in Geomydoecus) were variable (the number of variable positions is inflated by autapomorphies at 27 and seven of these positions in Thomomydoecus and Geomydoecus, respectively). Of 126 first positions, 9 ('Thomomydoecus) and 27 ( Geomydoecus ) were variable. Only one

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second position was variable in Thomomydoecus (an autapomorphy), and ten second positions were variable in Geomydoecus. Uncorrected sequence divergence values ranged from 1.9% to 17.4% in Thomomydoecus and from 7.7% to 27.0% in Geomydoecus. Phylogenetic trees were rooted between the two genera ( Geomydoecus and Thomomydoecus), such that each genus was used to root relationships within the other genus. Maximum-likelihood analysis of the COI sequence data was conducted with transition:transversion ratios of 1:1,2:1,3:1, and 4:1. The 3:1 ratio produced the tree (Fig. 3.1) with the best log-likelihood score (-4803.74511). Parsimony analysis, which was conducted using the same ratio (by giving third position transitions one- third of the weight of other character-state changes), produced a single most parsimonious tree (Fig. 3.2) that is largely congruent with the maximum-likelihood tree. One-thousand bootstrap replicates, performed under the same parameters, indicated good support for most of the nodes in this tree (Fig. 3.2). All phylogenetic analyses of these COI data indicated the existence of two clades within the genus Geomydoecus; these clades correspond to the subgenera Geomydoecus and Thaelerius (Table 3.1), which were recognized by Hellenthal and Price (1994) on the basis of morphological data. The placement of G. chihuahuae (locality 15) basal to all other members of its genus (Figs. 3.1-3.2) was unexpected based on taxonomy, given that this species is currently placed in the subgenus Geomydoecus (Table 3.1). Nevertheless, a dadistic analysis of morphology (Page et al., 1995) also indicated th at G. chihuahuae belongs to a lineage that diverged from other members of the genus prior to the main divergence

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G. thaeleri—5 G. thaeleri—1 G. thaeleri—2 G. craigi—3 G. craigi—6 G. thomomyus—4 G. thomomyus—7 G. idahoensis—12 G. idahoensis—13 G. oregonus—11 G. shastensis—9 G. welleri—20 G. welleri—17 G. welleri—19 G. warmanae—16 G. aurei—8 G. crovelloi—18 G. musculi—14: G. centralis—10 G. chihuahuae—15 T. minor—8 T. mznor—10 T. asymmetricus—15 T. asymmetricus—16 T. zacatecae—6 T. neocopei—20 T. bimeyi—18 91 r—— T. barbarae—7 I— T. arlenae—4

Fig. 3.1—Maximum-likelihood estimate of relationships among two genera of chewing lice, Geomydoecus and Thomomydoecus. Specimen names are followed by locality numbers (Appendix D). Bootstrap values are indicated above each branch present in more than 50% of 100 replicates.

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100 G. thaeleri—5 G. thaeleri—1 G. thaeleri—2 G. craigi—3 G. craigi—6 G. thomomyus—4 G. thomomyus—7 100 G. idahoensis—12 G. idahoensis—13 G. oregonns—11 G. shastensis—9 G. welleri—20 G. crovelloi—18 G. warmanae—16 84 G. aurei—8 G. welleri—19 G. welleri—17 G. musculi—14: G. centralis—10 G. chihuahuae—15 991— T. minor—8 _ l I— T. minor—10 | r — T. asymmetricus—15 ■— T. asymmetricus—16 — T. zacatecae—6 —————— T. neocopei—20 — — T. bimeyi- 18 861— T. barbarae—7 T. arlenae—4.

Fig. 3.2—Results of parsimony analysis of COI sequences of two genera of chewing lice, Geomydoecus and Thomomydoecus. Specimen names are followed by locality numbers (Appendix D). Bootstrap values are indicated above each branch present in more than 50% of 1,000 replicates.

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between the two subgenera. Therefore, these COI trees (Figs. 3.1-3.2) are largely congruent with the major divisions previously hypothesized w ithin Geomydoecus . Likewise, monophyly of the two subgenera of Thomomydoecus (Jamespattonius and Thomomydoecus; Hellenthal and Price, 1994) was supported by analysis of the COI data. These COI data seem to be most informative phylogenetically in those areas of the trees for which morphology (Page et al., 1995) is least informative. For example, relationships among members of the Geomydoecus subgenus Thaelerius were supported by relatively high bootstrap values at every node of the COI tree, whereas analysis of morphological data for these taxa produced an unresolved polytomy (Page et al., 1995). Similarly, morphological data did not resolve relationships among most Thomomydoecus species, except in supporting monophyly of the subgenus Thomomydoecus. In contrast, the COI data seemed to be informative with respect to most Thomomydoecus relationships. This suggests that future study of chewing louse relationships based on COI sequences, with the inclusion of more taxa, will further clarify relationships among species of chewing lice. Relationships among certain members of the subgenus Geomydoecus were not well resolved by analysis of the COI data. Parsimony and maximum-likelihood analysis provided different hypotheses of relationships of six of these taxa, and corresponding bootstrap values were low (Fig. 3.3a, b). Only the maximum-likelihood tree depicted Geomydoecus welleri as a monophyletic group. The cladistic analysis of morphology by Page et al. (1995; Fig. 3.3c) indicated a third hypothesis of

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G. idahoensis —12 G. idahoensis —13 G. oregomzs—11 G. shastensis—9 G. welleri—20 G. welleri—17 G. welleri—19 68 G. warmanae—16 G. aurei—8 G. crovelloi —18 G. mMSCw/z—14 G. centralis—10 G. idahoensis —12 G. idahoensis —13 1 * ^ . G. oregonus—11 G. shastensis—9 / D G. welleri—20 52 80 r— G. crovelloi —18 _ l L ~ G. warmanae—16 G. aurei—8 M 85 j------G. welleri—19 G. welleri—17 G. musculi—14 G. centra/is—10

c G. idahoensis G. oregonus G. welleri G. shastensis G. croveloi G. warmanae G. aurei G. centralis G. musculi

Fig. 3.3—Areas of disagreement between COI maximum-likelihood (a) and parsimony (b) trees. Bootstrap values are indicated above each branch present in more than 50% of 100 (a) and 1,000 (b) replicates. Relationships among Geomydoecus estimated horn morphology (c; from Page et al., 1995) are in conflict with the relationships estimated from COI data.

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relationships. Although Page et al. sampled taxa within this lineage more thoroughly than was done in the present study, the relationships proposed by Page et al. are strongly contradicted by the COI data. For example, monophyly of the oregonus species complex (G. idahoensis , G. oregonus, and G. shastensis; Price and Hellenthal, 1980), which was supported by the COI data in 87% to 94% of bootstrap replicates, was not supported by the morphological analysis. Moreover, imposing the arrangement of taxa inferred from the morphological analysis on the COI sequence data produced a far less parsimonious result than the most parsimonious COI tree; a minimum of 13-31 additional character-state changes were required (depending on whether G. shastensis was eliminated or retained in comparisons) to make this region of the tree congruent with that estimated from morphology. In summary, relationships indicated by parsimony and maximum- likelihood analysis of the COI data are very similar, and only disagree with respect to relationships among certain Geomydoecus populations shown in Fig. 3.3 (localities 8,14, and 16-20). To account for these differences between results of the two analyses, both the parsimony and maximum-likelihood COI trees (Figs. 3.1 and 3.2) were used in tests of cospedation (below) to accommodate ongoing uncertainty in the estimation of phylogenetic relationships among these spedes. Phylogenetic Relationships of Socket Gophers The COI sequence data collected for pocket gophers (Appendix G) included 111 positions that were potentially phylogenetically informative. Of 127 third positions in codons, 118 were variable (15 of these positions consisted of autapomorphies). Of 126 first positions, ten

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were variable; only one second position was variable (an autapomorphy). Uncorrected sequence divergence values ranged from 5.3% to 19.3% in Thomomys. Phylogenetic trees of pocket gophers were rooted between the two subgenera, Megascapheus and Thomomys. These two groups are distinct in both morphological and chromosomal characteristics (Thaeler, 1980). Furthermore, parsimony analysis, using the COI sequence of another pocket gopher (Pappogeomys; Hafner et al., 1994) as an outgroup, indicates that this is the appropriate rooting for relationships within Thomomys. Maximum-likelihood analysis of pocket gopher relationships was conducted with transition:transversion ratios of 1:1,2:1,3:1, and 4:1. A 3:1 ratio produced the tree (Fig. 3.4a) with the best log-likelihood score (-2755.64056). Parsimony analysis, which was conducted using the same ratio (by giving third position transitions one-third the weight of other character-state changes), produced a single most parsimonious tree (Fig. 3.4b). Maximum-likelihood and parsimony bootstrap analyses (Fig. 3.4a, b) indicated support for most of the regions of agreement between the two trees. Both maximum-likelihood and parsimony analyses indicated the same relationships among pocket gophers of the subgenus Thomomys (T. mazama, T. monticola, and T. talpoides). Likewise, both analyses indicated a close relationship between two of the T. bottae populations (8 and 10) and T. townsendii. Many of the relationships among populations of T. umbrinus, and the phylogenetic position of T. bottae from locality 9 (Jackson Co., Oregon) were not as well resolved, however. Hafner et al. (1987) used allozymic and chromosomal data to

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T. bottae—8 T. bottae—W T. townsendii—12 T. townsendii —13 T. umbrinus—15 T. umbrinus—16 T. umbrinus—17 T. umbrinus—18 T. umbrinus—19 T. umbrinus—20 T. bottae—9 T. umbrinus—14 T. bulbivorus—11 T. monticola—3 T. talpoides—4 T. talpoides—7 T. talpoides-6 T. talpoides—5 T. mazama—1 T. mazama—2

T. bottae—S T. bottae —10 T. townsendii—12 T. townsendii —13 T. umbrinus—15 T. umbrinus—16 T. umbrinus—17 T. umbrinus—18 T. umbrinus- 19 T. umbrinus—20 99 T. bottae—9 T. umbrinus—14 T. bulbivorus—11 T. monticola—3 T. talpoides-4 T. talpoides-7 T. talpoides-6 T. talpoides—5 T. mazama—1 T. mazama—2

Fig. 3.4—Results of maximum-likelihood (a) and parsimony (b) analysis of COI data from seven species of pocket gophers ( Thomomys ). Specimen names are followed by locality numbers (Appendix D). Bootstrap values are indicated above each branch represented in more than 50% of 100 and 1000 replicates, respectively.

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explore relationships among T. umbrinus populations and obtained results similar to those presented here. For example, T. umbrinus populations 15 and 16 appear to be closely related based on the COI data; these populations correspond with the Southern and Northern Sierra Madre populations examined by Hafner et al. (1987), which share a diploid chromosome number of 76. Likewise, populations 17-20 appear to be closely related based on COI data; these populations correspond with the Northern Desert and Central Plateau populations examined by Hafner et al., which share a diploid number of 78. Parsimony analysis of the COI data (Fig. 3.4b) and phenetic analysis of allozyme data (Hafner et al., 1987) both indicated that T. umbrinus atrovarius (locality 14) is the most divergent form of T. umbrinus examined. In contrast, maximum- likelihood analysis of COI sequences depicted the Southern and Northern Sierra Madre populations (15 and 16) as the most divergent representatives of T. umbrinus. Therefore, in this respect, the results of parsimony analysis of the COI data are slightly better corroborated by the allozyme data than are the results of maximum-likelihood analysis. Both parsimony and maximum-likelihood hypotheses of relationships indicate that several species of pocket gophers are not monophyletic (i.e., T. bottae, T. talpoides, and T. umbrinus). However, Patton and Smith (1994) have demonstrated that biological species boundaries in pocket gophers may encompass paraphyletic and polyphyletic groups because allozyme and DNA sequence data both suggest that certain populations of T. bottae are more closely related to T. townsendii than to other T. bottae populations (Patton and Smith, 1994). Similarly, allozyme data suggest that some populations of T. bottae are

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more similar to T. umbrinus populations than to other T. bottae populations (Patton and Yang, 1977). Therefore, although several spedes of pocket gophers do not appear as monophyletic groups in Fig. 3.4, these trees nevertheless may portray an accurate history of population divergence. Several uncertainties remain concerning relationships among these populations of pocket gophers. In light of these uncertainties, both phylogenetic trees (Fig. 3.4a, b) were considered as possible explanations of gopher relationships in subsequent comparisons with parasite phylogenies. Historical Assotiations Between Hosts and Parasites Individuals of Thomomydoecus minor horn localities 8 and 10 (9 km apart) showed only 1.9% sequence divergence (measured using maximum-likelihood distances with a transition:transversion ratio of 3:1). This level of divergence is similar to that observed between different individuals of Geomydoecus from the same locality (1.4-2.4%), suggesting that these two representatives of Thomomydoecus minor likely represent the same population. Although the hosts (T. bottae connectens and T. b. opulentus) from which these chewing lice were collected are more dosely related to each other than to other pocket gophers induded in this analysis (Fig. 3.4), they differ in 6.2% of their COI sequence. Additionally, these pocket gophers show a number of allozymic and chromosomal differences that suggest a long history of divergence followed by relativdy recent secondary contact (Demastes, 1996; Smith et al., 1983). Therefore, these pocket gopher subspedes probably share the same population of Thomomydoecus through recent

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host switching. To prevent the false appearance of cospeciation between these pocket gophers and their chewing lice, only one Thomomydoecus minor individual (locality 10) was retained in comparisons of tree topology. The exclusion of T. minor (locality 8) from the analysis does not afreet the results of parsimony or maximum-likelihood analysis of relationships among Thomomydoecus species. Both estimates of chewing louse relationships (Figs. 3.1-3.2) are more similar to both pocket gopher trees (Fig. 3.4a, b) than expected by chance (P < 0.001 for all comparisons using Component [Page, 1993a]; P < 0.02 for all comparisons using TreeMap [Page, 1994b]). For each of the four possible tree comparisons, 15 cospedation events were estimated by TreeMap. For three of these comparisons, this maximum number of cospeciation events was obtained only by assuming that some switching of hosts has occurred in the history of the chewing lice. One of these presumed host switches involves Thomomydoecus zacatecae (locality 6); the fit between host and parasite trees in each of the four comparisons is either unaffected or is improved by making this assumption. Thomomydoecus zacatecae most commonly lives on Thomomys bottae ; however, a few populations of Thomomys talpoides are known to host this louse. Hellenthal and Price (1991) suggested previously that the Thomomydoecus zacatecae that occur on Thomomys talpoides are the result of host switching among distantly related pocket gophers, with Thomomydoecus zacatecae invading Thomomys talpoides some time after the divergence of Thomomys talpoides and Thomomys bottae . Other presumed host switches are unique to only one of the four reconstructions. All other cases of incongruence between the host and

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parasite phytogenies can be accounted for by "duplication" and "sorting" events (Page, 1996), suggesting the possibility that the general pattern of parallel cladogenesis in this host-parasite assemblage has been perturbed on occasion by multiple lineages of chewing lice inhabiting a single pocket gopher lineage ("duplication" event) followed by random extinction of certain members of these chewing louse lineages ("sorting" event). The Mantel test for association between the maximum-likelihood distance matrices for chewing lice and pocket gophers (corrected for multiple substitutions using a 3:1 transition:transversion bias) indicated greater association than expected by chance in 1,000 permutations (r = 0.66 and 0.49, P = 0.001 and 0.02, for Geomydoecus and Thomomydoecus comparisons, respectively). The results of this test provide further evidence for a widespread history of cospeciation among these hosts and parasites. Evidence for cospeciation between pocket gophers and chewing lice seems to come predominantly from a few lineages. For example, the host and parasite phytogenies are most congruent between members of the Thomomys talpoides species group and their lice ( Geomydoecus , Fig. 3.5 and Thomomydoecus, Figs. 3.6-3.7). There also is evidence of parallel cladogenesis involving some members of the Thomomys bottae group; Thomomys townsendii populations form a dade, as do their lice of the genus Geomydoecus (G. idahoensis ; Fig. 3.5), and Thomomys umbrinus populations 15 and 16 form a dade as do their lice of the genus Thomomydoecus (Figs. 3.6-3.7). The maximum-likelihood tree for pocket gophers (Fig. 3.6) indicates a cospedation event involving

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craigi thaeleri thaeleri welleri-, welleri- G.aurei- welleri- G. G. G. craigi musculi— G. G. G. G. thaeleri G. oregonus- crovelloi— G. G. G. G. idahoensis- G. G. thomomyus- centralis- thomomyus- chihuahuae- q G. shastensis G. G. G. q

G. G. G. warmanae- G. G. G. G. G. ---- G...... • ----- ...... indicated by maximum-likelihood analysis ofCOI ...... / - -/v •/ ' \ \ % \ % / \ . G. J>

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- 4 10 - 8 Geomydoecus umbrinus— talpoides-6 m azam a-l mamma-2 ---y-monticola townsendii—ll*'-,4---' idahoensis— G. and T. T. umbrinus-19 T. talpoides T. talpoides—5 T. T. bottae T. T. umbrinus-18 T, umbrinus~\7--i-? T. bottae-9 T. T. umbrinus—\5 T. umbrinus~l6 T. umbrittus-20 T. bulbivorus-lV T. T. bottae- T. T. townsendii—1 T. talpoides-7 T. T. T. T. T. Thomomys cospeciation events. Parsimony analyses indicate thesame setof cospeciation events. Maximum-likelihood Fig. 3.5—Relationshipsof sequencedata. Specimen namescorrespondingare followedhost by forlocalityeach parasite. numbers (Appendix Letters D). identifybranch Dashed lengths analogous lines are indicatebranches given in Table the resulting3.2. from potential

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vC 4— (right) indicated by 6 16— 18 10 Thomomydoecus arlenae-4 minor- birneyi- barbarae-7 zacatecae- neocopei-20 T. T. T. T. T. (left) and flsywmetricMs-15 asymmetricus- T. T. T. v, - T . - --

---- T.

Thomomys ...... 4 1 0 talpoides-7 umbrinus—15 talpoides- umbrinus-20 umbrinus-18 -y--- umbrinus-18 bottae- T. umbrinus-16 T. T. talpoides-6''' T. ■T. ■T. ■T. ■T. B B ■ ■ 1— T. £ T. identify analogousbranches resulting from potential cospeciation events. Maximum-likelihood Fig. 3.6~Comparison ofrelationships of maximum-likelihood analysisnumbersof COI sequence (Appendix data. D). Specimen Dashed names lines indicateare followed the by corresponding locality host foreach parasite. Letters branch lengths for these branches are given inTable 3.2.

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16 15 18 Thomomydoecus barbarae—7 neocopei-20 T, arlenae-A T, T. minor-10 T. T. birneyi- T. T. T. T. zacatecae—6 T.

(left) and T. T. asymmetricus- T. T. asymmetricus- T. -y--- ...... Thomomys ------y * '- — - - — ...... 18 ...... 4 1 0 umbrinus-15 umbrinus-16 talpoides-6''' talpoides- T. T. talpoides—7 T. T. T. bottae- T. T. umbrinus- T. T. T. umbrinus—20 T. T. T. E H .. A fHt HZ H H Fig. 3.7--Comparison ofrelationships of by parsimony analysisof gopher analysisCOI sequencesof the louse andCOI sequences. by parsimony(Appendix Specimen and D). maximum-likelihoodnames Dashed arelinesidentify followed indicate analogous by localitythe corresponding branchesnumberslikelihood resulting host branch fromforeach lengthspotential parasite. for cospeciationthese branches Letters events. are given Maximum-in Table 3.2.

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Thomomys bottae and its louse ( Thomomydoecus minor) that is not indicated in the comparison using the parsimony tree for pocket gophers (Fig. 3.7). Potential cospeciation between Thomomydoecus and pocket gophers in each of the two subgenera ( Megascapheus and Thomomys ) leads to the inference of cospeciation deep in the history of the groups, possibly beginning at the time that the two subgenera of pocket gophers diverged (branch G, Fig. 3.6; branch H, Fig. 3.7). Although allozyme data presented by Hafner and Nadler (1988) for two of these hosts and their parasites suggest that these pocket gophers spedated prior to the corresponding spedation event in their lice, analysis of COI sequences (Hafner et al., 1994) suggests that spedation events in these hosts and parasites were approximately contemporaneous. If Thomomydoecus has been cospedating with Thomomys since the divergence of the two pocket gopher subgenera, many extinction events must have occurred in the history of the Thomomydoecus lineage to result in the current situation, in which many Thomomys populations do not host lice of the genus Thomomydoecus. Gaps in the distribution of Thomomydoecus (relative to that of its host, Thomomys ) are limited primarily to populations of pocket gophers occurring in the northwestern U. S. (Hellenthal and Price, 1984a, 1989), possibly indicating widespread extinction of Thomomydoecus in this region due to some aspect of the environment in these areas. The patchy, isolated distribution typical of pocket gophers would certainly increase the possibility of local extinction of parasites. It is not dear, however, why Geomydoecus in this region of the country seem not to have been similarly affected.

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Species of the two subgenera of Geomydoecus occur on different groups of pocket gophers (Hellenthal and Price, 1994). Members of the subgenus Thaelerius inhabit primarily pocket gophers of the Thomomys talpoides group (subgenus Thomomys ). In contrast, species of the subgenus Geomydoecus inhabit gophers of the Thomomys bottae group (subgenus Megascapheus) plus all other genera of pocket gophers. Subgenera of Geomydoecus seem to be reciprocally monophyletic based on both morphological (Page et al., 1995) and molecular data (this study). Because the subgenus Geomydoecus inhabits members of all genera of pocket gophers, whereas its sister group (subgenusThaelerius) inhabits only members of the Thomomys talpoides group, it appears that there has not been a perfect pattern of cospeciation among pocket gophers and their lice. This pattern of closely related lice inhabiting distantly related pocket gophers suggests the possibility that lice of the genus Geomydoecus currently hosted by Thomomys represent one or more lineages that moved onto Thomomys relatively recently from other genera of pocket gophers. According to this scenario, divergence between subgenera of Geomydoecus (branch S, Fig. 3.5) probably occurred before the divergence between subgenera of Thomomys, and therefore does not involve cospeciation. Nevertheless, there are several possible explanations for current host-parasite associations, and the sequence of events that has lead to present distributional patterns of pocket gophers and chewing lice must have been complex (Page et al., 1995). A more comprehensive study of pocket gopher and chewing louse associations, relative to the phylogeny of both groups, will be required before this history can be elucidated.

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Relative Rates of Evolution Within Each Group Before comparisons of rate of evolution can be made between pocket gophers and chewing lice, it is important to ensure that these comparisons will not be confounded by heterogeneity in rate of evolution w ithin either group (Page and Hafner, 1996). Accordingly, several relative-rate tests were conducted both within and between subgenera of pocket gophers (using bothPappogeomys and Thomomys taxa as outgroups) and within and between subgenera of chewing lice (using Geomydoecus as the outgroup for Thomomydoecus, and vice versa, and also using more closely related lice as outgroups). These relative-rate tests included, but were not limited to, all possible comparisons between sister taxa of pocket gophers and between sister taxa of chewing lice thought to have cospedated (lettered branches in Figs. 3.5-3.7). Among pocket gopher taxa, overall rate of substitution did not differ significantly in eight different comparisons, regardless of which outgroup taxon was used (binomial test, P > 0.26). Similarly, among chewing lice, no significant difference in rate of substitution was detected, regardless of which outgroup taxon was used (binomial test, P > 0.26 in 10 different comparisons). Results of the Mantel test (described above) provide further evidence for homogeneous rates of evolution within each group (gophers and lice). For example, if rate of evolution varied among members of one group, but was not mirrored by rate heterogeneity in the corresponding members of the other group, then the chance of obtaining a significant result is decreased (Hafner and Nadler, 1990). Therefore, the results of these tests provide no evidence for heterogeneity in rate of evolution in this region of the COI gene

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among pocket gophers or among chewing lice. Because rate of evolution in both pocket gophers and chewing lice does not depart significantly from clock-like behavior, direct comparisons of rate of evolution between these hosts and parasites are appropriate (Page and Hafner, 1996). Do Chewing Lice Have a Higher Rate of Evolution? Only those pairs of hosts and their parasites that have a common evolutionary history can be inferred to have diverged at approximately the same time. It follows that associations that are the result of host switching, duplication events, or lineage sorting are not relevant to comparisons of relative rates of molecular evolution (Page, 1993b). Analysis of historical associations between these pocket gophers and chewing lice, using TreeMap (Page, 1994b), indicated many possible reconstructions that could describe the events that have lead to current associations between these hosts and parasites. Because different reconstructions suggest different cospeciation events, I have attempted to limit branch-length comparisons to those branches (lettered in Figs. 3.5-3.7) that are most obviously orthologous within the limits of resolution provided by phylogenetic analysis of each data set. This is not to say that all of these apparently orthologous branches definitely represent cospeciation events. For example, some closely related pocket gophers may host chewing lice that are also closely related through some process other than contemporaneous cladogenesis. However, given the history of widespread parallel cladogenesis indicated by similarity in tree structure and similarity in sequence divergence (from the Mantel test), the majority of branches that appear orthologous between the gopher

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and louse trees should be the result of contemporaneous divergence, and therefore, may be informative in comparisons of branch length. Table 3.2 lists branch lengths of potentially cospedating pocket gophers and chewing lice. Branch lengths are based on maximum- likelihood analysis of all positions and of third positions, alone, using a 3:1 transition:transversion bias in each case. These branch lengths do not indicate any significant difference in rate of evolution between pocket gophers and chewing lice, either in overall rate of substitution or in rate of third-position substitution (Wilcoxon signed-ranks tests, P = 0.496-0.758). In contrast, a similar analysis performed by Hafner et al. (1994) on different taxa of pocket gophers and chewing lice indicated a significantly higher overall rate of evolution in chewing lice relative to pocket gophers (Wilcoxon signed-ranks test, P < 0.003). Reanalysis of data for the pocket gophers and chewing lice studied by Hafner et al. (1994), using the weighting strategy described above (for comparison with data herein), also indicates significantly different rates of evolution in chewing lice and pocket gophers (Wilcoxon signed-ranks test, P = 0.009). Regression analysis, performed through the origin (because cospedating pocket gophers and chewing lice should begin divergence at approximately the same time), indicates that the chewing lice studied by Hafner et al. (1994) are evolving at a rate about 1.5 times faster than the rate of pocket gophers (Fig. 3.8; Model I regression, slope = 1.45, P < 0.001; Model II regression, slope = 1.62). A similar difference in rate of evolution is indicated by regression analysis without constraining the line through the origin (Model II regression, slope = 1.51, y-intercept = 0.54).

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Table 3.2—Branch lengths from maximum-likelihood trees (Figs. 3.1 and 3.4a) based on COI nucleotide sequences for pocket gophers and chewing lice. Letters correspond to branches labeled in Figs. 3.5-3.7, which indicate cospedating taxa as suggested by maximum-likelihood and parsimony analyses. All codon positions and third positions of codons are considered separately.

Maximum Likelihood (Fig. 3.6) Parsimonv (Fia. 3.7) Chewing Louse Pocket GoDher Chewina Louse Pocket Gocher Branch Length Branch Length Branch Length Branch Length A 5.6 A 1.5 A 5.6 A 1.5 B 2.0 B 3.9 B 2.0 B 3.9 C 1.5 C 8.3 D 4.3 D 3.6 E 4.8 E 5.1 E 4.8 E 5.1 F 4.6 F 2.3 F 4.6 F 2.3 G 15.5 G 18.2 H 17.0 H 22.9 I 4.4 I 4.4 I 4.4 r 4.4 J 3.9 J J.6 J 3.9 j 3.6 K 6.6 K 2.3 K 6.6 K 2.3 L 6.2 L 5.1 L 6.2 L 5.1 M 5.3 M 6.6 M 5.3 M 6.6 N 2.5 N 5.9 N 2.5 N 5.9 0 4.1 0 4.0 0 4.1 0 4.0 P 4.3 P 1.5 P 4.3 P 1.5 Q 4.2 Q 5.3 Q 4.2 Q 5.3 R 12.6 R 4.4 R 12.6 R 4.4

Third Positions Maximum Likelihood (Fig. 3.6) Parsimonv (Fig. 3.7) Chewinq. Lsuss Pocket Gooher Pocket GoDher Branch Length Branch Length Branch Length Branch Length A 16.1 A 4.9 A 16.1 A 4.9 B 6.5 B 10.6 B 6.5 B 10.6 C 4.6 C 38.1 D 13.2 D 11.3 E 19.6 E 15.8 E 19.6 E 15.8 F 14.6 F 9.0 F 14.6 F 9.0 G 59.2 G 93.1 H 63.9 H 117.1 I 19.5 I 17.2 I 19.5 r 17.2 J 4.4 J 11.4 J 4.4 j 11.4 K 19.6 K 9.0 K 19.6 K 9.0 L 30.3 L 15.8 L 30.3 L 15.8 M 13.6 M 22.8 M 13.6 M 22.8 N 11.6 N 34.0 N 11.6 N 34.0 0 10.5 0 19.6 0 10.5 0 19.6 P 11.8 P 0.0 P 11.8 P 0.0 Q 31.3 Q 21.5 Q 31.3 Q 21.5 R 84.9 R 14.8 R 84.9 R 14.8

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ls

2 '33 Vw «C l. oC 10 S £ slope = 1.622 -a .£ “ I ■o -S u U JO

Fig. 3.8—Reduced major-axis regression (through the origin) of pocket gopher and chewing louse maximum-likelihood branch lengths (data from Hafner et al., 1994; Geomydoecus comparisons = closed squares, Thomomydoecus comparisons = open squares). This reanalysis was performed assuming a transition:transversion bias of 3:1. All nucleotide positions were given equal weight in both the gopher and louse data sets. Circled points represent Orthogeomys comparisons based on the best maximum-likelihood trees for pocket gophers and chewing lice (the louse tree is congruent with tree A of Hafner et al., 1994; the gopher tree also is congruent with tree A, except relationships within Orthogeomys are congruent with tree B).

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Plotting host divergence compared to parasite divergence for the taxa included in the current study (Table 3.2; Fig. 3.9) produces a wide scatter of points, most of which occur near the origin. This wide scatter of points, coupled with a paucity of information at greater levels of sequence divergence, provides little basis for comparing relative rate of evolution of these gophers and lice. More importantly, these points show no obvious linear structure consistent with a pattern of cospeciation (Hafner and Nadler, 1990) and suggest a lack of any predictable relationship between genetic divergence in these host and parasite lineages. It is possible that the scatter of points seen in Fig. 3.9 is the result of the inclusion of several host and parasite pairs that did not diverge contemporaneously. Most of the comparisons made here are based on apparent cospeciation within species of pocket gophers and within species of chewing lice (Figs. 3.S-3.7). Demastes and Hafner (1993:529) proposed that studies focused on taxa at lower taxonomic levels (below the level of the species) "are likely to encounter problems associated with reticulate evolution of host taxa (hence, mixing of parasite lineages) and retention of ancestral ('pleisiomorphic') parasite taxa on recently evolved host lineages." A mixture of these processes in the divergence of populations of species of Thomomys and their parasites may explain the somewhat diffuse array of points provided by analyses of sequence divergence (Fig. 3.9), in that some parasite pairs may have diverged prior to their hosts and others may have diverged later than their hosts.

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All Positions 20

15

oo 10

5

0 1 o --- 0 10 155 20 0 10 1520 255 Pocket gophers Expected number of substitutions per site (xlOO)

Third Positions Only

3 . 100 120 ’35 100 a.o> 80 CO .2g w

« «S S 40 o u fa* U E 20 3 C T3 41 J 8 Xa. 100 0 20 40 60 80 100 120 ui Pocket gophers Expected number of substitutions per site (xlOO)

Fig. 3.9—Plots of maximum-likelihood branch lengths (from Table 3.2) for pocket gophers and chewing lice (Geomydoecus comparisons = closed squares, Thomomydoecus comparisons = open squares) that may have cospeciated, as suggested by maximum-likelihood and parsimony analyses (left and right, respectively).

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Because of the statistically significant level of congruence in host and parasite phylogenies and the significant result obtained from the Mantel test, it seems more likely that the scatter of points in Fig. 3.9 is due to heterogeneity in rate of evolution among pocket gophers and among chewing lice. As discussed previously, there is no significant heterogeneity in overall substitution rate within these groups. Nevertheless, 379 base pairs of sequence may not be enough to demonstrate statistically any but the most dramatic differences in rates of evolution. For example, differences in rates of evolution among the most rapidly and most slowly evolving rodents examined in Chapter 2 cannot be detected based on analysis of only the first 402 base pairs of sequence. Because sister taxa, by definition, have diverged over the same length of time, the lengths of branches leading to sister taxa (e.g., branches A and B in pocket gophers, Fig. 3.6) should be roughly equal if DNA substitutions have accumulated at the same rate in the two taxa. Inspection of branches leading to sister taxa of pocket gophers and of chewing lice indicates large, but mostly statistically insignificant differences in branch length (Fig. 3.10). Only branches A and B of chewing lice are significantly different in length when expected numbers of substitutions per 379 base pairs are compared between sister taxa (binomial test; P = 0.024). This apparent heterogeneity in rate of evolution among pocket gophers and among chewing lice has undoubtedly produced much of the wide scatter of points in Fig. 3.9. Recently diverged taxa may show different rates of evolution simply as a result of stochastic factors. Given enough time, these taxa may eventually show more equivalent levels of DNA substitution. It is also

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o O' Chewing lice Pocketgophers Fig. 3.10~Comparison ofmaximum-likelihood branch lengths (consideringall substitutions; Table3.2) of sister taxa of pocket gophersbranches and in Figs.chewing 3.5-3.7. If lice. rates should ofevolution Letters alsobe correspond roughly areequal equal. betweenwith lettered sister taxa, branch lengths 6.0

H

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possible that rate of accumulation of non-neutral DNA substitutions can differ among populations of a species due to historical differences in effective population size resulting from founder events and differences in frequency and intensity of population bottlenecks. It is noteworthy that heterogeneity in branch lengths of sister taxa seems to be more widespread in pocket gophers than in chewing lice (Fig. 3.10). Pocket gophers are known for characteristically small effective population sizes, and effective population size may be a determinant of rate of substitution in other mitochondrial genes (see Chapter 2). Because chewing lice generally have larger population sizes than do the pocket gophers they live on, lice may undergo less frequent or intense population bottlenecks than their hosts. These larger population sizes in lice may be responsible for what appear to be more homogeneous rates of evolution exhibited among lice than among pocket gophers. Although reanalysis of data presented by Hafner et al. (1994) confirms a higher rate of evolution of the COI gene in chewing lice than in pocket gophers (Fig. 3.8), comparative data on rates of evolution of the taxa included in the current study do not show evidence of a difference in rate of evolution of these groups (Figs. 3.9-3.10). The reason for different results in these two studies is unclear, but heterogeneity in rate of evolution within the pocket gophers and within the chewing lice included in the current study (Fig. 3.10) appears to be greater than the heterogeneity in rate of evolution observed in the gopher and louse taxa studied by Hafner et al. (1994:1088, Fig. 2). It is possible that H afner et al. were not troubled by this within-group rate heterogeneity because they made comparisons primarily among species, rather than within species.

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CONCLUSIONS The majority of the evidence for a higher rate of evolution in chewing lice compared to pocket gophers comes from comparisons of Orthogeomys and their lice (Fig. 3.8 and Page, 1996). Page (1996) has discussed the possibility that some of these comparisons are based on dadogenic events in the chewing lice that predate dadogenic events in the pocket gophers. If this is the case, then rates of evolution in chewing lice must be much lower than previously reported relative to rates of evolution in pocket gophers. This issue can be resolved by induding missing taxa of Orthogeomys and their lice in future studies and by examining more nudeotide sequences to determine if presumed phylogenetic relationships among the pocket gophers are correct. A more likely explanation for the discrepancy in the results of this analysis and the analysis of data from Hafner et al. (1994) is that rate of COI evolution in chewing lice is generally higher than in pocket gophers, but the current study did not recover any evidence for rate heterogeneity because the taxa that happened to show a pattern of parallel dadogenesis induded many conspedfic gophers and conspedfic lice. Despite statistically significant congruence between the host and parasite trees examined here, many gopher-louse comparisons in this study may not be the result of contemporaneous dadogenic events. Demastes and Hafner (1993) discussed potential problems with analyses of cospedation below the level of the spedes (reticulate evolution of host taxa and retention of ancestral parasite taxa on recently evolved host lineages). In addition to these problems, it appears that there may be problems in determining relative rate of evolution between hosts and

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parasites when recently diverged host and parasite taxa may show differences in substitution rate within each group. It would be surprising if pocket gophers and chewing lice showed equivalent rates of evolution given the dramatic morphological, physiological, effective population size, and life-history differences between them. In fact, one might predict a much greater difference in overall rate of evolution than the approximately 1.5-fold difference in rate of evolution observed (Fig. 3.8). Perhaps the study of other mitochondrial- or nuclear-DNA regions will reveal more dramatic differences in rate of substitution. Findings to date based on comparisons of apparently cospedating taxa are enticing, but are still relatively incomplete, even for the COI gene. There is certainly a need for more detailed examinations of cospedation and relative rates of evolution in pocket gophers and chewing lice based on more taxa, longer DNA sequences, and other mitochondrial and nudear genes. These future studies may be most productive if they indude many comparisons between different spedes of pocket gophers and their assodated chewing lice in lieu of comparisons within spedes of gophers and lice.

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Index to scientific and common names of codents included in Chapter 2.

Taxon Common Marne Order Lagomorpha Oryctolagus cuniculus Domestic Rabbit Order Rodentia Suborder Sciurognathi Family Sciuridae Sciurus carolinensis Gray Squirrel Marmota flaviventris Yellow-bellied Marmot Spermophilus tridecemlineatus 13-Iined Ground Squirrel Family Geomyidae Cratogeomys castanops Yellow-faced Pocket Gopher Pappogeomys bulleri Buller’s Pocket Gopher Geomys bursarius Eastern Pocket Gopher Thomomys bulbivorus Western Pocket Gopher Family Heteromyidae Chaetodipus penicillatus Coarse-haired Pocket Mouse Family Muridae Mus musculus House Mouse Rattus noroegicus Norway Rat Spalax ehrenbergi Blind Mole-rat Suborder Hystricognathi Family Bathyergidae Heterocephalus glaber Naked Mole-rat Family Hystriddae Hystrix africaeaustralis Old World Porcupine Family Erethizontidae Coendou bicolor Prehensile-tailed Porcupine Family Caviidae Cavia porcellus Guinea Pig Family Dasyproctidae Myoprocta pratti Acouchi Family Ctenomyidae Ctenomys boliviensis Tuco Tuco Family Echimyidae Echimys chrysurus Arboreal Spiny Rat Euryzygomatomys spinosus Guiara Proechimys simonsi Spiny Rat ______Thrichomys apereoides______Punare

127

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CO S’ > to 9 CL TJ ATT ATT 555 567 ATT ATC ATC ATC ATT CTA ATT TTC ATT TTC TTT TTC TTC ATT 555 CTA TTT ATC TCT TTC ATT TCT TTC ATC TCT TTC ATT TCT TTC ATT TCC TCA TCA 000 000 000 TCC TTC ATT CAC CAC TCG CTC 678 901 234 CAC GCT TTC ATT CAC TCA TTC 000 CAC TCC TTT ATT (Appendix con'd)B AAC CAC TCA TTT AAC CAC AAC CAC AAC CAC TCC AAT CAC AAC CAC 000 000 000 000 000 345 AAC AAC CAC TCG TTC ATC AAC CAC 444 444 455 AAT CAT GCCAAT TTT CAC ATT GCT TTC ATT AAC CAC TCC 000 AAT AAC AAC CAT GCCAAC TTC CAC ATT GCC AAC CAC TCC AAT CAC TCT AAT CAC ATT ATT AAC ATT ATT ATT ATT GTC ATC GTC ATT GTT GTT ATT ATC ATT ATT ATT ATC ATC ATT GTC ATC GTA ATC ATC ATT GTT ATT GTC ATT gene of 21 rodents and one rabbit.

b AAA AAA ATT GTA AAT CAC 000 000 000 AAA ATT AAA AAA ATT ATT AAT 000 000 000 AAA AAA ATT ATT TTC AAA 333 333 333 444 TTT TTT AAA ATA AAA ATT ATC AAA TTA AAA ATT CTA ATC AAA ATC CTT ATC AAA ATC CTA TTA ATC AAA TTA ATC AAA ATT CTA 000 000 890 123 456 789 012 TTA TTA TTA ATA AAA ATA CTA ATC CTC ATT AAA 000 000 TTA TTA ATA AAA CTA CCA CCC CTA ATT AAA ATT 000 CCT CTT CTC CCA 567 CCA TTA CCA TTA ATACCA AAA CCA CTA CTA CAT CCC CAC CCC CAC CCC 234 CAC CCT TTA ATT CAT CCG CAC CAC CCC CAC CCC CAT 122 222 222 223 000 000 000 901 TCA ACA TCC CAT AAC CAC ACC TCA ACC CAC AAA AAA TCA AAA AAA TCA CAC CCC CGA 345 678 CGT AAA CGA CGC AAA 000 000 000 CTA CGA AAA TCA CAC CCA CTC ATC AAA ATC CTA CGA AAA TCA CAC ATA CGA AAA TCT CAC CCT GTA CGA AAA TCC CAC CCT ATA ATA CGA ATA CGC AAG TCC ATA CGA ATC CGA AAA TCT CACATC CCC CGA AAA TCC CTA CGT AAA TCA ATC CGA AAA ATT CGA AAAACA TCT CGA CAT AAA CCT ATT ATC ATT CGC AAA ACC ACC CGC AAA ATA CGA AAA TCA CAT ATA ATA ATC CGA AAA TCT CAC ATT CGT AAA ACT CAC CCT 000 789 012 ATT ATT ATT AAC AAC AAC AAC AAC AAC ATT AAT AAT ATC AAC 000 000 111 111 111 ACA ACC CAC ACC AAC AAA ACA ACA ACA 000 000 000 000 000 000 000 000 000 000 123 456 ATG ?CC AAC ATG ACA ATG ATG ATG ACA ATG ACA AAC ATG ACC AAC ATG ACA ATG ATG ATG ACC CAC ATG ATG ACC AAC ATG qcA ATG ACA ATT ATG ACC ATG ACC ATG ATG ACA ATG ACA t otnys ATG t Position: 000 Thomomys Proechim ys Pappogeomys Chaetodipus Coendou Cavia Euryzygoma Thricom ys Oryctolagus Spermophi1us Cratogeomys Geomys R a ttu s Heterocephalus S palax CtenomysEchimys ATG ACC AAT S ciu ru s Marmota H y s tr ix Mus M yoprocta Specimens examined correspond with Table 2.1. The first 1140 nucleotide positions of the mitochondrial cytochrome to oo

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GGC GGA GGT GGT GGA GGT GGA GGA GGC GGA 000 TTA TTA GGT CTG GGT TTA TTA GGC CTA TTA GGT TTA GGA CTA TTA GGT TTA CTA GGA CTA 901 234 TTA GGA CTA GGA CTA CTA GGC CTA GGA CTA TTA GGA TTA TTA CTA CTT TTA CTT CTC CTA CTA CTA CTA TCA TCT CTC TCT (Appendix B con'd) B (Appendix GGT TCC TTA GGC TCC GGT TCT TTA GGC TCC CTC GGC GGA TCC TTA GGG TCC GGA TCA 000 000 000 Oil 111 012 345 678 GGT TCT CTA GGA TCC CTA TTT TTC TTT GGC TCT CTC 000 000 000 000 000 999 000 111 111 111 111 111 789 TTT GGC TCC CTC TTC GGG TTT GGC TCT CTA AAC TTT AAT TTT GGC TCC AAT AAC TTC GGCAAC TCC TTC CTC AAT TTT GGA TCC AAC TTT AAC TTC AAT AAC TTC AAC AAC TTT AAC TTT AAT TTT GGC AAC TTT GGC TCC AAT TGG AAC TTT GGC TCT TGG AAC TTC TGA TGA 123 456 TGA 000 000 TGA AAC TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA 889 999 999 890 TGG TGA TGA TGA TGA TGA TGA TGA TGA 000 TGA TGA TGA TGA AAC TTT GGC TCC CTC CTT TGA TGA TGA TCA ACG GCC GCA TCA TGA TGA 888 GGT TCA ACA TGA TGA AAC TTC GGA TCC 000 567 AGC GCC ACA TCA TCC GGC TCA 234 TCA TCA 000 000 000 000 000 TCT GGT TCT GGT TCT GCC 788 888 ATT TCA AAC ATT TCA AAT ATC TCAAAT GCA ATT TGA TCC TGA GCA AAT AGC ATT TCA AAC ATC TCA GCA AAT ATT TCA GCA AGC ATC TCC TAC 000 000 AAC ATC AAT ATC AAC ATT TCA AAC ATC TCC GCA TGA TGA AAC TCC TCT TCT TCC TCT AAC ATT CCT AAT ATT TCA 000 TCC TCA AAC ATC CCC TCT CCA CCA CCA CCT CCA TCT AGT ATC 012 345 678 901 CCC CCC 777 777 777 CCC TCC GCT CCA TCC GTC CCC TCT ACC CCA TCT AAC ATC TCA ACA ACC GCT ACC CCA TCC AAC ATC TCA GCC CCA TCC AAC ATT TCA ACC CCA TCC AAC ATC ACA CCC GCC CCA TCT AAC ATC ACA 789 ACT CCC CCT AAT ATC GCA CCT GCG CCA GCC CCA ACC CCA CCA CCT CCA CCT CCA CCC CCT CCA CCG 456 CCT CCT CTA TTA CTA CCA GCA CCA CTG CCC 666 666 666 123 CTT 000 000 000000 000 000 000 000 000 000 000 000 TTA CCA ACA TTA GAC GAT GAC GAC CTC GAC CTC GAT TTG CCC ACT GAT TTA GAC 890 GAC TTA GAC GAC CTA GAC CTC GAC CTA GAC CTT 000 000 GAC GAC CTA CCA ACG CCC CCA GAC TTA GAC GAC TTA CCA GAC CTT CCC GCT CCT TCA GAC CTT CCT GCT CCA GAT TTA Position: 556 Thricom ys Thomomys Echimys Proechim ys Euryzygomatomys Geomys Chaetodipus Cavia Coendou Ctenomys Pappogeomys Cratogeomys Spermophilus R a ttu s HeterocephalusH y s tr ix S palax Oryctolagus S ciu ru s M yoprocta Mus Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O LO GCA GCA GCA GCA GCA GCT TCC GCA GCA TCC TCT 000 111 TCA TCC ACT ACT ACT GCC ACA ACG TCT ACA TCA ACA TCA ACA ACC TCT TAT ACT TAT ACT GCA TAC ACC TAC ACT TAC TAC ACC TAT TAC TAC TAC TAC ACT TAT ACA TCA TAC TAC ACA TAT 000 000 TAT ACG TCT CAC CAC CAC CAT CAC CAC TAC ACA TCC CAC 012 345 678 901 000 (Appendix con'd)B ATA ATG CAC ATA ATA CAC ATA CAT ATG ATA ATA 789 ATA CAT ATA CAC TAC ACA GCC GCA GCA TCC GCC ATA CAC GCA ATA CAC GCA GCA GCC ATA CAC TAC ACC CTA GCT ATA CTA CTA GCA CTA CTA GCA CTA CTA GCT ATA CAT TTA CTA CTA GCA ATA CAC TAT CTA GCA ATA CAC 000 000 000 555 555 555 666 666 666 677 CTA TTG GCT ATA CAC TTA GCC ATA CAC TAC TTA CTA TTT TTT TTC CTC GCC TTT TTC TTC TTC 000 TTT TTC TTC TTA GCC ATA CAC 445 TTT TTC TTT TTA TTC TTA GCT ATA TTA TTA TTC CTA CTG TTT TTA 567 890 123 456 TTA CTA CTA TTC CTA GCT CTA CTA CTA 444 CTA TTA TTT TTA TTC CTA CTA GGA TTA GGC GGA CTC TTC CTA GCA ATA CAC TAT ACT GGC GGC 234 GGC 444 GGA ACC GGA ACT GGG ACA ACA 901 ACA GGC ACT GGC ACT GGC ACC ACT GGC ATT TTA ATC ACA ATT 678 TCA 000 000 000 000 111 111 111 111 111 111 111 111 111 111 111 CTC ACT GGG TTC ATC ATC CTT ATT ATC CTT ACA GGT CTA TTC 345 000 333 333 344 ATC ATC TTA ATC TTC ACA GGC CTA CAA CAA ATC 012 CAA CAA 333 CAA CAA ATC CAA ATT ATC ACC GGG CTC CTT CTA ATC GTC CAA ATC ATT ACA GGT CTT CTA CAA ATT ATC ACA GGT 789 CTA CAA CTT CAA ATC CTA ACA TTA CAA ATC GTT ACC GGA CTA TTC CTA CAA ATC ATC ATT CAA ATT GCA CTT CAA ATC ATT ACT GCA GCA GTC ATA ATA CAA ATT 000 000 000 ATA ATC ATT ATT CAA ATC GCT TCA GGC GGA ATC CTC CAA ATT TTA ACT GGC ATA GTA CAA ATC CTC ATT ATC ATA CTA ATC CTC CTC 000 TTA CTC CTT TTA ATC TTA CTA GCC TGC CTC TGT CTA GGC CTG CAA ATT ATT ACA 000 TGC TGC CTA TGC TGC TGC TTG TGC CTT TGC CTT GTA GTA TGT GTA TGT TTA GTA TGT GTA TGC CTA GGA TTA CAA ATT 567 890 123 456 ATC TGC CTA ATC TTA TGC 000 111 111 111 111 111 111 111 GTC TGC CTG CTG GCC TGC GTC TGC CTA GTA TGC GCC TGC CTA TTA ATA CTA TGC CTA CTG TTA CTT TGC TTA GTC ATC CAA ATC CTT ATA CTC CTT u Position: 111 112 222 222 222 Thricontys Proechim ys Echimys Euryzygomatomys Cavia Ctenomya Spalax Coendo Pappogeomys Chaetodipus Thomomys H y s tr ix Cratogeomys Spermophilus Geomys M yoprocta Oryctolagus Mus R a ttu s Heterocephalus S ciu ru s Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GGT GGA GGA GGA 678 GGG GGC TAC GGT TAT GGT TAT TAC GGT TAC GGG TAC TAC GGC TAC TAC TAT GGT 000 000 AAC TAC AAT AAC AAC AAC TAC GGC AAC 222 222 222 000 012 345 AAT TAT GGT AAT TAC GGC AAC TAT GGC GTA GTA GTA GTA GTA AAC TAC GGC GTA AAC TAT GGC GTA GTC GTG AAT TAC GGC GTC AAC TAC GGC GTT GTT GTA AAT TAC GGC (Appendix B con'd) B (Appendix GAC GTA AAC GAC GAC GTA AAC GAC GAC GTA AAT GAC GAC GAC GTC AAT GAC GTA AAC GAC GTT GAT GAC GAC GTC AAT TAT GAC CGA CGT GAC CGA CGA CGA CGA CGA GAT TGC TGT TGC CGA TGC CGA GAT TGC CGA TGT CGT GAC TGC CGA TGC 001 111 111 111 TGC CGA TGT CGA TGC 000 000 000222 000 222 222 222 222 222 222 TGT CGA GAC TGC CGA TGC CGA TGT CGA GAT TGC TGC CGA GAC GTA AAC TAC GGC TGC CGA TGT CGA ATT ATT TGC ATC ATT TGT ATC ATT ATC CAT ATT CAC CAT ATT* CAC ATC CAC ATC CAC CAT ATT CAC GCA ACC ACA CAC ATC GCC ACC ACC CAT ATC GCC CAT ATT 000 000 000 ACA CAT ATC ACC CAT GTC GTC GTA GTA 678 901 234 567 890 123 456 789 GTA GTG GTA GTA ACA 999 999 900 000 000 345 TCA 000 000 TCA TCA GTC ACG CAC ATC TCT TCA TCA GTA ACA TCA TCA GTT ACA CAT TCA 000 TCA TCT TCA GTG GCT CAT ATC TCA TCA GTC ACC CAC ATC TCC TCA TCA GTA ACA CAT ATC TTT TCA TCA TTC TCG TCT GTC GCCTTC CAC TTT 111 111 111 111 122 222 222 TTC TCA TCA GTC ACC CAC ATC TTC TTT TTC TTC TCA TCA TTC TCA TCA GTC ACC GCA GCC GCA TTC TCA TCA GCA GCA GCA GCA TTC GCC ACA GCC TTC TCA TCA GTC ACC CAT ATC TGC CGG GAC GTA AAC ACC ACA GCA TTC TCA TCT ACC ACA GCA TTC TCA TCA GTA 111 111 ACA ACA GCC TTC ACA GCC ACA ACA GCC ACA GCC AGC ACC 111 ACC ACT ACA 890 123 456 789 012 CTA ACA GCT TTC TCA TCA GTG GCC CAT ACT CTC ATC TCA ACA ACA GCA TTA ATA ATA ACC ACC ACT ACC ACA GCT TTC TCA TCA ACA 000 000 000 000 000 777 778 888 888 888 999 ACC ACA ACT GAC ACA ACC ACA GCC TTC TCA TCT GTT GAC ACA GAT GAC TAC ACA GAT 111 111 GAC ACC CTT ACA GCC TTT 000 GAT ACA ATA ACA GCC TTT TCA TCA GTA ACA GAC ACA GCC GAC ACA GAC ACA GAC ATC TCC ACG 234 567 GAT ACC CTCGAC ACA ACC GCCGAC TTC ACT TCA GAT ACC ATA ACA GAT GAC GAC ACC GAT us GAT ACT ATA urus GAC ACA Position: 777 Proechim ys Thricom ys Ctenomys Bchimys Thomomys Coendou C avia Euryzygomatomys Pappogeomys Chaetodipus S palax Sperm ophi1 R a ttu s Heterocephalus OryctolagusS c i Geomys Cratogeomys M yoprocta H y s tr ix Mus Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 132 TAT TAC TAT TAT TAT TAT TAC TTC 888 TAC TAT TTC TAC 222 CTT CTA CTT TAT CTT CTC TAT CTA CTA ATC CTG TAT CTT TAC CTA 012 345 TTA TTA CTA CTG TTC CTT TTC CTA 222 TTG TTC CTC TTT CTC TTC TGC TGC 789 TGC 222 ATT TTC ATT CTC ATC TGT ATT 777 777 888 ATC TGC ATT TGC ATT TGC (Appendix con'd)B TTT ATT TTC TTT 123 456 TTC ATC TGC TTT TTC ATC TGC TTC ATC TTC TTC ATC TTC TTC TTC ATC CTC TTT TTC TTT 667 777 890 TTT TTT TTT TTC ATC TGC 000 000 000 000 000 000 TTT TTC TTC ATTTTT TGC TTC TTT ATT TGT CTT TAC TTT ATA TTT TTC ATG TTT TTCATA ATT TTC ATC TTC ATA ATA TTC ATA ATA 666 567 CTA TTC TTC ATC TGC CTG TAC 000 ATA TTT TTC ATC ATA CTA TTA 222 222 222 222 ATA TTC TTT TTA TTC TTT ATA ATA TCC TCC TCC TCA TCC TCA ATA TCT GCA TCC ATA TTC TTT ATT TTC 566 666 GCC TCC GCT TCC GCA GCC TCC GCA GCC GCA TCT GGA GCA TCA ATA TTC TTC ATT GGA GGA GCC TCC 678 901 234 000 000 000 GGT 222 222 222 GGT GGT GCA TCA AAT GGA GCA AAC GGA GCA AAC GGA GCA TCA AAC GGA GCA AAC AAC GGA GCA 345 555 555 000 222 AAT AAT GGT AAC GGA GCT TCA AAC GGA AAT GGA GCA TCA CTA TTC TTC ATC TGC AAC AAT AAC GGC AAC GGA GCT TCA AAC GGA AAT GCC GCC 222 GCT GCT CAC GCC CAC GCC CAT GCC CAC GCC AACCAC GGC GCC GCA TCC CAC GCT AAT GGT GCT TCC CTT CAT GCC CAC GCA GCA GCA 444 444 555 000 000 000 456 789 012 CTA ATA CTC CAC GCT TAC TAT 123 444 TAT GCA CAC GCC 000 TAT TAC ATA CAC GCC AAC GGT TAT ATA TAC CTA CACTAT GCC AAC GGC GCC TCA CGA CGA CGA 890 334 CGA CGA TAC TTA CGC TAT ATA CAT GCT CGA TAT ATA CGA TAT TTT CAT GCT CGA CGA CGA TAC ATC ATC ATT CGA TAT CTA CAC ATC ATC ATC CGA ATC ATC CTC ATC TTA ATT 000 000 000 TTG ATT CGC TAT GCA CAC GCT CTA ATT CTT ATC CGT TTA ATC CGA TAT ATA CAT GCA CTT ATC CGC TAT ATA CAT GCT CTT ATC TGA 901 234 567 TGA TGA CTA TGA TGA TTA 000 222 222 222 222 222 222 222 TGA CTG ATCTGA CGA TTA TAT CTA CAT GCC AAC TGG CTA ATC CGT TAT ATA CAC TGA TGA TTA TGA CTA ATT CGT TAC CTC CAC TGA TGA TGA TGA CTT TGA TTA ATT CGA TAC ATA CAC TGA CTA TGA CTA TGA TGA CTA ATC TGA ATA TGA CTG ATC CGG TAT ATT us 1 Position: 233 333 333 Thricom ys Thomomys Ctenomys Echimys Proechim ys Sperm ophi Pappogeomys Cavia S ciu ru s EuryzygomaContys Geomys Chaetodipus Cratogeomys Coendou Oryctolagus R a ttu s S palax Heterocephalus Marmota M yoprocta H y s tr ix Mus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AAC AAC AAT AAC AAC AAT AAC TGA AAT TGA TGA AAT TGA AAC TGA AAT TGA AAC 333 333 333 444 TGA TGA 789 012 TGA AAT TGA AAC TGA TGA TGA TGA AAC ACT ACC ACC ACC ACC ACT 333 ACT ACC 456 ACC ACA ACA TGA AAC ACC ACA GAA GAA 333 GAA GAA GAA 000 000 000 000 GAA ACA TGA GAA GAA ACA TGAGAA AAT GAA ACA TGA AAC GAG GAA GAA (Appendix B con'd) B (Appendix TCA ACA GAA ATA AAA ATA GAA ACC TGA AAC TTT GAA ACATTT TGA AAC ACA GAA ACA TGA AAT CTA GAA ACC TGA AAC TTC TTA 567 890 123 TTT ACA TAT TTC CTA GAG ACA TTC ATA GAC ACA TAC TAC TTC TAC CTA TAC TTT TAT TAT ACA TAT AAA TTC ACA TCC TTC ATA ACA ACA TTA TAT TTA ATA ATA ACA TTA TTA ACT TAC ACC TTC ATA TAC ACT TAC 000 000 000 000 TAC CTT TAT AAG TAC TAC TAC TAC TCA TCC TAC ACT TTT ATA TCT TCA TCC TAT TCC TAC 000 TCT TCA TCA TAC ACC TCA TCA TCC TCC TAC ACT 345 678 901 234 GGG 000 GGG GGA TCT TAC GGC GGA TCA TAT ACA TTT ATA GAA GGA TAC GGA TAT TAT GGA TCC TAC TAC GGA TAC GGA TCT TAC ACT TAT GGG TAC TAT GGA TAC GGA TCG TAT TAC 000 000 333 333 333 333 333 333 333 333 TAC TAC TAT GGG 789 012 TAT TAC TAC TAC TAC GGA TAC TAC GGA TCT TAC CAC TTC AAA GAA TAC TAT GGC TAC TAT GGA 333 CTT 000 CTT TAC TAC GGC TCT TAC ACT TTA ATT TAC TAC GGC TCC TAT ATG ATT TAC ATC TAC ATC TAC ATA TAC TAT GGC ATC ATT TAC ATT TAC 000 GGA GGA GGA GGA ATC GGA GGT GGT GGC GGA ATT GGA GGA TTA GGA CTA 990 000 000890 000 123 111 456 111 111 122 222 222 223 333 333 000 CGA CGA GGG ATT TAC CGC CGA CGA CGA CGA CGA CGA GGA CGA GGT ATT TAC 567 GGA CGA GGT ATT TAC 000 GGT CGA GGA GGA GGA CGA GGC GGA GGC GGC GGA GGC CGA GGG GGC CGA GGC ATT TAT TAT GGT TCT TAC GGT CGA GGA GGA GGA GGA 000 ATT ATT GTA GTA GTA GTA GGA CGA ATT GTA GTA ATC ATC ATT 901 234 222 222 222 223 333 CAC CAC GTA GGC CAT CAT ATC CAC ATT GGT CGT GGT ATC TAC TAT CAC ATC CAC CAT CAT CAC ATT GGC CGA CAC ATC GGA CGA CAT GTG GGA CGA 888 899 999678 999 000 000 222 CTT CTA CAC CTA TTT CAC ATC TTT TTC CAT TTT CAC TTC ATA ATC CAT CTT CAT GTA CTT CAT ATT CTC CTC CTT CAC CTT CAT GTC GGA CGA GGC TTA TAT TAT CTA ATC CAC CTA ATA CTC Proechim ys Oryctolagus S ciu ru s Pappogeomys Thomomys Echimys Thricomys Spermophilus Cratogeomys Cavia Euryzygomatomys Ctenomys Geomys Chaetodipus Coendou Spalax Marmota H y s tr ix Heterocephalus Mu s Mu R a ttu s M yoprocta

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. £ CTG CTT CTT CTT TTG CTC CTC CTT TTA 789 CTA TTA CTT CTC ?TA GTC GTC GTA GTA GTC CTA GTC CTT GTA CTA GTA GTT 456 GTC GTA TTA GTA TTA GTA GTT GTT CTT GTA GTC GTC TAC TAC TAT TAT TAT TAT TAC 999 999 999 TAC 000 000 000 GGG GGG GGG TAC GGC TAC 889 GGT GGC TAC GTC CTT GGA TAT GGA TAC GTC CTC GGT TAT GGA 890 123 GGC TAT GGT TAT GGC TAT GGT TAT (Appendix con'd)B ATA ATA ATA GGA ATA ATA ATA 888 ATA ATA ATA ATA GGA 567 GTA GTT GGT GTA ATA ATA ATC TTC TTC TTC TTT TTC TTC TTC ATG GGC TAT TTC GTT GGT TTT TTT TTT ATA TTT GCT TTC ATA GGA TAC GCC GCA GCC CGA TTC GCA 000 000 000 000 788 888 GCA GCA GCT TTT GCC TTC ACA ACA ACA GCA TTT ACT ACA ACC GTT 000 777 ACC GCA TTT ACA ACA ACT GCC GCA GCA ACT GCA GCT ACT GCC TTT ATA GGG TAT GTC CTC GCT ACC GCT ACC GCA ACT GCT TTC GCT GCT ACC GCC TTC ATA GGG TAC GCC GCA ACT GCC GCT GCT GCT GCC GCC ATA ATA ATA ATG ATA ATA ATA ATA GCA ACA GCA ATA 012 345 678 901 234 ATA ATA ATA 777 777 ATA ATA GTT GTG GTA GTT GTT GTC ACT GTC GTA ATG GCT ACT GCC TTC 000 000 000 789 ACT ATA ACC ATA GCC ACC CTA GTA ATA GCA ACT ACA ATA ACT 333 333 333 333 333 333 333 333 333 333 333 333 666 666 GCA GCA GCA TTC ACA CTT GCA TTC ACA GTACTA ATA TCA GCA ACC GCA TTT ATA 333 666 TTT CTA TTC CTA CTA TTA TTA 556 CTG TTC TTA CTG TTT CTT TTA CTT CTG CTC TTA TTG GCA CTC TTA 555 567 890 123 456 CTA CTA TTA TTA TTA TTC ACA GTT CTT ATT ATC ACC GTA CTC CTA TTC TTG CTA CTA TTT CGA CTA CTA TTCCTT CTT CTA CTA TTT CTA CTC CTA TTT GTA GTA ATA CTC CTA TTC GCA GTA ATA GCC ACA GCA GCT ATC CTT ATA ATC CTC ATC ATC CTA 234 ATC ATT 333 333 333 333 455 555 ATC CTA CTT TTA GTA ATC ATC ATT ATC CTG ATC ATT CTTATT CTC GTA GTA ATT TTA TTA TTC GCA GTA 000 000 000 000 000 000 ACC TTA ATT CTA ATC TTA GTT ATT GTT ATC CTC CTA TTT GTA GGA GGC GGA GGA GGA GGA GTT GGA 000 GGA GGG ATC GGA GGC GGT GGC ATC TTG CTC CTA TTC TTA TCA ATA GGA GGA 333 333 ATT ATA GGA GTT 000 345 678 901 ATT ATT ATC ATC ATC GGA ATT ATC ATT ATT GTA GGA ATT GGC ATC ATT ATT ATC ATT GGT ATT ATT ATT ATT GGA ATT GGC ATC ATT Pappogeomys Proechim ys Thricom ys Position: 444 444 S ciu ru s S palax Cavia Chaetodipus Coendou Echimys Thomomys Cratogeomys Spermophi1us Oryctolagus Ctenomys Heterocephalus R a ttu s Euryzygomatomys Geomys Mu s Mu M yoprocta H y s tr ix Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U 1 GCT GCC 555 GCC GCA GCT GCA GCC 444 456 GCT GCT TCA GCC TCA GCA TCA GCT TCA GCC TCA TCA GCA TCA GCA 555 TCT TCC TCA TCA GCT TCC TCA CTA CTA 890 123 CTA TCT GCG TTA CTT CTC CTT CTT CTC CTA CTA TCA GCT CTA CTT CTC CTT TCA GCA CTC CTA TCA 567 CTC TTA CTA CTA TCATTA GCT TTA TTA CTT CTA TCA CTT CTA CTA TTA TCA CTA TTC 444 445 CTT TTA TTA CTA TCA GCT CTC CTA TCACTC GCA CTA (Appendix con'd)B AAC AAT AAT 234 AAT AAC AAC 000 000 000 000 000 AAC AAC ACC ACA ACA ATT ACT AAC ATT ACG AAC 678 901 ATT ATT ATT ACC AAC ATT ACT AAC ATT ACT AAT GTC ATC ACA AAC CTG CTT GTT ATT ACT GTA GTT ATT ACA GTC 345 333 333 344 444 000 000 000 444 444 444 444 444 444 444 GTT GTC ATT ACA AAT GTC GTA ATC ACT GTA ATT ACC AAT CTT TTA GTC ATT ACT AAT GTA GTT ACA ACA 333 012 ACC ACG 000 ACA ACC ACA ACC GCA ACC 444 444 GCC ACT GTA ATT ACT AAC 222 GCG ACC GTC ATT ACT AAT GCT GCT ACA GTA ATT GCA GCA ACT GCA ACT GGA GGA GCA GGG GCA 456 789 222 000 000 GGA GGC GCT ACAGGG GTC GCT ATT ACA ACC GTC AAT ATT ACT GGA GGA GGA GGA GGC TGA GGA GCG ACA GTC ATC ACA 123 000 TGA GGG GCA TGA TGG TGA TTC 112 222 890 TTC TGA TTT TGA GGC GCT ACC GTA ATT ACA AAC TTC TGA GGT GCC ACA GTC ATC ACA AAC 000 TTC TTC TGA TTC TGA TTC TTT TGA GGG GCC TTT TTT TGA CGG CCG ACC GTT TTT TGA TTC TGA TCT TTC TGA 567 444 444 444 444 TCA TCA TCA TTA TCT TTT TGA ATA TCT TTC TGA GGT GCA ACA ATA TCC 444 ATA ATA ATA TCA ATA ATA TCA ATA TCA ATA TCA ATG ATA 901 234 CAA CAA CAG CAA ATA TCA 444 444 GGA GGA GGA CAA ATA TCA TTC TGA GGT GCC GGA CAA GGT CAA GGC CAA ATA TCA GGA CAA GGA CAA ATA TCA GGA CAA GGA CAA ATA TCT TTT TGA GGG GCT ACT GGA CAA ATA TCA GGA CAA ATA TCA TGA GGA CAA 444 TGA 000 000 000 000 000 TGG GGT CAA ATA TCCTGA TTT TGA GGT GCT TGA GGA CAA TGA TGA TGA TGA TGA TGA TGA CCC TGG GGA CAA ATA TCA TTC TGA CCC CCC TGA GGA CAA 012 345 678 444 000 CCT TGA GGA CCA CCA TGA GGA CCA CCA CCA TGA GGA CAA CCA CCC TGA CCC CCA TGA CCA CCA CCA TGA GGA CAA ATA TCA CCA CCA TGA GGA CAA ATA TCA TTCCCA TGA GGA CCA CCA Pappogeomys Thricom ys Spermophilus Thomomys EuryzygomatomysProechim ys Chaetodipus Spalax Position: 000 000 000 011 111 111 Oryctolagus Cratogeomys Coendou C avia Ctenomys Geomys Echimys Sciurus CCA TGA Mus H y s tr ix R a ttu s Heterocephalus Marmota M yoprocta

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n O u> GAC GAT GAC GAC GAC 111 GAC GAC GAC GAC GAC GAC 890 123 GTA GTA GAC GTA GTA 001 GTA GAC GTA GTT GAT 000 000 GTA GAC GTA GTG GAC GTT GTA GAC GTA TCA GTA TCA 555 555 555 567 TCA GTA TCA GTA GAC TCA GTA TCA GTA GAT TCA TCA TCA TCA GTT GAT TCA GTA GAC TCA TCA GCA TCA GTA TCA TCT TCA GTT GAT TTT TCC 555 000 000 234 TTC TCC TTT TTC TTC TTC TTT TTC TCT TTC TCA (Appendix con'd)B 901 GGA GGC TTC GGC GGC GGT TTC 000 000 000 GGA GGG ??A TTC GGG GGG TTT GGG GGT TTC GGC GGA GGT GGG GGA GGA GGG GGG TTC GGA GGA GGA GGC GGC GGT GGA TTT TGA TGA GGA GGA TGA TGA TGA TGA TGA GGA TGA TGA GGA GGA TAT TGA TGA TGA GGA GGA TTT TGA ATC TGA ATC ATC 999 999 999 900 ATT ATC ATC ATC ATC ATC ATC ATC ATC ATT TGA GGT GGT TTC ATC TGA ATC TGA TGA TGA ATC TGA GGT GGA TTT TGA TGA TGA 000 000 000 000 TGA GAG GAG GAG GAG GAA TGA ATC TGA GGA GAG GAA TGA ATT TGA GGT GAA TGA 123 456 789 012 345 678 GTA GTA GAA TGA GTA GTA GAA TGA GTA GAA TGA GTA GTA GAA GTC 890 CTA GTT CTA GTA GAA TGA CTA CTT CTA CTA CTG CTC 444 444 444 444 444 444 444 455 CTA GTC TTA GTA GAG TGA CTA GTC GAA TGA ATT TGA GGG GGC TTC CTA GTG GAA CTA GTC GAA TGA TTA GTT GAA TGA TTA CTA GTA CTA GTA GAG TGA ATC TGA GGTCTA GGA GTA TTC CTA GTA GAG TGA CTA GTA GAA TGA ATT TGA 567 ACC ACC ACA CTG 777 778 888 888 888 ACC ACA ACT ACC ACC GAC GAC GAC ACC ACC 234 CCC CCC 444 444 ACA ACC ACA ACC CCT ACC ACA ACA ACC ACA 444 GGA GGA GGA CAA GGA GGA ATT GGT CCT ATC ATC GGT ATC GGA ACA 000 000 000 000 000 000 000 000 TAC ATT TAT ATC GGC CCT ACT TAC ATC GGC CCT TAT TAT TAT ATC GGCTAT ACT ATT GGC TAC ATT GGA CAA GAC TAT ATT TAC ATC GGG CCC TAC ATC CCA CCT TAC ATT 444 444 444 444 000 000 789 012 345 678 901 ATC CCA TAC ATT CCT ATC CCC TAT ATT GGC ACA ATC CCA TAT ATA GGA ATC CCC ATT CCC ATC CCT ATC CCA TAC ATC CCT ATT ATC CCC TAC ATC GGAATT CAA CCT TAT ATC GGT CAA ATC CCT TAC ATT GGG ACT ATT CCT ATT CCC TATATC GTA CCA GGA CAA GAT CTA GTA GAA TGA ATC ATC CCT TAC ATT GGCATC CCA CCT ACA TAT GTC GGT ACA ACT ATC ATC ATT CCA ATT CCA Spermophiius S ciu ru s Pappogeomys Thomomys Position: 555 666 666 666 677 777 Cratogeomys Oryctolagus Geomys Chaetodipus Spalax Proechim ys Thricomys R a ttu s H y s tr ix Coendou Cavia Euryzygomaomyst Mus Ctenomys Echimys Marmota Heterocephalus M yoprocta

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. u> ■ v i ACA ACA GCA 890 ACA ACA GCC GCA GCA ACA ATT ACA ATT ACA ATT ATT ACA ATC ACC 666 667 567 000 000 ATC GCA ATC ATT ATT ATC GCC ATC ATC ACA ATT ATC GTT 000 ATC ATC ACT ATC ATT ATT ACT ATT GTT GCA TTT ATT ATT ACC TTC 555 555 555 555 TTT ATC ATC TTT ATC (Appendix con'd)B CCA TTT ATC ATC CCT TTT ATC CCC CCA TTC ATC CCA TTT ATT CCA TTT ATT CCA TTC ATC ATC CCA TTC ATC ATC CCA TTC 555 566 666 678 901 234 CCC TTT ATT ATT CCA 000 000 555 CCA CCA TTC CCT CCC TTT ATT CCA TTT CCA TTC CCC TTT TTG CTC CTA CTA CCA TTC ATA CCA TTC ATT CTT TTA CCA TTT ATT ATC GCG 555 CTA CCA TTCCTA ATT ATT TTA ATC CTG GTA GTA 012 345 ATC ATT CTC ATC TTC TTT ATC CTT TTT ATT 444 555 000 000 000 789 TTC ATC TTG CAC TTT ATT CTC CAC CAC TTT ATT GTT CAC CAT TTC CAT 456 CAT TTT ATC CTC CAT TTT GTT CTT CAC TTC GTT CTC CAC TTC ATC CAC TTT ATC TTC TTT TTC TTT TTC CAT TTC ATC TTC CAC TTC ATC CTC TTC TTC 000 000 555 555 555 555 555 TTT CAT TTC 444 444 TTC GCA GCC 000 GCA GCC GCA 555 GCA GCA GCA TTC GCC TTC CAC TTT TTT GCT 333 334 000 TTC TCT GCC TTC TTC TTC GCC TTT CAC TTC TTC TTC 333 555 555 TTC TTC TTC TTT GCT TTC CAC TTC AGC CTG TTC TTC GCA TTC TTC TTC GCT TTT CAC TTC TTT CGA 233 901 234 567 890 123 CGA TTC TTC CGA TTC TTC GCT TTC CAC TTC ATC CGA CGA ACA CGA ACA 222 ACA ACC ACC CGA ACC CGA TTC TTC GCA TTC CAT TTT CTA ACA CGA TTC TTT TTA TTG CTA ACA CGA TTC TTC GCA TTC CTA ACA CGA TTT TTA ACT ACC CTT 555 555 555 555 ACA TTA ACC CGA TTC TTT GCC TTC CAT TTC ATT CTT ACA CTC ACCACT CGA CTG ACCACT CGA TTA TTC ACAACC TTC CGA TTA GCC ACT TTC CGA CAC TTC TTC TTC ATC GCC ACC CTA ACA CGA TTC TTT ACC TTA ACC CGA TTC TTC ACC TTA ACC CGA ACA ACT CTT ACA CTA ACA CGT TTC ACT CTA ACA GCT GCC GCA ACC GCA ACC CTA ACA CGC TTC TTC GCC GCT GCT 789 012 345 678 GCC GCA GCG GCC GCT GCA ACA GCT AAA 555 555 AAA AAA GCC AAA 456 000 000 000 000 000 000 000 AAA GCC AAA AAA AAA AAA AAT AAG GCT ACCAAA CTT TCA CGT TTC TTT GCA TTC AAA GCC AAA GCC ACC AAA AAA AAA GCT ACC CTA ACT AAA GCA ACT AAA AAA Thricomys AAA EuryzygomatomysProechim ys Pappogeomys S ciu ru s Thomomys Chaetodipus Spalax Coendou C avia Ctenomys Echimys Spermophi1us Cratogeomys Geomys R a ttu s HeterocephalusH y s tr ix Position: 111 111 222 222 Oryctolagus AAA GCC ACT CTT M yoprocta Marmota Mus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TCA TCA TCA TCC TCG TCA TCA ACA ACA TTA TCA CTA 567 TCT ACA CCA TCA CCA CCA CCA CCC CCA CCA CCT CCC TCA AAC AAC CCT TCA AAC CCA TCA AAC CCC AAC AAC CCA AAC AAC CCA AAC 122 222 222 AAC AAC CCA TCA AAC CCA CTA AAC CCC 666 666 666 AAT AAC CCT TTA AAT CCA CTA AAC AAC AAC AAC AAC AAC AAC AAT AAC AAC 111 AAC AAC AAC AAC AGC AAT AAT AAT AAC CCA AAC (Appendix B con'd) (Appendix B TCC TCA TCC AAC TCA TCC AAC AAC TCA TCA AAT AAC CCC TCA 666 666 TCA TCT GGA TCT GGA GGA TCA GGA TCA GGA GGA 000 000 000 000 000 000 111 111 GGG TCA 666 GGC TCC AAC AAC CCA GGA TCA GGG ACA GGA ACA TCA ACA GGA ACC ACA GGA 789 012 345 678 901 234 ACA GGA TCG ACT ACT ACA GGT GAA GAA GAA ACA GAA GAA ACT GGG GAA ACA GGG TCA 456 GAA ACA GGA TCA GAA GAA ACA GAA GAA CAT CAC GAA ACA GGA CAC CAC CAC 666 666 666 123 CAT 000 000 000 CAT TTA CTT CAC GAA CTA CTA CAC CTT CAC GAA CTA CAC CTC CTC 890 000 556 CTA CTA CAT GAACTT ACA CAT GGA GAA TCC ACA CTC CAT CTT CAT GAA TTC TTT TTC TTT TTC CTC CAC GAG ACA TTT TTC TTT TTA CAC TTC 555 999 990 000 000 000 567 TTC CTT CAC GAA ACA GGA TCA TTT TTC 000 TTC CTT CAT GAA TTT CTT TTT CTC CAT GAA ACT GGC TCC AAT 999 234 CTT TTC CTT CAT GAA ACT CTA TTC TTA CTA CTA CTC TTT CTG TTT CTT TTC CTT TTA CTC TTA CTA CTA TTA TTA CTC TTA CTT TTA TTC CTT CTG CTA CTA CTA 899 CTT TTA CTT 901 CTC TTA CTA CTC CAC CAT CAC CAT CTT CTT CAC 888 CAC 678 CAT CAC ATT CAC ATC CAC CTA CTA ATT CAT ATC CAC ACC GTC CAT 555 555 555 555 GTT CAC GTA GTT CAT GTT CAC CTC CTC GTA GAC GTT GTC CAC CTT GCC ATA ATA ATA ATA ATA GTC ATA ATA CTA GTT 555 ATA GTA ATC 012 345 ATA ATA TTA ATT CAC CTC ATA GTC ATA ATA ACA GTC ATA GTT CAC TTA ATA GTC CAC CTA TTA GTT GTT GTA 555 GTG 789 GTC GTT GTC GCT GTT GTC CTG GTA CTA GTG ATA GTA ATA ATC CAC CTC CTA TTT CTC CAC ATA GTA CTA ATA GTA CTT ACA CTT GCA ATT CTA GCA 000 000 000 000 000 000 000 CTA ACA GCT CTA GCC 777 777 888 888 TTA GTC TTA GCA GCA GCA ATA GCC GCA GCA 123 456 ACC ATG ATT GCA GCA 555 555 GCC GCC CTA GCT GCC CTA 000 GCT CTA GCC CTA GCC CTC ACT GCC GCT CTA GCC GCC omys t Thricoinys Proechim ys Echimys Cavia Ctenomys Euryzygoma Pappogeomys Spalax Coendou Chaetodipus H y s tr ix Thomomys Heterocephalus M yoprocta Oryctoiagus Pactus Cratogeomys Geomys Spermophilus Position: 777 S ciu ru s Mus GCC Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VO GAT GAC GAT GAT GAC GAC GAT 000 GAC AAA GAT AAA GAC AAA GAC AAA AAA GAT AAG AAA AAA AAA GAC AAA GAC AAA AAA AAA 788 888 AAA ATC ATC ATT ATC AAA GAC TTT CTT ATT 678 901 234 666 666 666 777 ATC AAG GAT ATT ATC AAA GAC ACA ATT ACA ACA ACA ACA ATT TCA ACA ATT ACA 345 TCA ACC AAGACT GAC 777 ACA ACC AAA GAA ACA ACC ACC ACT (Appendix con'd)B TAC TAT TAT TTC TAC TCA TAC ACA ACCTAT AAA GAT TAT TAT ACA ATC AAA GAT 012 000 000 000 000 777 TAC ACA ATT AAA GAC TAT TAC TAC TAC TAT TAC TAC TAC TAT TAC TAC TAT TAT ACATAT ATT TAC TAC TAC TAC TAC 000 666 666 666 666 789 TAC TAT TAC TAC ACA TAT CCC CCA CCA CCA CCC CCC CCC CCC CCA 456 000 666 CCA 666 CCC TAC TAC CCC CCA CCA CCC CAC CCT TAC TAT ACA ATC CAT CCT TAC CAC CAC CAC CAC CAT 666 CAC CAC CAC CAC TTT TTC TTT 890 123 556 TTT CAC CCT TAT TTT TTT CCA TTC CAC CCT TAC TAT ACA ATC CCA TTT CCA TTC CAC CCT TAT TAT CCA CCA TTT CCA TTC CAC CCT CCA TTC CAC CCA TTC CAC CCA TTT CCA TTC CAT 567 CCA CCA TTC CAC CCA TTC CCA TTT ATT ATC ATC ATT ATC CCA ATT 555 555 ATC 000 000 000 000 ATC ATC ATC CCT TTCATT CAC ATC CCC AAA AAA ATC AAA AAA AAA AAA ATC CCA AAA AAA AAA 455 AAA GTA CCA TTC CAC CCA AAA AAA AAA AAA AAA GTA CCA TTC GAT GAC GAC GAT GAC 666 666 666 666 666 666 GAC 678 901 234 AGC GCT GAT GAT GAT AAA GAC AAG ATC CCC GAC AGT TCA TCA TCA 666 GCA GCA 444 444 TCA TCA TCG GAC AAT TCA GAC AA? ATC GAT GAC TCC GACGAC AAA TCC ATC GAC AAA ATT AAC GAC TCG GAC 012 345 666 000 000 000 000 GAT TGC GGA AAA GTT GAT TCA GAC GAT GAC AAC TAT GAC TGC GAC GAC TGC AAC GAC TCA TCC AAC TCA TCA TCA TCA TCA TCC TCA TCA 000 TCA 789 TCA TCA TCA TCA AAT AAC AAC AAC AAC TCC GAC TCT GAT AAC AAC GAC 666 666 AAC AAC AAC TCT GAC TCA GAC AAA ATT CCA TTC CAC 456 000 CCG AAC CCT ATC TCT ATC TCT TTA TTA ATA CTA CTA ATC 666 TTA ATC CCA TCA CTT CCA CTA CTT ATT TCT GAC TCA GGA GGA TTA GGA GGC GGA CTA GGA GGA CTA 890 123 GGC ATT GGC GGC CTA GAC TCC AAT GCC GGC 000 000 666 GGA GGC GGA TTA GGC ATC CAA GGA ATT CCA GCA GGC ATC GGA ATT GGC GGC CTT GGA GGA T h ricon ys Euryzygomatoitys Proechim ys Cavia Thomomys Ctenomys Echimys Spalax Coendou Pappogeomys Geomys Chaetodipus R a ttu s H y s tr ix Heterocephalus Oryctolagus Position: 223 333 333 333 444 Cratogeomya Mus M yoprocta S ciu ru s Spermophilus Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CCA CCA CCA CCA CCA CCA CCA CCA CCT CCA CCA 777 CCA CCT TCA CCA TCA CCA TCA CCT TCA CCA TCA CCA ACA CCC ACA CCA TCA CCA CAC TCC 678 901 TTC TTC TTC TCC CCA TTT TTT TTC TTC TTC TTT TTC TTC TTC TTC TTT TTT TTC 333 333 344 TTT TTT TTC TTT TTC TTT TCA CTA CTA TTA CTT TTT CTA ATA CTA TTT TTC CTA 012 345 CTA 000 000 000 000 TTA CTA TAT CTA TTT TCA TTA TTT TTA TTA CTA (Appendix B con'd) B (Appendix ACC ATT GTC ACA CTA GTC 222 333 GTA TTA GTC GTC TTA ACC CTA TTA TCA CTA ATC TTA CTA GTA CTA GTA CTC CTA GTA CTA GTA CTA GTA CTA 456 789 TTC GTG TTG TTC CTA CTA GTC TTA GTG CTA GTA CTA TTA GTC CTA TTT TCA TCC ATT TGC ATA TTC AGC ATA ATC TCC ACA CTA 000 000 000 ACC TTA ACC ACC ACC 222 222 777 777 777 777 777 777 ATT ACC ACC ACT ACC CTA CTC CTA 112 CTA ACA 000 CTA 777 ATA ATA CTT ATA CTA ACA CTC CTA CTA CTC TTA ATA CTA CTA CTA 567 890 123 CTC ATA CTA ATA TTT TTC ATG ACA TTA GTA 777 TTC ATA GTA GCA CTA ATA ATA CTA ATT TCT ACT GCT CTT CTA ATT CTC 234 ATT CTG TTA TTT CTT CTG ATT CTA CTA TTC TTA TTA TTC CTC ATT ATT 000 000 000 011 111 111 ATA TTA TTC GCC CTA ATA ATA TTT CTT CTC CTC CTA ATA ATT ACA ATA CTA 000 000 ACG CTC TTA ATT ATC ATA CTC CTA ACT ATA ATC CTC ACA ATA GGG ATA ATA TTA ATA ATA CTA CTA ATA TTC TTA TTA CTA CTA CTA CTA CTT CTT ATT CTA GCT CTG TTA CTT 000 000 TTC TTA ATA CTA ACA GCC 777 777 777 777 777 ATA GTA GCC ATT CTC ATA ATA CTA CTC CTA TTT TTA CTA CTT TTA TTA TTC ATA CTC TTC ATT CTC ATA TTA CTAATA ATC CTT CTA CAG ATC 789 012 345 678 901 666 ATA CTG TTG TTA CTT CTA CTA CTC GCC TTA TTT TTC ATT CTC 456 CTC GCT 000 000 000 000 ATC CTA ATC TTG GTC CTC CTT GCT ATT TTA CTA CTC ACA CTA TTC GTG GTT GTT ATC GCA GTA ATT CTA GTC TTC CTT GTT GGA 999 999 999 000 GGA GGT GGA ATA GGA CTA GGA CTA GGC CTA GGA CTA GGA 000 CTA GGA CTC GGA ATA GGA CTA GGA ATC ATT CTA GGT ATT CTA GGC 567 890 123 888 889 ATC CTA GGT ATT TTA ATC CTT GGC CTC ATT ATC 000 666 666 666 666 TTT TTT CTA GGA ATC CTA ATT CTC CTA GGTATC GTA TTTTTC ATA ATA TTT CTA TTG GGT GTC CTT CTC TTT TTC TTG GGA ATC CTT ATC ACC CTA GGT us Proechim ys Thricom ys Cavia Euryzygomatomys Ctenomys Bchimys Pappogeomys Coendou S palax Position: Cratogeomys M yoproeta Geomys H y s tr ix Thomomys Chaetodipus OryctolagusS ciu ru s Spermophilus R a ttu s Heterocephal Mus Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CCA CCA CCA 678 CCC CCT CCA CCC CCA CCA CCT 345 CCA CCT CCA CCC CCA CCA CCA CCA 777 777 CCA CCA CCA CCT CCA CCT CCA CCA CCA CCA CCA CCA ACC ACT 000 000 000 012 ACG 777 ACC ACT ACT ACC ACT ACT CCT ACC AAC ACG CCC AAC ACC CCT CCC 000 789 AAT AAT AAT ACA AAT AAC ACC AAT ACC (Appendix B con'd) B (Appendix CTC AAC TTA AAT ACA CCC CCA TTA TTA AAT ACACTG CCC TTA AAC ACA CCT CCT TTA 888 888 999 999 999 CTA 777 777 CTA CTT AAT CCG CCA TTA CCA CCC CTC AAC ACC CCA CCC CCA CTA AAC CCA 890 123 456 778 888 AAC CCC CTT AAT ACC CCT CCC AAT AAC CCT CTT 567 GCC AAC CCC GCC AAC CCA GCC AAC 777 GCT GCT GCA AAT CCT TTA AGT GCT AAC CCA GCC AAC CCT CTA AAT ACT CCC GCC GCA AAT CCT CTA AAC CCG CCC CCA GCA AAC CCC CCA CCT GCC AAC CCC CCG GCT AAT CCT GCA AAC CCC CCA GCA AAC CCC 777 CCT CCA GCA AAC CCA CTA CCC 901 234 677 ACC CCT GCC AAC ACA ACA ATA CCC GCA AAC CCA CTC AAC ATA CCA ACT CCC GCC AAC ACC CCC ACA CCC 678 TAC TAT ACC TAC ACC TAC TAC TAC TAC ACC TAC ACC TAC TAT TAT ATA CCT 345 AAC TAC ACA 666 666 AAC 777 777 777 777 777 777 777 AAT TAT ACA CCC GCT AAC AAT TAT TCT CCT AAC TAT ACT AAT TAC AAC AAT TAC ACA AAC TAT 012 GAT GAT AAC TAT ACT CCT GCC AAC CCA CTT AAC GAT GAC AAT GAC AAC GAC 000 000 000 000 000 000 000 000 000 000 CCA CCC GAC AAC TAT ACT CCA GAC CCA GAC CCA CCC CCC CCA GAC AAT CCA GAC GAC CCA GAC AAC GAC CCA GAC CCA GAT AAC GAC CCG GAC CCT GAT AAC TAC ACA CCA GCA AAC CCC ATG AGC ACG GAC GAC GAT GAT GAT GAC CCA GAT GAC CCA GAC 000 000 GGA GAC GGA GAC GGG GGT GGA GAT GGA GGC GGA GGA 445 555 555 555 666 CTA CTA TTA GGA GAC CCA GAC AAC CTA TTA CTA GGA CTA CTA GGG CTA GGA GAT CCA GAC CTA GGA CTA GGA GAC CCA GAC AAC TTA GGA CTA CTT CTA CTA GGA GAC CCA GAC AAC TAC ACC 111 111 111 111 111 111 CTA CTA GGA CTT CTT CTA GGA GAT CCT GAT AAT CTA CTA AAA AAA AAA CTA 000 000 000 234 567 890 123 456 789 444 444 111 GAC CTC CTA GAC CTT GAT GAC CTC GAC TTA GAT CTC TTA GGA GAC GAC CTA GAC GAC CTA TTA GGA GAC GAT AAA GAC CTT GAC GAC CTC GAC ATA CTA GGA GAC CCA GAC AAC GAT CTT GAC GAT GAC GAC CTA GAC GAC urus Position: Pappogeomys Proechim ys Oryctolagus S c i Spermophilus Cavia Thomomys Coendou Ctanomys Echimys Euryzygomatomys Thricom ys Cratogeomys Spalax R a ttu s Chaetodipus Heterocephalus Geomys M yoprocta H y s tr ix Marmota Mus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CCT CCC CCC CCA CCC CCA CCC CCA CCA 888 555 345 CCC CCA CCA CCT CCC ATT ATC ATC CCT ATC CCC 888 ATC CCT 555 ATT ATT ATC 000 000 012 ATT ATT ATC CCT ATT TTT ATC CCT TCC ATC TCA TCT ATT TCT TCA TCA 444 TCC ATC TCA ATC CCT TCA TCT CGC CGA TCA CGC TCA CGC TCC CGC TCC ATC CGT 888 888 CGA CGC CGC TCA 000 000 444 CGA TCA CGA CGA CGA TCA CAA CGA (Appendix con'd)B CTA CTA CTA CTC CTA CGC TCC ATC CCT CTA CGA TCC CTA CTA 123 456 789 CTA CTA CGC TCT ATC ATT ATC ATC 000 000 888 888 890 ATC CTA CGC ATC CTA ATT ATT ATT CTA GCA ATC GCA ATC GCA ATC CTC CGC TCT ATC CCT 888 GCA ATT CTA CGC GCA GCC GCT GCT 567 GCA ATC CTA GCC GCA ATC CTA TAC GCA TAC TAC GCA ATT CTA CGC TCC ATT TAC TAG TAC GCC ATC TAC GCC TAT GCT TAT TAC GCC TAT TAT TAC GCT ATC CTT GCA GCA GCA GCT GCA GCA GCC TAC GCA GCT TAC GCA GCA GCA GCA TAC GCG TAT GCA ATT CTA GCA GCA GCA TTC TTT TTC TTT GCC TAC TTC TTC TTT TTT TTT 000 000 000 000 222 233 333 333 334 444 678 901 234 TTT TTC TTT TTT TTT GCC TAT GCC ATC CTC TTT GCC TAC GCT ATC CTA CTA TTT TTA TTA CTA CTA TTA CTC TTT GCC TAC GCT ATT CTA CTA CTA CTG CTA CTA 222 000 CTA TTA CTA CTA TTC TTC TTC 000 TTT TTC TTC 012 345 TTT TTT TAT TTC TAT TTT TTATAT TTC TAC TTT TAC TAC TTC TTA TTT TAC TTC CTC TTC 888 888 888 888 888 888 111 222 TAC TTC CTGTAT TTT TTC TAC TTC TAT TTC 789 TAT TTT TAT TAC TAC TTC TAT TTC TAC TTC TGA TGA TGA TGG TGA 888 TGA TGA TGA TGA TGA TGA TGA TAT TGA TGA TGA TGA TGA GAA GAA 888 111 111 GAA GAG TGG TAT GAA GAA GAA GAA TGA GAA GAA GAA GAG CCA GAA CCA CCG GAA CCA CCA 888 CCA GAA TGACCA TAC CCA CCA CCT CCA CCT GAG CCA CCA AAA AAA 000 001 AAA AAA AAA AAA CCA AAA CCC GAA 888 888 000 000 000 000 000 000 ATC ATC ATT AAA ATT AAA CCCATC GAA ATC AAA ATC ATC ATC AAA CCA GAA ATC ATT AAA CCAATC GAA AAA TGG TAC TTC TTA TTT GCC TAC ATT AAA CAT ATT AAA CCA GAA CAT CAT ATT AAA 000 CAC 788 901 234 567 890 123 456 CAC ATC AAA CAC CAT CAC ATT AAA CAT CAT ATC AAA CAT CAT ATT AAA CAT CAC CAT CAT CAT ATC AAACAT CCA ATT GAA TGA TAC CAC CAT Thomomys Thricom ys Pappogeomys Echimys Proechim ys Position: 900 000 Spermophilus S palax Chaetodipus Cavia Ctenomys Euryzygomatomys OryctolagusS ciu ru s Cratogeomys R a ttu s Heterocephalus Coendou CAT ATT AAA CCA Geomys CAC H y s tr ix M yoprocta Marmota Mus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TTC ATG TTC TTC TTT 012 CTC TTC 000 ATC CTA TTC CTA TTA CTA TTA CTA CTA ATA TTC CTA CTT CTA CTA ATT ATT 000 789 TTC ATA GTC GCC CTA GCC GCC GCC 456 GCT GCC GCC GCT 999 999 999 GCC GCC GCC CTA CTT GCC TTC CTA ATA CTC TTT GCC ATA CTC TTT CTA TTA ACA CTA TTC CTA CTA GCA ATCCTA ATT CTA CTA CTA ATA TTA 123 CTA TTA TTA CTA 999 TTA TTA CTA CTA TTA CTA CTT ATA CTATTA TTC (Appendix B con'd) B (Appendix ATC ATC ATC 890 ATC ATC GTC GTC ATC GTC ATT ATC 990 000 000 000 111 GTA ATT GTT GTT ATT CTC CTA CTA TTA CTA ATC CTG CTT CTA 000 000 000888 000 889 999 567 TTA CTA CTA CTA CTA GTC CTA CTT CTC CTC CTT ATT ATC ATC CTA ATT ATC ATT CTT ATC CTC ATA ATT 888 ATC ATC 234 ATT CTA ATC ATC ATC ATC ATT ATC TCT TCC ATT CTATCC ATC TTA ATA CTA TTC TCA TCT TCA 901 888 TCC ATC 899 999 TCT TCA TCA TCA TCT TCA TTC TTT 678 GCC CTA TTA TCA ATA TTC TTA GTT ATA TCA ATC GTT CTT ATC TTA TCT ATC ATC ATC TTA GTC CTA TCC CTA GTT CTC CTA TTA ACT TTC TCT ATC TTA GTC CTA 012 345 888 888 888 CTA CTT ATC TTC TCT CTA ATC CTC CTA CTA GTA TTT CTA TTA GTC TTC TCA ATC CTA GTA TTC CTA GTA GCG CTA GTA GCC GCC 888 GCC TTG GCC GCC GCC 777 888 888 888 GCC TTA GCC GCC CTA GCC GCC GCC GCC GCT CTA GCT TTA ATT TTC GCC CTA CTA TTA CTA GCA CTA GTA CTG GCC ATA 777 CTA GTA CTA CTA GTA CTA ATA GTA GCA CTA GTC CTA TCT ATT TTA GTG CTA 888 888 GTA GTA CTA GCA CTA ATA TTC TCC ATC GTC GTT 000 000 000 000 000 000 000 000 777 GTA GTT GTT ATC GCT GTA TTA GTC GTC GTC GTC 890 123 456 789 GGA 667 GGC GTA TTA GGA GTA GGA GGA GGA GGT GTA GGT GTC GGA GGA GGA GGC GGA GGT GGA GGA GGA GTC 666 GGA GGG GGA GGC GGA GGA GGT GTC GGA GGA GGA GGA GGA GTA ATA GGT TTA CTA CTA GGA GGA CTA CTA CTA CTA CTA CTA CTA CTA CTA 901 234 567 000 000 000888 000 888 888 888 AAA 566 666 AAA AAA AAA AAA AAA AAA 678 000 888 AAT AAA CTT GGC GGG GTA AAT AAA AAC AAA AAT AAA CTC GGA GGA GTA TTA AAC AAA CTA AAC AAA CTAAAC GGA GGG AAT AAT AAC AAA AAT AAA CTT GGC GGA AAT AAA CTA AAC AAC AAA CTA GGA GGG AAT AAT AAC AAC AAA AAT AAA AAT AAA CTC AAC AAA TTA GGA AAC AAA CTA GGA Chaetodipus Coendou Ctenontys Proechim ys Thricomys Position: 555 Oryctolagus Cratogeomys Pappogeomys Thomomys Spalax Heterocephalus H y s tr ix Cavia Echimys Euryzygomatomys S ciu ru s Geomys Spermophilus R a ttu s Mus M yoprocta Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 144 TGC 999 TGC TTC ATT TGT ACT CAA TGC CAA TGT CAA TGC 999 456 789 CAA CTA CAA CAG CAA CAA TGC CAA CAA ACC AGC AGC CAA TGC AGT 123 AGC CAA TGT AGT AGC ACC CAA ATC ACA CAA AGT CAA TCC AGT CAA ACC AGC TTA AGT CAA TGC CTT ATA TTC 890 ATC CTT 000 000 000 000 ATC ATC AGC CTA AGT TTA TTA AGT CTG (Appendix con'd)B CCT CCC CTC AGC CAA TGC CCA CCA CCT CCA 000 999 999 999 567 CCA CCA CCC CCA CGC CCT CGA CCT TTA CGA CGC 555 555 556 666 666 666 CGA CCC TTACGC AGC 000 CGA CCG CTA AGC CGA TTC TTC CGC CCT ATT AGC CAA TTT CGC TTC CGC 000 TTC CGA CCT TTC TTT TTC CGA CCC ATT AGT CAA GTC ACA TTC CGCTCA CCA TTC ATT AGT GACACA TGT TTC CGC CCT ACA TTC TCG TAC CGT CCT ATC AGC CAG TGC ACA TTC CGC 444 455 TCA 000 ATG TTC ATA ATA ATA ATA ATA TCA ATG ATA ACA CTA ATA ATA CTA 444 ATG ATA TTC CGA TTA CTC TCA TTCCTC CGA GCA CTA ATA ATA ATA TTC AGC AGT AGC AGC 999 999 999 999 999 AGC ATA ATA TTC CGAAAT CCT ATG ATC AGC ATA CGA TTC 012 345 678 901 234 AGC AGC AGC AGC AGC TTA AGC CGC CGC CGC 999 CGC CGT CGA AGC CTG ACC TTC CGA 000 000 000 CGC CGA 789 CGT AGC CAA 999 CAA CGA CAA 000 CAA CGA AGC CAA CAA CGA AGC ATA ATA TTT CGA CCC ATT CAA CGT AGC ATG ATA CAA CGA CAA CGA CAG CGT ACG CAG CGT AGC 999 AAA AAA CAG 000 AAA CAA AAA CAA CGA GCC AAG AAA CAA AAA AAA AAA AAC CAG TCC AAA CAA CGC AGC TCA AAA CAA 999 TCC AAA TCA TCA AAA CAA CGA AGC TCA AAA CAATCA CGA TCA TCT TCA TCT AAA TCA AAA TCA ACT 999 000 000 ATA ACA TCC TCC ACC TTA ACC ATG CAC 999 CAT ACA GCC CAC CAC ACA TCA AAA 234 567 890 123 456 CAC ACA TCA 000 CAC ATA CAC CAT ACA CAC TTA TCT AAA CAA CTA 999 CTT 122 222 222 223 333 333 333 444 CTA CAC TCT TCC CTA CTC CTA 000 CTT CAT CTT CTT CAC ACA CTT CTC CTT 999 GCC 111 000 ATT GTA TTA CAT TTC CTT CAT TTC TAT TAC CTA CAC ACA TCA ATC CCT GCC CCT 999 345 678 901 CCT ACC CTC CAC ATA 000 CCT CCA TTC CCC CTT CTTCCA CAT CTC ACA CTA CAT CCC CCC ATA CCT CCC ATA CCT TAT CTC CAC ACA TCA AAA CAA CGA CCA CCT CCA ATC CTA CAC TTA CCA CTA CCC CTT CTT CAT ACTCCA GCC TTC AAT CTG CAT CCC TAC CCA CCC T hriconys Proechim ys Pappogeomys OryctolagusS ciu ru s Ctenomys Euryzygomatomys Spermophilus CCA CTG Cavia Position: 111 Spalax Chaetodipus Cratogeomys Thomomys Echimys HeterocephalusH y s tr ix Coendou R a ttu s Geomys Mus M yoprocta Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CCA CCA CCC CCA CCG CCC CCG CCA CCC CCC CCA 000 CCA CCC CAA CAA CCA CAA CCA CAA CAA CAA CAA 111 111 CAA CCA CAG CAA CCA 000 CAA CCA CAA CAA GGT GGC 890 123 456 GGC GGA CAA GGC GGC CAA AGC GGC GGT GGC GGA GGA CAG CCC GGA GGA GGA CAA 567 GGA GGA GGA GGG GGA GGA GGA GGA GGA CAAGGA CCA GGA CAA GGA GGA CAA CCC (Appendix B con'd) B (Appendix ATT GGA ATC ATT GGA GGT 111 111 111 ATT ATT GGA GGT 000 000 000 ATC GGA ATC ATC TGA TGA TGA 901 234 TGA ATT TGA ATT TGA ATT TGA ACA TGA ATC GGA ACA TGA ATC ACA TGA ATC GGC GGT CAA ACC ACT TGA ACC ACC ACT ACA CTT ACA TGA ATC CTT ACA CTA ACA TGA ATC TTA TTA TTA CTC GTC ATC TTG ACA TGA ATT GGT 012 345 678 ATC TTA ACA CTG ACA TTA ACC TGA ATC ATT 999 000 000 000 Oil 111 111 112 222 222 CTT ATT ATT CTC ACA TGA ATC GGT GGC CAA CCA 999 000 000 000 000 CTT ATC TTT ATT ACC CTA ACA TGA CTA CTT GTC CTG CTG CTA GTA CTA CTA ATT ACC CTA ACA TGA CTA TTA GTT ATC CTA CTA TTA ATC CTC ACACTA TGA ATC ATT ATT GGA CTC GGA ACA 456 789 CTC CTA GTC CTC TTT ATC CTA CTG CTA CTA CTA ACA TGA ATT GGA GGT CTA CTT CTC ACA CTC ACA TGA CTC TTT ACG CTA CTA CTC CTA TTC ATA ATT CTC CTA ACA TGA ATT GTA 999 999 AAT CTC CTC ATC CTC AAT AAC AAC AAT GAT AAC GAT GAT GAC GAC 890 123 GCC GCC GCC GCA GCC TCA GAC GCA TCT GTA GCT GCT GTC ACA GCA AAC GTAGAT GCA GTA GCC AAC GTG 000 000 000 000 000 000 111 111 111 111 CTA GCA GCA AAT CTA GTA GCA GAC CTC GTC GCA GAT CTA CTA 901 234 567 788 888 888 889 TTA TTA CTA GCA 999 999 999 999 999 999 ATT CTA ATC ACC TTA GTC TCA GAT ATT CTA ATA GTT CTA GTA GCT ATT TGA ATC CTA TGA TTA CTA GTA GCC 000 000 999 TGA TGA GTT TGA ATT TTA TGA ACG TTC ATC TCC ACC CTC CTC ACC TGA TGA ATC CTA GTA TGA TGA ATC CTA GTA GCA TGA TGA TGA TGA ACC CTA TGA ATT TTA ATT 345 678 TTC CTA 999 CTA 000 TTG TGA ATA CTA ACA TTA TGA TTC TTT TTC TAC TTC TTT TTT TTC 777 777 777 012 CTT 000 999 TTA CTT CTA TTA ATA TGA ATT CTA GTG GCC AAC CTA CTA TTC TGA TTA TTA TTC TTA TTG TTA TTA TGA CTA CTA CTA TTA CTA CTA TGA ATC TTA GTA GCA AAC CTT TAC ATA CTA TTC CTA CTA TTT TGA ATT TTA GTT ATA Thricom ys Pappogeomys Proechim ys Oryctolagus Position: S ciu ru s Cavia Thomomys Ctenomys Echimys S palax Chaetodipus R a ttu s Coendou Spermophi1us Geomys Cratogaomys Euryzygomatomys Heterocephalus H y s tr ix M yoprocta Marmota Mus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 123 ACC ATC ATT 111 ATT ATC ATT ATC ATT ATC ATT ATC ACC TGT ATT TGT ATT 111 CTA TGC ATT TGC TCT TTC TCT ATT TCT ATT TCA TCA TCA TCC CTA TTA GCA 778 888 GCT ATT TTC TTT TTC TTC ATT TTC CTC ATC 111 TTC TTC ACC ATC TTT TTC TTC 777 TAC TAC 111 TAC TTC TAC TAC 234 567 890 TAT 777 (Appendix B con'd) B (Appendix TCT TCT ACC TAT TTCTCC TTT TAC ACC TTC CCC TCA CTA TAT 677 CTA TAC CTA TAT ATA 111 111 000 000 000 000 000 000 678 901 ATC ATC CTA ATC TTA TAC TTC GTC ATT ATC CTA TAT GTC CTA TAC TTT GTC CTC TAC TTC ATC CTC TAC TTT 111 000 345 TCC TCA TCC TCA TCC TCG TCT TCC 111 GCA TCC GCA TCA ATC GCA TCC ATC 000 GCA GCC TCC CTA GCA TCT ATC TCC TAC TTC 111 789 012 TTA GTA GCC ATA GTA GCG GTA GCT CAA CTA CAA CTA CAA CTA GCC TCA ATC CAG TCG GCC 456 555 555 666 666 666 CAA GGC GGA CAA GGA GGA CAA 555 GGC CAA ATCGGC GCC CAA TCT CTA ATC GCC CTG TCC TAC ATC TTT GGC CAA CTA GCA TCA ATC TCC TAT TTC GGC CAA TTA GGT CAA CTA GCA TCC ATC TCC TAC TTC GGC CAA GTA GCC TCA ATC CTA TAT TTT ACT GGC CAA CTA GCA GGA CAA GTA GCA GGT CAA GTA GCA 000 000 000 000 890 123 ATC ATC ATC GGT 445 ATT ATC ATT ATT GGT CAA CTA GCC TCC ATC AGT ATC ATT ATT GGA CAA ATT GGT CAA ATT ATT GGT CAG ATT ATC ATT 567 TCA 000 444 ACC ACA CTC ACA ATT ATC CTC ATT GGT CAA CTA GCC TCC ATT ACT TAT TTT ATT ATT ATT ATC ATT ATT ATT GGT ACC ATT ATC 000 234 444 ATC ATC ATT ATC ATT ATC ATT ATT ATT ATC ATT TTC 000 901 TTC ATC ACA ATT TTC ATT TAC ATC ACC ATT GGC TAC TTC TAT TTC TAC TTT 678 CCC 000 CCA CCA CCA CCA TTT ATT ATC ATT CCT TAT CCC CCA TTT ATC CCA CCC 345 TAC CAT CCC CCT CCA TTT CAC CAC TAC CCT 111 111 111 111 111 111 111 111 111 333 333 333012 344 000 000 GAA GAA TAC CCA GAA TCC CCAGAA TTT ATT GAA GAA CAC CCA TTT GAA CAC CCG TTC ATC ACC GAA TAT CCA TTT ATC HI GTA GAA CCC CCA TTC ATC 000 789 GTC GAA GTC GTT GTA GAA CAC GTA GTA GAA CAC GTA GAA TAC CCA TTC ATC GTT GAG GTA GAA GTT GAA CAC GTT GTA GTA GAA CCA GTT GTT GTT GAA CCA GTA GTT GAA CAC GTA GAA CCA CCA TTC GTT GAA TAC GTT GAA CAC CCG TAT ATT ATC ATC GGC CAA CTA GCA TCA ATC Pappogeomys Thricomys Thomomys Proechim ys Position: 222 Chaetodipus Ctenomys Echimys Sciu ru s Spermophilus Cratogeomys Cavia Oryctolagus Geomys Euryzygomatomys R a ttu s Spalax HeterocephalusH y s tr ix Coendou Mus M yoprocta Marmota

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 147 AGA AGA AGA AGA 334 890 AAT TAA TAT AGG TGA AGA TGA AGA TGA TGA AGA TGA 567 TGA AGA CTC TGA AGA AAA TGA AGA AAA TGA AGA AAA TGA AAA TGA AAA TGA AGA 333 333 AAA TGA AGA AAA TGA AGA AAA TGA AGA AAA TGA AGA CTC AAA CTT AAA CTC CTA AAA CTT AAA CTT TTA CTC ATA 901 234 CTT ATA AAA CTT CTT AAA CTA TTA TTA TTA CTA ATG TTA AAA TGA ATC ATA CTA AAA TTA 222 233 678 ATA CTC CTT AAA TGA AGA ATG CTT AAA TTA AAA AAA TTT CTT AAA 111 111 111 111 111 111 AAA ATA 111 111 111 111 111 111 ATA ATC AAA ATA ATA AAA AAA CTT AAA ATC CTC AAA TGA AAA CTT CTT AAA TGA AGA AAC AAA AAC AAA ATA AAT AAA AAC AAA AAC CAT AAT 111 AAC 111 GAC AAA GAC AAT AAC AAG AAT AAT AAT AAG GAA AAC AAA CTA CTT 111 222 222 GAA AAC AAG CTT GAA GAA GAA 111 789 012 345 GAA GAA GAA GAA GAG ATA 111 ATA ATT GAA AAT AAA ATC ATT GAA AAC ATT TTT ATA GAA ATA ATA ATT CTA GTA GAA ATA 123 456 ATA CTC AGC CTC ATA GAA AGC CTC ATA GAA AGC AGC GGG TTA GTA GCT TTA ATA GAA 001 111 111 GGA CTC ATT GAA AACGGA AAA ATT GTT GAA GGC ATC ATTGGG GAG TTA AACGGC AAA TTA AGT CTA AGC AGC CTA ATT ACG AGC CTA TTA 567 890 ACA GCA GCA GGT 111 111 111 GCA GCG GCT GCA AGC CTA ATC GAA AAC ACA ACA ATA CTA 111 000 000 ATA CTA GCA ATC TCT CTC CCA CCC CCC CTA ACT 900 CCA CCA ACC ATC CCC AGC ATC CCA ATT GCA GGT CTC ATC CCA ACC ATT TTC CCC CTG ATA 111 111 111 111 111 111 111 111 000 011 678 901 234 CTT CCC ATC CTC ATA CCA CTA CTT ATA CCT CTA ACA AGC CTC ATA CCA ACA ACA GGA TTT ATA GAA AAC CTC ATA CTT ATA CCA CTC ACTATC AGT ATA CCC ACA ATT 111 CTA ATA CCC CTA 999 999 CTA CTT 000 CTC ATA CCA CTG CTC ATA CCA CTT ATA CCC CTA ATA ATT TTA ATT ATC ATC ATT CTT ATA CCT TCA ACA 111 ATT ATC ATT 012 345 ATC ATT 000 TTA TTC GCC ATT ATT TTA CTT GTC CTA CTC CTT CTT ATT 789 CTA CTA ATC TTA ATC CTT ATA ATT CTA 111 111 ATC CTA ATT TTA ATC TTA ATC ATT ATT 000 000 ATC CTT ATT 456 ATC 888 888 999 TTA CTA ATC CTC ATA CCA TTA ATC CTC CTC TTA TTA TTA ATA ATC CTA ATC ATT CTT TTA ATC TTA ATT CTT ATA CCA ATC TCA GGA ATT ATC ATC ATT CTA ATT CTC ATT CTC ATC CTA Thricom ys Coendou Proechim ys Pappogeomys Echimys Ctenomys H y s tr ix Thomomys Chaetodipus S palax C avia Euryzygomatomys Spermophilus R a ttu s Oryctolagus Heterocephalus Geomys Position: S ciu ru s M yoprocta Cratogeomys Marmota Mus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix C

.50 (Appendix C con'd) C (Appendix gene of 21 rodents and one rabbit.

b MTNMRKSHPLIKIINHSFIDLPAPSNISAWWNFGSLLGVCLALQI ITGLFLAMHYTA MTNMRKSHPLIKIINHSFIDLPAPSNISAWWNFGSLLGVCLALQI MTNTRKNHPLIKIINHSFIDLPAPSNISAWWNFGSLLGVCLALQIITGLFLAMHYTA MTNIRKSHPLIKIINHSFIDLPTPSSISYWWNFGSLLGACLILQIITGLFLSMHYTA MTNIRKTHPLIKIVNHSFIDLPAPSNISAWWNFGSLLGLCLAIQILTGLFLAMHYTS MTIMRKSHPLMKMVNHAFIDLPAPSNISSWWNFGSLLGLCLIIQIASGLFLAMHYTS MTIMRKSHPLMKIVNHAFIDLPTPPNISGWWNFGSLLGLCLILQILTGLFLAMHYTS MTNIRKTHPLLKIVNHSLIDLPAPSNISAWWNFGSLLGLCLMIQIFTGLFLAMHYTS MTIMRKSHPLMKIVNHAFIDLPTPPNISGWWNFGSLLGLCLILQIFTGLFLAMHYTS M7NVRKSHPLIKIINHSFIDLPTPSNISAWWNFGSLLGVCLVIQIITGLFLAMHYTA ______MTHLRKSHPLIKIINHSFIDLPTPSNISAWWNFGSLLGICLMMQILTGLFLAMHYTA MTNMRKSHPLIKIVNHSFIDLPVPSNISAWWNFGSLLGVCLGLQILTGLFLAMHYTA MKIMRKSHPLFKIVNHAFIDLPTPPNISGWWNFGSLLGMCLILQISTGLFLAMHYTS MTNIRKSHPLLKIINHSFIDLPTPSNISTWWNFGSLLGACLIIQILTGLFLAMHYTA MTNIRKSHPIIKIINHSFIDLPTPSNISSWWNFGSLLGVCLALQIITGLFLAMHYTA MTNIRKSHPLIKIINHSFIDLPTPSNISAWWNFGSLLGVCLLLQIITGLFLAMHYTA MTNIRKTHPLLKIVNHSFIDLPAPSNISAWWNFGSLLGLCLLIQILTGLFLAMHYTS MTNIRKTHPLIKIINHSFIDLPAPSNISTWWNFGSLLGLCLVIQILTGLFLAMHYTS MTNLRKSHPLIKIINHSLIDLPTPSSISTWWNFGSLLGMCLGLQIVTGLFLAMHYTS MTNIRKSHPLFKIINHSFIDLPAPSNISSWWNFGSLLGVCLMVQILTGLFLAMHYTS MAIMRKSHPLMKIVNHAFIDLPTPPNISGWWNFGSLLGLCLILQILTGLFLAMHYTS MTHLRKSHPLIKIINHSLIDLPAPSSISTWWNFGSLLGICLGLQIITGLFLAMHYTA MTNMRKTHPLFKIINHSFIDLPAPSNISSWWNFGSLLGVCLMVQIITGLFLAMHYTS Thricomys Proechimys ctenomys Echimys Euryzygomatomys Coendou Cavia Spalax Thomomys Myoprocta Heterocephalus Chaetodipus Spermophilus Hystrix Eattus Oryctolagus Sciurus eratogeomys Pappogeomys Mus Geomys Amino acid sequence of the mitochondrial cytochrome Marmota Positions are marked in intervals of 50 amino acids. Specimens examined correspond with Table 2.1. - o5 o5

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100 (Appendix C con'd) C (Appendix • DTTTAFSSVTHICRDVNYGWLIRYAHANGASIFFIFLYFHIGRGIYYGSYTFMETWN DTTTAFSSVTHICRDVNYGWLIRYAHANGASMFFIFLYFHIGRGIYYGSYSFMETWN DTTTAFS SVTHICRDVNYGWLIRYAHANGASMFFIFLYFHIGRGIYYGSYTFMETWN DTTTAFS DTSTAFSSVTHICRDVNYGWLIRYFHANGASMFFILLYLHVGRGIYYGSYTYTETWN DTTTAFSSVTHICRDVNYGWLIRYMHANGASMFFIFLYFHIGRGIYYGSYTFMDTWN DTTTAFSSVAHICRDVNYGWLIRYAHANGASMFFIFLYFHIGRGLYYGSYTFMETWN DISTAFSSVAHICRDVNYGWLIRYLHANGASMFFIFLYLHIGRGIYYGSYTFLETWN DTTTAFSSVTHICRDVNYGWLIRYLHANGASMFFILIYLHIGRGIYYGSYTLSETWN DTATAFSSVAHICRDVNYGWLIRYLHANGASMFFICLYLHVGRGMYYGSYMFMETWN SVAHICRDVNYGWLIRYLHANGASMFFICLYLHVGRGLYYGSYMFTETWN YTTTAFS DTLTAFSSVTHICRDVNYGWMIRYLHANGASMFFXCLFLHVGRGIYYGSYHFKETWN DTMTAFSSVTHICRDVNYGWLIRYMHANGASMFFICLFLHVGRGLYYGSYTFMETWN DTMTAFSSVTHICRDVNYGWLIRYLHANGASMFFICLFLHVGRGLYYGSYTFLETWN DTLTAFSSVTHICRDVNYGWLIRYMHANGASLFFICLYIHIGRGIYYGSYLYTETWN DTISAFSSVAHICRDVNYGWLIRYIHANGASLFFICLYLHIGRGIYYGSYLYKETWN DTLTAFSSVAHICRDVNYGWLIRYMHANGASLFFICLYMHIGRGIYYGSYLYKETWN DTLTAFSSVTHICRDVNYGWLIRYMHANGASMFFICLFLHVGRGMYYGSYTYFETWN DTLTAFSSVTHICRDVNYGWLIRYMHANGASLFFICLYIHIGRGIYYGSYLYTETWN DTMTAFSSVTHICRDVNYGWLIRYMHANGASLFFICLFLHVGRGLYYGSYTYFETWN DTMTAFSSVTHICRDVNYGWLIRYMHANGASLFFICLYIHIGRGIYYGSYLYKETWN DTTTAFSSVTHICRDVNYGWLIHYLHANGASMFFICLYMHVGRGIYYGSYTYLETWN DTMTAFSSVTHICRDVNYGWLIRYMHANGASMFFICLFLHVGRGLYYGSYTYFETWN

______1us Thricomys Coendou Ctenomys Proechimys Cavia Echimys Thomomys Spalax Euryzygomatomys Chaetodipus Heterocephalus Hystrix Oryctolagus Sciurus Spermophi Cratogeomys Pappogeomys Rattus Myoprocta Geomys Mus Marmota

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(Appendix C con'd) C (Appendix >150 IGVILLFMVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGPTLVEWIWGGFSVD IGIALLFTVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGTTLVEWIWGGFSVD IGTLLLLAVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGPTLVEWIWGGFSVD IGVILLFTVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGPTLVEWIWGGFSVD IGILLLLAVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGPTLVEWIWGGFSVD IGILLLFTVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGTTLVEWIWGGFSVD IGILLLFMTMATAFMGYVLPWGQMSFWGATVITNLLSAIPYVGTTLVEWIWGGYSVD IGIILLLSVMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGPTLVEWIWGGFAVD IGILLLFLSMATAFVGYVLPWGQMSFWRPTVITNLLSAIPYIGQDLVEWIWGGFSVD IGVLLLFAVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGTTLVEWIWGGFSVD IGIILLFRVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGTTLVEWIWGGFSVD IGIILLFTVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYMGTTLVEWIWGGFSVD MGVILIITVMATAFMGY 7LPWGQMSFWGATV1TNLLSAIPYIGPTLVEWIWG ?FSVD 7LPWGQMSFWGATV1TNLLSAIPYIGPTLVEWIWG MGVILIITVMATAFMGY IGILLLFLTMATAFVGYVLPWGQMSFWGATVITNLLSAIPYIGQDLVEWIWGGFSVD IGILLLLLTMATVFVGYVLPWGQMSFWGATVITNLLSAXPYIGQDLVEWIWGGFSVD IGVILLFAVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGTTLVEWIWGGFSVD IGVILLFWMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGTTLVEWIWGGFSVD IGVILLFALMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGTTLVEWIWGGFSVD IGILLLFLTMATAFVGYVLPWGQMSFWGATVITNLLSAIPYIGQDLVEWIWGGFSVD IGIMLLFLTMATRFMGYVLPWGQMSFWGATVITNLLSAIPYVGQDLVEWIWGGFSVD VGVILLFAVMATAFMGYVLPWGQMSFWGATVITNLLSAIPYIGPTLVEWIWGGFSVD IGIILLFAVMATAFIGYVLPWGQMSLWGATVITNLLSAIPYIGTTLVEWIWGGFSVD

______Thricomys Ctenomys Thomomys Cavia Echimys Proechimys Spalax Coendou Euryzygoma tomys Euryzygoma Spermophilus Pappogeomys Chaetodipus Sciurus Cratogeomys Heterocephalus Hystrix Oryctolagus Geomys Mus Rattus Myoprocta Marmota

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(Appendix C con'd) C (Appendix *200 KATLTRFFAFHFILPFIITALVAIHLLFLHETGSNNPSGLNSNSDKIPFHPYYTIKD KATLTRFFAFHFILPFIIVALVMTHLLFLHETGSNNPSGLNSDSDKIPFHPYYTIKD KATLTRFFAFHFILPFIITAMVMIHLLFLHESGSNNPSGLNSDSDKIPFHPYYTIKD KATLTRFFAFHFVLPFIITAMVMIHLLFLHETGSNNPSGLNSNSD?IPFHPYYTIKD KATLTRFFAFHFSLPFIITALVLVHLLFLHETGSNNPSGIDSNSDKIPFHPYYTIKD KATLTRFFAFHFVLPFIITAMVMIHLLFLHETGSNNPSGLNSDSDKIPFHPYYTIKD KATLTRFFAFHFIMPFIIATMIMVHLLFLHETGSNNPSGLNSDSDKIPFHPYYSIKD KATLTRFFAFHFIVPFIITALVMVHLLFLHETGSNNPSGLNSDSDKIPFHPYYTIKD KATLTRFFAFHFILPFIITAMVMIHLLFLHETGSNNPSGMNSDSDKIPFHPYYTIKD KATLTRFFAFHFILPFIITALVMVHLLFLHETGSNNPSGLDSNADKIPFHPYFTIKD KATLTRFFAFHFILPFIITALTMVHLLFLHETGSNNPSGINSDSDKIPFHPYYSFKD KATLTRFFAFHFILPFIITALAMVHLLFLHETGSSNPLGIPADCGKVPFHPYYTTKD KATLTRFFAFHFILPFIIAALAIVHLLFLHETGSNNPTGLNSDADKIPFHPYYTIKD KATLTRFFAFHFILPFIIAALAIVHLLFLHETGSNNPTGLNSDADKIPFHPYYTIKD KATLTRFFAFHFILPFI IAATAMVHLLFLHETGSNNPLGIPSDSDKIPFHPYYTLKD KATLTRFFAFHFILPFI KATLTRFSAFHFILPFIVAALVMVHLLFLHETGSNNPSGLISDSDKIPFHPYYTIKD KATLTRFFAFHFVLPFIIAALVMVHLLFLHETGSNNPSGLISDSDKIPFHPYYTIKD KATLSRFFAFHFILPFIITALATVHLLFLHETGSNNPLGIPSDCAKIPFHPYYSTKD KATLTRFFAFHFILPFIIATLVLIHLLFLHETGSNNPTGIPSNSDKIPFHPYYTIKD KATLTRFFAFHFVLPFIIAALVMVHLLFLHETGSNNPSGLISDSDKIPFHPYYTIKD NATLTRFFAFHFILPFIITALVMVDLLFLHETGSNNPLGIQSNYSKVPFHPYYTTKD KATLTRFFAFHFILPFIITALVMVHLLFLHETGSNNPLGLPSDCSKVPFHPYYTTKE

______Thricomys Proechimys Ctenomys Cavia Echimys Coendou Thomomys Euryzygomatomys Pappogeomys Spalax Hystrix Spermophilus Chaetodipus Cratogeomys Geomys Rattus Heterocephalus Myoprocta Oryctolagus Sciurus Mus Marmota

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(Appendix C con'd) C (Appendix • 250 • ILGLLFMLLSLTMLILFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP IMGFMFMGFTLLFLVLFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGLLLMILTLLMLILFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGLLLMLTALLILVLFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGILLMMITLMSLVMFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGLLFMLFALMMLILFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGLLLMLLTLLSLTLFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGALFMMLALLCLVLFTPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGAMMLTIILTSLVLFFPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP ILGLILMLLILLISTLFSPDLLGDPDNYTPANPLNTPPHXKPEWYFLFAYAILRSIP ILGVLLLILALMTLVLFSPDLLGDPDNYTPANPLSTPPHIKPEWYFLFAYAILRSIP FMGLQIMLLILLTLTLFHPDLLGDPDNYTPANPMSTPPHIKPEWYFLFAYAILRSIP ILGVLLLILILMTLVLFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILQSFP ILGILIMFLILMTLVLFFPDMLGDPDNYMPANPLNTPPHIKPEWYFLFAYAILRSIP FMGAILLLTLFMTLVLFFPDKLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP FLGALLLIMFFMTLVLYFPDKLGDPDNYMPANPLNTPPHIKPEWYFLFAYAILRSIP FLGVILVLALFLTFVLFFPDLLGDPDNYSPANPLNTPPHIKPEWYFLFAYAILRSIP FLGWMLLMLFLTLVLFFPDKLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP FLGVIMLLMLFLTLVLFFPDKLGDPDNYMPANPLNTPPHIKPEWYFLFAYAILRSIP LLGVFMLLLFLMTLVLFFPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP LLGVLLLLLLFMVLVLFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP TLGFLVAILLLLILVLFSPDLLGDPDNYTPANPLNTPPHIKPEWYFLFAYAILRSIP us ______Thomomys Cavia Proechimys Thricornys Sciurus Pappogeomys Spaleuc Coendou Ctenomys omys Echimys t Euryzygoma Oryctolagus Spermophi1 Cratogeomys Geomys Chaetodipus Heterocephalus Rattus Hystrix Myoprocta Marmota Mus

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(Appendix C con'd) C (Appendix *300 NKLGGVLALVFSILILTLFPALHTSKQRSMTFRPLSQCLLWLLVANLIILTWIGGQP NKLGGVLALVLSILILMLFPALHTAKQRSMTFRPMSQCLLWILVANLIILTWIGGQP NKLGGVLALVMSILILVALPFLHTSKQRSMMFRPXSQCLFWTFISTLLTLTWIGSQP NKLGGVLALVFSILILMLFPLLHLSKQRSMMFRPLSQCMFWILVADLFTLTWIGGQP NKLGGVIALVLSILVLALFPLLHTANQRSMMFRPISQFLFWTLVSDLFILTWIGGQP NKLGGVMALVFSILVLALLPYLHTSKQRSLSFRPLSQTLFWILVSDVITLTWIGGQP NKLGGVMALVFSILILALLPYLHTSKQRSLAFRPLSQTLFWILISDMILLTWIGGQP NKLGGVLALVLSILVLAFIPFLHMSKQRSMMFRPISQVLFWVLVADLLTLTWIGGQP NKLGGVLALVFSILILMLFPVLHMSKQRSMTFRPISQCLLWILTANLVILTWIGGQP ______NKLGGVMALLASILVLALFPILHLSKQRSMTFRPISDCLLWMLTANLWLTWIGGQP NKLGGVLALTFSILILMLFPILHSSKQRSMSFRPLSQCLMWILVANLLILTWIGGQP NKLGGVLALIFSILILAIIPLLHTSKQRSMLFRPFSQCLFWILAANLLILTWIGGQP NKLGGVLALMFSILILMLFPTLHMSKQRSMSFRPLSQCLLWILVANLIILTWIGGQP NKLGGVLALILSILVLALMPLLHSSKQRNMSYRPISQCLLWLLAANLLVLTWIGGQP NKLGGVLALVFSILILMLFPLLHLSKQRSMMFRPLSQCMFWILVADLFTLTWIGGQP NKLGGVLALIFSILILMLFPILHMSKQRTMMFRPLSQCLFWILVADLFTLTWIGGQP NKLGGVLALILSILILAIIPMLHTSAQRSLTFRPISQLLFWMLVADLITLTWIGGQP NKLGGWALLMSILVLALLPYLHTSKQRSLSFRPLSQTLFWVLVADLLLLTWIGGQP NKLGGWALILSILILAFLPFLHTSKQRSLTFRPITQILYWILVANLLVLTWIGGQP NKLGGVLALVLSILILALFPMLHTSKQRSMRFRPLSQCLLWLLAANLLILTWIGGQP NKLGGVLALILSILILALMPFLHTSKQRSLMFRPITQILYWILVANLLILTWIGGQP Thri cornys Thri Euryzygomatomys Proechimys Pappogeomys Thomomys NKLGGWALVLSILVLAFLPYLHTSNQRSLMFRPLSQSLFWTLVADLLLLTWIGGQP Coendou Cavia Ctenomys Echimys Spermophi1 us Spermophi1 Chaetodipus Rattus Spalax Cratogeomys Hystrix Geomys Heterocephalus Oryctolagus Sciurus Myoprocta Marmota Mus

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• 350 • VEHPFIIIGQLASISYFSIILILMPISGIVEDKMLKWN VEHPFIIIGQVASILYFSIILILMPLAGLVENMIMKL* VEPPFIIIGQIASILYFSIILLLLPIAGLIENKILKW* VEHPFIIIGQLASISYFSIILILMPISGIIEDKMLKLY VEYPFITIGQLASISYFCIILILMPMTSLMENHLLKW* VEYPFITIGQLASISYFCIILILMPTTGFMENKLLKW* VEHPYILIGQLASISYFLTILVLMPLTSMMENKFLKW* VEYPFITIGQLASMTYFFTILILMPSTALMENKLLKW* VEHPFITIGQLASISYFCIILIIMPTISFMENKLLKW* VEPPFISIGQLASISYFCIILILMPTTSLMENKLLKW* VEHPYIIIGQLASILYFAIILLILPTISLIENKLLKW* VEYPFIIIGQLASVLYFTIILLILPTISLIENKLLKW* VEYPYIIIGQLASILYFLIILILMPLAGLVENKMMKW* VEHPYITIGQLASISYFSILLIIMPLTSIMENKLLKW* VEHPFILIGQLASITYFFIILILMPLTSMMENKLLKW* VESPFIIIGQVASILYFTIILILMPLAGLIENKMMKW* VEYPFIIIGQVASILYFAIILFALPSISMLENKLLKW* VEPPYIIIGQVASVLYFLIILILMPIAGLIENKMLKW* VEPPFIIIGQMASILYFSILLILMPLAGIIENKMLKW* VEPPFIIIGQVASILYFLILLLLMPMAGLIENKLLKW* VEHPYITIGQSASIPYFFIILILFPLTSLLENKMLKW* VEHPFITIGQVASVLYFSTILILMPLASLIENKILKW*

______Pappogeomys Thomomys Thricomys Proechimys Spalax omys t Euryzygoma Spermophi1 us Spermophi1 Cratogeomys Chaetodipus Sciurus Oryctolagus Geomys Rattus Hystrix Coendou Cavia Ctenomys Echimys Heterocephalus Marmota Mus Myoprocta

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix D

Collection, localities for pocket gophers used in this study. Locality numbers correspond with Table 3.1. Host and parasite vouchers are deposited in the Louisiana State University Museum of Natural Science mammal (LSUMZ) or tissue (M) collection, the New Mexico Museum of Natural History (NMMNH), or the University of South Dakota Natural History Collection (USDNHC). Other initials are those of individual collectors.

1 Washington: Mason Co.; 2 mi. N Shelton on Hwy. 101, Shelton Airport (LSUMZ 34383; TSD 567) 2 Oregon: Lane Co.; 1.1 mi. N, 5.5 mi. E McKenzie Bridge (LSUMZ 31398; TSD 504) 3 Nevada: Washoe Co.; 2.2 mi. N, 2 mi. E intersection Hwy. 28 and Hwy. 431 (LSUMZ 31411; TSD 493) 4 New Mexico: Sandoval Co.; 11 mi. E, 3 mi. N Jemez Springs (LSUMZ 34031; DLR265) 5 Washington: Klickitat Co.; 5 mi. S Trout Lake, on Forest Rd. 051 (LSUMZ 34387; TSD 571) 6 Utah: Sevier Co.; 5 mi. S, 2 mi. E Monroe (Mud Springs Flat) (LSUMZ 31301; TSD 454) 7 South Dakota: Lawrence Co.; Timon Campground (USDNHC 1091 and 1094; PDS 453 and SCM 4) 8 New Mexico: Socorro Co.; 3.5 mi. S La Joya, W side Rio Grande (LSUMZ 29548; JWD 39) 9 Oregon: Jackson Co.; Ashland, near intersection of Hwy. 66 and Interstate 5 (LSUMZ 34331; TSD 576) 10 New Mexico: Socorro Co.; 2.0 mi. N, 0.5 mi E Polvadera (NMMNH 1260; JWD 68) 11 Oregon: Benton Co.; 4 mi. N Corvallis (Benton Co. Courthouse) (SW 1/4, NE 1/4, sec. 13, T11S, R5W) (LSUMZ 31306; TSD 535) 12 Nevada: Lander Co.; 9.8 mi. N, 0.3 mi. E Battle Mountain (LSUMZ 31264; TSD 459) 13 Oregon: Malheur Co.; 15 mi. W Vale (by road) on Hwy. 20 (LSUMZ 34330; TSD 583) 14 MEXICO: Sinaloa; 1 km N Siqueros, 50 m elev. (M 3042; MSH 1452) 15 MEXICO: Durango; 12 km E El Salto, 2490 m elev. (M 3040; MSH 1450) 16 MEXICO: Chihuahua; 13 km E Tomochic, 2100 m elev. (LSUMZ 34346; MSH 1442) 17 MEXICO: Durango; 50 km N, 20 km W Bermejillo, 1140 m elev. (M 3034; MSH 1444) 18 Arizona: Santa Cruz Co.; 10.5 mi. S, 0.6 mi. E Patagonia (0.4 mi. N, 0.6 mi. E Crescent Spring) (LSUMZ 33869; TSD 546) 19 MEXICO: Michoacin; 6.5 km S P4tzcuaro, 2200 m elev. (M 3044; MSH 1459) 20 MEXICO: Mexico; 9 km N Valle de Bravo, 2370 m elev. (M 3047; MSH 1463)

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix E 234 ATG ATG ATG ATG ATG ATG ATG ATG ATG TTA CTT CTT CTT 567 000 CTT CTT TTG 111 111 ATG ATA ATA 000 ATC ATC CTT ATT 111 ATT ATT 000 ATT ATC ATC ATT ATT individuals. 111 000 000 011 111 TAT GCT ATA TAT GCT ATA 000 678 901 234 CAA CAA ATT ATC CTT CAG ATT ATC CAG ATT ATT 444 455 555 555 CAG ATT CAA CAA CAA (Appendix E con'd) E (Appendix 000 012 345 678 901 ATT 345 000 TCT TCC TCT 999 ATG ATT TAT GCC ATG ATT TAC GCT ATA ATT TAT GCT ATA ATA ATT TAT GCT ATA ATA ATG ATT TAC GCT ATG ATG ATT TAT GCT ATG ATT ATC TCC ATC TCT 444 444 ATT ATC TCC CAGATC ATT TCC ATC TCT ATT TCC 999 GGA 456 789 GGA GGA 000 000 111 GGA GGA GGA TTA 333 TTA CTA TTA CTA CTA TTA TTA ATT Thomomydoecus 999 123 ATT 000 ATT ATT ATT ATT GGG GGG GGT GGG GGG GGT 889 890 ACA ATT ACA ATT GGA ACG ATT GGA ATG ATT TAT GCT ATG ACT ATT GGG 123 456 789 012 333 333 TTT GGA TTGTTC ATC 000 000 000 000 TTT GGA TTT 567 GGG GGA GGG ACA GGG ACA GGG ACA GGG ACT GGG GGG TTC 000 GGA TTT GGG GGA GGA GGG TTT GGT GGA GGG TTT TTT GGG ACA TTT GGG TTT 567 890 CCT CCT CCA 901 234 GTT GTT 788 888 888 GTT CTT CTT CCT 222 222 223 000 000 CAA 777 CAG 901 234 122 ATC CTC CCT GGG TTT ATC CTC ATC CTT CCT 345 678 AAA CAG AAG CAG GTT TTT 000 000 000777 000 000 000 AAG CAG GTT TTT AAA AAG CAG GTTAAA TTT CAG GTT TTT TTA AAG CAG GTT TTT 111 678 TTA ATT TTG ATC CTTTTA CCC CTA TTA ATT TTA CCA 000 AAG 345 ATT ATT ATT GGG AAA GGG AAG GGT AAG GGA AAG 012 TAC ATT TTG ATC TAT ATT TAT ATT TAT ATT TTGTAT ATT ATT TTA TTG ATC CTT CCT TAT ATT 666 666 777 AGA AGA 000 000 AGA GGA AAA AGA AGA AGA GGG 000 111 111 789 GTT GTT GTT GTT GTT GTT 000 666 GAG 000 000 000 000 000 000 000 GAG GAG GAG GAA ??A GAA GTT TAT 890 123 456 789 012 556 ??C GAG 000 TAT GAA TAT GAA AGA GGG AAA AAA TAT GAA TAT GAA TAT GAA ??A ??C GAG TAT GAG ??C 123 456 ??T GAA GTT TAT 000 000 — 15 TAT GAG AGA GGG AAA AAA CAG GTT TTT — 15 ??G — 16 — 7 — 20 ??G GAG GTT TAT — 7 — 4 TAT GAA A?? GGA AAG — 18 --10 --10 Position: Position: zacatecae— 6 neocopei--20 asymmetricus— 16 TAT Jbirneyi--18 barbarae zacatecae— 6 ??A arlenae minor--8 asymmetricus b irn e y i barbarae neocopei asymmetricus arlenae— 4 T. minor T. asymmetricus T. T. T. T. T. T. T. T. T. minor T. T. T. T, T. T. m inor--8 T. Locality numbers correspond with Appendix D. Nucleotide sequences of a 379 base-pair region of the COI gene of nine

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. u * ACT ACT ACA ACG 111 677 901 ATA ATA 345 678 CCA CCT CCT ACG CCT ACT 111 666 GGG ATA GGG ATG 222 222 222 GTT CCT ACT 222 222 222 GTT CCT GTT CCT ACT GTT GTT GTT GTT CCC ACT GTT 666 GTT GGG ATG GTC GGT ATG GTT GTT GGA ATA GTA GGT ATA GTT GGT ATG GTT GGG 789 012 GCT 111 111 (Appendix E con'd) E (Appendix 456 222 222 ATT GCC ATT GCT ATT GCT ATT GCT ATT GCT ATT ATT GCT ATT GCT TTC ACC 111 555 666 TTC ACC TTT ACC GTT TTC ACC TTT ACC TTC ACC TTC ACC GTT GGG TTC ACT TTT ACT 123 222 GTA GTA GTA GTA 111 555 ATA 456 789 012 345 678 ATA ATA ATA ATA ATA ATG ATA 890 ATG ATG ATG GTA ATT GCT GTC CCT 222 222 ACT ATG ACT 890 123 CAT CAC ATG CAT CAC CAT CAC 445 555 CAT CAC CAT CAC CAT CAC CAT CAC 000 000 001 111 111 111 222 GCC ACT ATG GTA GCC ACT GCC ACT GCT ACC ATA GTA GCT 444 567 GCA GCA GCA GCA GCA 900 AGT AGG AGG AGG 234 TGA GCA TGA GCA 999 ACT AGT 344 444 901 111 111 111 111 111 GTT TGG GTT TGA GTT TGA GTT TGA GCTGTT CAC CAT GTT TTT TTT ACT 333 678 GTG GTT TGA GTA GTA GTG GTG GTA GTG 999 999 TAT TAT TTT ACC 333 345 TTT TTT TTT TTT GTG GTT TGA TTT TTT GTG GTT TGA GCT CAC CAC TTT GCA TAT TTT ACT GCA GGT TTC GGT TTT 888 888 111 111 111 111 111 122 456 789 012 345 678 901 234 567 CGA GCA TAC TTT ACC AGG GCT ACT ATA GTA CGG GCA TAT TTC ACCCGG AGA GCA GCC TAT CGG GCA TATCGG TTT ACC CGG GCA 222 333 TTG GGT TTA GGT 789 012 TTG TTA TTG GGA AGT AGT AGT 222 GTT AGT GTA CTA GGT GTA GTA GTG GTA TTA GGT 890 123 111 111 GAT AGT 778 888 GAT AGT CGG GAT AGC 123 456 GAT AGT CGA GCG TAT TTT ACC AGG GCT ACTGAT ATG AGT CGG GCA TAT TTC ACC AGA GCC ACT ATG GTA GGG GTA GGT GGT GGT 567 111 GTC 777 GTT 112 222 GTA GAT GTC GTT GAT GTA GTC GAT GTA ATT ATT GGT ATT ATT GGA ATT GGG GTA TTA GGT ATT ATT 111 234 777 GAT GAT 111 GAC GAT GAT GAT 111 111 111 111 111 111 111 111 567 890 GCG ATT GGT GCG GCT ATT GGT GTA TTA GTA GGT ATT GGT GCT GCG GCG GCT GCG GCT GCG — 16 GAT GTC GAT — 16 20 7 7 cus--15 18 4 GAT — — — --20 — 8 — — 18 — — 10 Position: Position: zacatecae— 6 GAT zacatecae--6 a s y m m e t r i n e o c o p e i minor— 10 arlenae--4 asymmetricus— 15 m i n o r b a r b a r a e T. T. T. T . a r l eT n . a e b a r bT. a r aT. e T.T asymmetricus . b i r n e y i T . m i n o r T. T. T .T b . i r n n e e o y c i o p e i T. T. asymmetricus T. T. minor— 8

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACT 345 TCA TCT 333 333 789 012 ATA ACT ATG ACT ATA ATG ACT ATA ACT ATA ACC 012 TAT TCT 888 888 222 222 TAT TCT TAT TAT TCT TAC TCA 333 333 444 333 GGT GGG GGG GGG ATG ACT GGT TCG 789 TCA TAT TCT TCA TAT TCT TCA TCA TAT 777 222 TCA TAT TCT 123 456 GGA GGT ATA ACT GGG GGA GGA GGA GGA GGG ATG ACT GGG GGG GGG ATG 456 ATG TCA 777 ATG ATA TCC (Appendix E con'd) E (Appendix 890 GTC GTC GTT GTT GTC GTC CGG GTC CGG ATG 123 CGG CGG ATG 777 222 222 CGG CGA 222 223 333 ACT ACC ACT GTC GGG GGT ACT ACT ACT ACT GTC 890 667 222 AGA AGA CGG ATG AGA TTC TTC GGG AGA GGA GGA AGA 567 222 GGG AGA GGA GGG TTA TTT TTT 234 222 333 333 333 333 333 333 TTC TTA TTC TTT TTA TTC TTT TTA TTC ACT TCT TTT 901 TCT TCT CTT 566 666 666 222 TCT TTT GGT AGA CGG ATG TCT TTC 111 111 122 222 333 GTA GTA GTA TTT GTG TTT TTA GTA TTT TTA TTT GTA TTT TTA TTT GTA TTT TTA TTC ACT GTG 678 555 ACA ACA ACA ACA TCT TTT GGT AGA 111 TTT TTT TTT TTT GCC GCC ACA GCC ACA TCT GCT GCT ACA TCT TTT GGT AGA CGG ATG TCT 000 333 333 789 012 345 678 901 234 567 GGG TTT GTA TTT TTA TTC GGA GGG GGG TTT GGA GGA TTA TTA GCC ACG TTA 333 ATT ATC ATT ATA ATT ATT TGG 000 000 123 456 GCA 444 444 555 555 222 222 222 222 222 AGT TGA AGG TGA TTG GCC AGG TGA TTG TGG GCA ATC GGG TTT TGG GCA TGG GCA ATT GGA TTT TGA TGG GCA TGG GCA TTT 999 990 567 890 CTT CTT ATG 890 123 456 789 012 345 TTC CTC 222 222 GTA TTT AGA TGA TTA GCC GTG TTT GTG AGT TGA TTA GTG GTG TTT AGA 999 234 ATG CTC TGG GCA ATC GGG TTT ATA ATG ATG ATA TGG GCA 567 333 334 444 ATA 222 AAA AAG GTA TTC AGG TGA AAA AAA TCC TCT TCT TCT 234 TCT 333 222 GTA GTA GTA 678 901 222 222 222 222 223 333 ATT TCT ATG TTT 901 ATT 233 ATT 222 GGG GTA GGG 16 ATT TCT ATA 15 GGG GTG AAG GTA TTT AGG TGA TTG -- — 7 ATT 20 ATT TCT ATG — 6 4 ATT TCC ATG ATA TGA GCA — — — 20 — 18 ATT 10 — 18 GGA 8 GGA GTA AAG GTA TTC — — Position: 888 899 Position: z a c a t e c a e asymmetricus— 15 ATT a r l e n a e - - zacatecae--6 asymmetricus— 16 GGG GTA AAG GTA TTT AGG TGA TTG GCC ACA TCT TTT n e o c o p e i b i r n e y i barbarae--7 GGG GTG AAA n e o c o p e i b i r n e y i minor— 8 minor— minor— 10 GGA GTA AAG GTA TTC T. T. T . b a r b a r a e T. T. asymmetricusT. T . m i n oT. r asymmetricus T. T. T. T. T . m i n o r T. T. arlenae— 4T. GGA T. T.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vO ui 333 788 TTA GTT CTA GTT TTA GTT 345 678 901 GTA GTT CTT GTG GTT CTT GTA 012 777 777 777 GAT GTG GTT TTA GAT GAT GTG GTT TTA GAT ?TG 666 GTG GTG TG? GTG GAT GTG GTT TTA TG? TGT GTG 333 333 333 333 333 333 TCT TGT TCT TGT GTA GAT GTG GTT TTA TCT TGT GTG GAT GTA TCT TGT GTA GAT ??T TCT 333 AAT AAT AAT ??? 555 556 666 666 GCA AAT G?A AAT TCT TGT GCA AAT TCT TGT GTA GAT GTG G?? ??T TCT 555 TTG TTA TTG GTT TTG GTT TTG GCA GTT TTG GTT GTT TTG GCA GTT 678 901 234 567 890 123 456 789 444 455 TTA TTA GTT TTG GCA TTA GTT TTA CTA TTA TTA GTT TTATTA GG? AAT TCT TTA 345 333 333 333 333 333 444 GGT GGT GGT GGT GGT — 16 — 20 — 7 — 18 GGT — 4 — 8 — 10 GGT Position: zacatecae--6 GGT b irn ey i asymmetricus— 15 GGT asymmetricus barbarae arlenae minor minor T. neocopei T. T. T. T. T. T. T. T.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. > no to 9 "0 TTA TTG TTA TTG TTG TTG TTG TTG TTG TTA TTA TTA TTA TTG 567 TTG TTG TTA 555 TTG ATT ATT ATT ATT 555 234 ATT ATT ATT TTG ATT GTC ATT ATT ATT ATT 000 000 000 ATA ATA ATA ATT ATA CAC ATT CAT CAC CAC ATT ATT CAT 000 CAT CAT (Appendix F (Appendix con'd) F TCA TCC CAC ATT ATT TCT TCC CAA ATT ATT TCC CAT ATT TCC TCT CAC TCC CAT TCC CAC ATG ATT TTG individuals. Locality ATC GTC TCC CAC ATT GTC ATT TCC CAC ATT ATT ATC TCT ATT TCC ATC ATT ATC ATT TCT ATT TTG ATT TTA TTA TTG ATT TTG CTA 333 444 444 444 455 TTG TTA TTG GGC TTA GGC GGC CTA ATT TCA CAC ATT ATT GGC GGG TTA ATT TCC CAT ATT ATT GGC 333 000 000 000 000 GGT Geomydoecus TTT TTC TTT TTC GGT TTT GGG TTA TTC GGT TTA ATT TCT CAC ATT GTT TTT GGT TTA ATT TCC CAC ATG ATT GGA GGA TTT GGA GGA TTT GGA GGA GGA TTC GGT TTA GGA TTT GGG TTA 000 000 GGA TTT GGT CCA CCA GGA CCA GGA TTC CCA GGA CCA GGA TTT 567 890 123 456 789 012 345 678 901 CCG CCT GGA TTT GGT TTA CTC CTC CCA 222 222 223 333 CTA ATC CTC CCA ATC ATT CTT ATT CTT CCA ATC CTT 122 901 234 ATT CTT CCA ATC CTC 000 000 000 ATC CTA CCC GGA TTT GGT ATC ATT TTA CCA GGA TTT ATC TTA CCT GGG TTT GGT TTA ATT TCC ATT TTA CCA GGA TTT GGT CTG ATT TCT CAT ATA ATT CTA TTA ATC CTT CCA GGA CTA TTA ATT CTT TTG ATT TTA CCA TTA TTA ATT TTATTA CCT TTA ATT TTA CTA 678 TTA ATC CTCTTA CCA GGA TTT GGA 000 TTA ATT TTA CCA GGA TTA TTA CTA CTA TTA ATT CTA TTA ATT ATC ATT ATC 345 ATT ATT ATT TAT ATT TAT TAT ATT TAT ATT 000 000 TAT TAT ATT GTT TAT ATT GTT TAT ATT GTT TAT ATT GTT GTT TAT ATC 000 GTT TAT ATT GTT GTA TAT GAA GAG GAG GTA GAA GTT TAT GAA GTT TAT GAA GAG GTT GAG GTT TAT 456 789 012 GAG GTT GAG 000 GAA GAG GAG GTG GAG GTT TAT ATT GAG ??G ??G ??C ??G ??? GAA ??T GAG GTT TAT ??G ??A ??A GAG GTT TAC ATT ??T GAG GTG TAT ATT ??T ??T 123 000 — 12 ??G — 9 — 18 — 10 — 4 — 16 — 5 ??A — 2 — 1 — 19 ??G — 17 ??A — 14 — 6 — 3 Position: 000 000 000 111 111 111 weileri--20 ??T idahoensis— 13 ??G GAA GTT TAT ATC th a e le r i w e lle r i oregonus--ll chihuahuas— 15 thomomyus— 7 ??T th a e le r i thomomyus c e n tr a lis aurei— 8 ??T c r a ig i musculi G. G. w e lle r i G. G. crovelloi G. G. G. G. warmanae G. G. idahoensisG. G. G. shastensis G. G. G. G. G. G. th a e le r i G. c r a ig i Nucleotide sequences of a 379 base-pair region of the COI gene of 20 numbers correspond with Appendix D. o ON

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a\ ATG ATA 111 TCT ACT 234 ATA ATG ATA ATG ATA ATA ATA ATA ATA ATA ATA ACT ATA ATA ATA ATA 011 ATG 901 ATA ACT ATA ACT ATA TCT ATA TCT GCA ATA ATG GCA GCA GCA ATA ATA GCA GCT GCT ATA ACT GCT TAT TAT GCA TAT GCA TAT TAT GCT ATATAT GCA 111 111 111 111 345 678 TAT TAT TAT GCT (Appendix F con'd) F (Appendix ATT ATT TAT ATT ATT ATT TAT GCT ATT 000 000 000 012 ATT TAT ATT TAT GCA ATG ATT ATT TAT GCT ATA ATG ATA ATC TAT GCG ATG ATT TAC GCG ATA ATA ATG ATG ATC TAT ATG ATT 789 ATA ATA GGA GGA GGG ATG ATT TAT GCA GGG GGG GGG ATA GGG GGC GGA ATA GGG ATA ATT ATT GGG ATG GTG TAT GCG GTT GGT GTA GTG GTT GGA ATA GTT GGA ATA ATT GTA GTG GGG ATG ACT GTT ACT 889 999 999 999 ACA GTT ACA ACC ACA GTA GGT GGC ACA GGG 888 GGT ACT GTT GGT GGT GGT ACC GTG TTT GGC ACC GTT TTT GGC ACA GTT GGG TTT GGT ACTTTT GTT GGC GGT TCA ATA TTT GGC ACA GTT GGC ATA ATT 888 TTT GGT TTT GGG ACT TTT TTC GTT TTT GTG GTT 788 GTT TTT GGT ACT 901 234 567 890 123 456 GTT TTT GTG TTT GGT ACG GTA GTA TTT GGG ACA GTT GGG ATA ATT TAT GCT GTA TTT GGA GTA TTT GGT ACC GTG CAG CAG GTC CAA CAG GTT TTT GGT ACT GTC 777 CAG CAA AAG CAG AAA 345 678 AAA AAA CAA GTG AAA CAG AAG CAG AAA CAG AAA AAA 777 777 AAA AAG CAA AAA GGT AAG AAA CAG GTC 000 000 000 000 000 000 000 000 000 000 000 111 789 012 GGT AAA AAA GCT GCC AAA AAA CAG GTA TTT GGT AGC GGA AGA GGA AAA AAA CAA GTT TTT AGG GGG AAA AAA AGA GCA AAA AAA GAG AGA GGG AAA AAG CAA GTT GAG GAG AGG GGA AAA 000 000 GAA CTT CTA GAA CTG GAG AGT GGA AAA AAA CAA GTT CTT 890 123 456 TTT GAA AGT GGA AAG TTG GAG AGA GGA AAA 000 TTA CTT TTT GAG AGA GCA TTC GAA 4 TTC GAA AGA GCA AAA 7 — 15 — 13 TTA GAA AGA GGA AAA AAA CAA GTT TTT — 12 CTA GAG AGA GGA AAA AAA CAA 1 TTT GAA AGA 5 TTT GAA AGA GCT AAA 2 TTT GAA AGA — 18 — 10 — — — 11 — — — 3 TTT GAG AGA GCA AAA 6 — 20 — 17— 19 CTT GAG AGA GGG AAA AAA CAA GTT 8 TTA GAA AGA — — Position: 556 666 666 666 c r o v e l lw o i e l l e r i t h o m o m y u s c h i h u a h u a e o r e g o n u s w e l l e r i w e l l e r i shastensis— 9 c e n t r a l i s warmanae— 16 a u r e i - - muscuii— 14 TTA GAG AGG G. G. G. G. G. idahoensis G. G. G. G. G. G. G. G. idahoensis G .G t . h aG e t . h l e a rG. c e i r l e a r i thomomyus g i i G .G t . h aG. c e r l e a r i g i i

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o\ N> ATA ATA ATA ATA ATG ATA ATA ATG ATA 677 ATA ATG ATG ATG ATA GGG GGA ATA GGG GGA ATA GGG 111 111 GGA GGA GGG GTA GGG GTA GTG GTA GGA ATA GTA GGA GTT GGG GTT GGA ATG GTA GGG ATG 111 666 666 GTT GGA ATA GTA GGG GTA ACT ACA ACA ACA GTG GGT ACC ACC ACT GTT ACT GTT 111 012 345 678 901 ACT GTT ACT GTA GGC ACT GTT GGT ACG GTA ACA GTA GGA ACG (Appendix F con'd) (Appendix F TTT ACA GTG TTC TTT TTT TTC TTC TTT TTT ACT TTT TTT TTT ACT TTT ATG ATG ATG TTC ATG TTC ATG TTC CAC ATG CAT ATA TTT ACT CAT ATA CAC ATA CAT CAC CAC CAC ATG TTT CAC CAT ATA CAT CAC ATA TTC ACT CAT CAT 890 123 456 789 CAC CAT CAC CAC CAC GCC CAT CAC ATA GCC GCT GCC 444 445 555 555 555 666 GCC GCC CAT CAT ATA TTT ACT GCT TGA GCC TGA TGA 444 TGA 234 567 TGA GTC 901 GTT TGA GCT CAC GTT GTC GTA TGA GCT CAT CAC ATG GTG TGG GTA GTA GTC TGA GCT GTG GTA TGG GCT 678 GTG 111 111 111 111 111 111 111 111 GTG GTA TGA GCT CAT CAC ATA GTG GTA GTA GTG GTG TGG GCT CAC CAC GTG TTT GTG GTG TGA GCC CAT CAC TTT TTT GTG GTT TGA GCT CAC CAC ATA 345 111 TTT GTA GTA TGG GCC CAC CAT ATG TTT TTT TTT GGG TTT GTA GTT GGT TTT GGA GGG GGG GGT GGC TTT GGG TTT TTA TTA GTT CTT GGG TTT GTA GTG TGA ATT CTT GTA TTG GGG TTT GTG GTG TGA GCC CAT CAC ATG GGG GTA CTT GGG GGG GGT GTT CTT GGG TTT 222 222 222 333 333 333 344 GGT GTT CTT GGG TTT GTG GTT TGA GGA ATT GGA GTT 890 123 456 789 012 ATT ATT GGA GTT CTA ATT GGG GTT CTA GGC ATT GCT ATT GGA GTT CTT GGG TTT GCA ATC GGG GTT TTA GGG TTT GTG GTC TGA GCC CAC CAC ATG TTT 111 112 GCA GCA ATT 567 111 111 111 111 111 111 GCA ATT GGT GTT CTT GGG TTT GTG GCA GCT ATT GGAGCA GTT ATT CTT GGGGCA GTT ATT CTA GGC TTT GTG GTC TGA GCC GCT ATC GGG GTT TTA GGG TTT GTT GCT GCT GCT GCT ATT GGG GTT — 15 — 12 — 13 GCA ATT GGT GTT CTT — 10 GCT ATT GGT GTA CTT GGG — 18 — 16 — 11 — 2 — 5 — 1 GCT ATT GGA GTT CTA — 19 — 17— 20 GCT ATT GGA GCT ATT — 14 GCA ATT GGA GTT CTT — 3 — 8 Position: r o v e llo i w e lle r i thomomyus— 7 chihuahuae idahoensis idahoensis thomomyus--4 c oregonus a u rei G. G. warmanae G. w e lle r i G. w e lle r i G. G. G. G. G. G. musculi G. G. G. shastensis— 9 G. centralis G. th aG. e le r i c r a ig i G. th a e le r i G. craigi--6 G. th a e le r i G.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. u> ON ACT ACT ACT ACT ACA ACG ACA ACA ACT ACT ACA ACA ACT CCC CCC CCA 222 222 CCT ACT CCT CCT CCC CCA ACT GTA CCG ACT 222 222 222 GTA GTA CCT GTA CCA GTA GTA CCC ACT GTG CCT ACT GTA CCT ACC GTA CCC GTA GTA CCT GTT CCT ACT GCG 789 012 345 678 GCG GCA GTA GCC GCT GCA GTA CCT (Appendix F con'd) (Appendix F ATT GCA ATT ATT ATT GCA GTT ATT ATT ATT GCT GTC 111 111 111 222 GTG ATT GCG GTA GTA ATT GCG GTA ATT GCG GTA GTA GTG ATT GTA ATT GCT 890 123 456 ATA ATA ATG GTG ATA ATG GTG ATG ATG ACA ATG GTA ATT GCG 222 222 222 222 222 567 ACA ACT ATG GTA ATT GCT ACT ATG GTAACT ATT ATA GCA GTA GTG ATT GCA GTC CCT ACT ATG ACA ACT ATG ACG ATG GTT ATT GCT GTC CCT ACC 234 GCT ACT ATA GTA GCA ACT ATGGCA GTA GCA GCT ACT GCT GCA GCT GCT GCA GCA GCA ACC ATG GTG ATT 122 222 900 000 000 001 AGG AGA GCA ACT ATG 901 AGG AGA AGA AGA AGT AGT GCA ACC AGA GCA ACT ATA GTT ATT GCT GTC AGA GCA AGA 999 ACT ACT AGT GCA ACT ATG GTA ATT GCA ACC AGA GCT ACT ACT ACT ACT AGA GCA ACT TTC 999 TTT ACC AGT TTC ACT TTT TTC TTT ACC TTT ACT AGG TTT ACT TTT ACT TTC ACT TTT TTT TTT TAC TTT ACC AGT TAC 111 111 111 111 GCT TAT GCT TAC GCA 789 012 345 678 GCA TAC GCA TAC 111 456 CGA GCA TAT TTT ACT AGT CGA GCT TAT CGG GCA TAT CGA GCA TAT CGA CGA GCA TAT CGA CGA GCA AGA CGA GCA TAT TTT ACT AGT CGG GCA TAT AGG CGG GCG TAT TTT ACC AGG AGT AGO 111 111 890 123 778 888 888 888 999 GAT AGC GAT GAT GAT AGT GAC AGT CGA GCA TAT GAT GAT AGT CGA GCA TAT 111 GTA GAT AGT CGG GCT TAT TTT ACCGTT AGT GAT AGC GCT ACC CGG GTA GTA GAC AGC CGA GCA TAT TTT GTT GAT GTA GTA 111 234 567 GAT GTG GAT AGT GAT GAT GTT GAT GAT GAC GAT GTA GAT GTA GAT AGC GAC GTA GAC GAT GTC GAT AGA GAC — 12 — 15 GAT GTT GAT AGT CGG GCG TAT TTT ACC — 9 --18 GAT GTA GAT AGC CGG — 4 GAT GTA — 10 — 7 — 16 — 2 GAC — 5 GAC GTT GAC AGT — 1 — 14 — 17 — 19 — 6 GAT GTA GAT AGC CGA GCA TAC — 3 Position; 777 777 idahoensis— 13 idahoensis warmanae welleri— 20 GAT GTG GAT AGT CGA thomomyus oregonus— 11 GAT GTT aurei--8 GAT w e lle r i c r a ig i G. G. G. G. m usculi G. centralis G. w e lle r i G. crovel1oi G. chihuahuae G. G. th a e le r i G. thomomyus G. G. c r a ig i G. G. th a e le r i G. G. th a e le r i G. shastensis G. G.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. i—* £ TCC TCC 345 TCC TCT TCC TCG TCG TCG TCG TCG TAT TCT 888 888 TAC TAT TCA TAC TCA TAC AGA TAT TCC TCA TAC TCA TAC TCC TCA TAC TCC 222 222 222 TCT TAC TCA AGA TAC TCC TCG GAA TTT ACC TCA TAC TCA TAC TCT TAC TCT TCA TAC ATT ATC ATC 222 GTT GTA ATT AGC GTG ATT TCG TAC TCC GTT GTG (Appendix F con'd) (Appendix F 123 456 789 012 CGG ATT CGA CGA CGA 222 AAG CGA 890 AGC CGG ATA 667 777 777 777 AGT CGA ATT TCC TTT TCT AGT CGG ATT TCG TAC TCC AGT CGA AGT 222 AGC AGT CGG GTC AGA CGA AGT AGT AGT CGG GTC GGA GGA 222 GGA GGG AGA GGT GGT GGA GGT AGT CGAGGA GCA TTT GGT AGT TTT GGA AGT CGG ATT AGA TAC TTT GGG 234 567 TTT GGA TTC TTT GGA TTT GGA AGA CGA 901 TCT TCC TTT TCT TCC TCT TTT TCC 222 222 TCT TCT TCT TCT TTT TCA TTT 222 ACT ACC ACT ACT ACT ACT ACA ACA TCA TTT GGT AGT CGG GTA ACA ACT ACA ACG TCA TTC GGG AGT CGT GTT GCC GCC ACC TCTGCC TTT ACC TCT TTT GCT ACG TCA TTT GGG AGT CGA GCC GCC ACC TCC TTT GGG AGC CGT GCC ACC TCT TTT GGG GCT ACC TCT TTT GCT GCA ACA CTC 012 345 678 222 222 CTT GCC CTT CTT CTT ATG CTT GCT CTT CTA GCC TTG CTT TTG GCT CTT GCT ATG ATG GCT ACGTTA TCA TTT 222 TGA TGA 789 TGG TGA TGA TGA TGA TGA TGG TGA TGA 456 AGG TGA AGG AGA TGA AGT TGA AGT TGA 123 TTT 222 222 TTT TTT AGG TTT TTT AGA TTT AGT TTT TTT AGA TTT AGT TTT AGG GTA GTT GTA GTT GTG 567 890 222 222 AAG GTA TTT AGT AAG GTT TTT AGG TGA AAA GTT AAG GTT TTT AGG TGG TTA GCT AAA GTG AAA GTC TTT AAG AAA GTA AAA GTA 234 222 GTA AAA GTT TTT AGG TGG CTA GCC GTA GTT GTG GTG GTA GTT AAG GTG TTT AGT GTT AAA GTG GTT GTT AAG GTT 233 333 333 334 444 444 444 555 555 555 566 666 666 901 222 GGA GTA AAA GTT TTC AGT GGG GTA AAA GTT TTT AGG TGG TTA GCT GGG GGG GTA AAA GGG GGG GGG GTA AAA GTT TTT AGC GGG GGG GTA 12 GGG GTG AAA GTT TTT AGG TGG TTG GCT -15 — 13 GGG — 10 — 4 — 11 — 5 GGA — 2 GGA 3 GGA — 6 GGA — 8 Position: th a e le r i thomomyus— 7 GGG thaeleri— 1 GGA idahoensis-- welleri— 17welleri--20 GGG GTA AAG GTT welleri— 19 GGG warmanae— 16 crovelloi— 18 GGG GTA AAG aurei c r a ig i- - musculi— 14 G. G. G. G. oregonusG. G. shastensis--9 G. idahoensis G. G. c r a ig i G. chihuahuae-G. G. G. G. G. centralis G, G. G. thomomyus G. G. th a e le r i

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. n O ACA ACA ACA ACA ACA ACG 444 ACT ACT ACT ACA ATA ATA ACA ATA ACA ATA ACA ATA ATA ATA ATA ACT 789 012 ATG ATG ATA ACT ATA ATA ATA ACT GGA GGA GGA 456 GGA ATA GGA ATA ACA GGT ATA ACT GGT GGT ATA GGT GGA GGT ATA ACT GGT GGT 333 333 333 GGC GGT GGG GGA GGG ATA ACT GGT GGG GGA GGA GGG GGT GGT GGA GGA (Appendix F con'd) (Appendix F 223 GTC GTT ATC GGC GGG GTA GTA GTT GGT GGT ATT GGC GGG ACC ATT 567 890 123 333 333 333 333 333 333 ACT ACT ACT ATT GGT ACT GTG GGT GGG ACA ACT GTA ACA 333 222 222 TTT TTT ACT ATC GGC TTT TTT ACT TTC ACA GTA GGA TTT TTT TTT ACT ATC TTA CTA TTT TTA CTA 333 CTA TTT CTA TTT ACT GTA CTC CTC TTA TTT ACG GTT TTT 333 TTT TTA TTC CTA TTT CTA TTC ACT GTG GGG TTT TTT ATC 111 111 122 ATT TTT CTA TTT GTA TTT GTA TTT ATC ATC TTT GTA GTA 111 TTT ATT TTC TTT ATT TTT TTA TTT ACT TTT TTT TTT TTT GTA TTT GGA TTT ATT TTT TTA GGA GGA TTT ATT TTC TTA TTT 000 GGA TTT GTG TTT GGA GGC GGA GGA TTT TTT 000 TCT TCA GGA TTT GTA TTT CTA TTT ACG TCT TTT GGT 000 TCT TCA GCT TTA GGG TTT ATC TTT GCA 890 123 456 789 012 345 678 901 234 990 TGG GCT TTT TGA GCA TTA GGC TTT TGG GCA CTT GGT TTT 223 333 333 333 333 333 TGA TGG TGG TGG GCT TTG TGG 999 567 CTA TGA GCC TTT GGC TTT ATC TTT TTG TTT ACT ATC GGT TTA TGG GCA TTT CTT TTA CTA TTA TGA TCTTTG TTA TGA GGA TCA TTT TCA GTA GGA TTT CTA TTT ACG GTT CTA TGA TCT TCA GGG TTC CTA TGA TCC ATG 999 TTA ATA CTA TTA TGA TCT TCA GGA ATA CTA ATA GTG TGG GCA TTA TCG ATG CTA TGG GCA TTT GGC TTT ATT TTC CTG TTT ACT ATC TCT ATG CTT TGG TCA TTT GGG TTT ATT TCC TCC CTC TCC TTT 678 901 234 GTG ATT TCT ATG CTC 222 222 222 222 GTC TCT CTT GTT ATC TCA ATT TCG GTA ATT TGA GCC TTA GTA TCC CTG GTG TCG TTA ATT TCG GTA — 15 TCT TCA ATG TTG — 12 — 13 ATT TCG ATA — 9 — 18 GTC TCA ATG — 4 — 10 19 — 16 GTC TCG ATG — 5 GTA — 2 — 20 — 17 ACC TCG ATA CTA TGG GCC — 14 — 6 GTA TCT — 8 GTT TCA Position; 888 899 thomomyus th a e le r i thaeleri— 1 thomomyus— 7 GTA warmanae craigi— 3 c r a ig i w e lle r i oregonus— 11 ATT TCG GTT ATG au rei m usculi G. G. G. G. crovelloi G. w e lle r i G. centralis G. G. G. welleri-- G. G. idahoensis G. G. chihuahuae G. th a e le r i a. G. G. G. shastensis G. G. idahoensis

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 166 ??? ??? TTA TTA 788 TTA GTG GTT GTT TTA GTT TTA GTT GTT TTG 777 ATT TTA GTG TTG GTC TTT ATT CTA GTT CTT GTA GTT CTT GTA GTG CTA GTA GTT TTA GTA GTG TTG 333 333 333 GTG GTA GTA GTA GTT CTA GTA GTT GTA GTA GTG TTA GTA GTT GTT CTT GTA GAT GAT GTA GAT 777 777 GAT GAT GAT GTT GTG GAT GTG GTA GAT 666 GTA GAT GCG GAT GTA TGC GTG GAT GTG TGT TGT TCA TGC TCT TGT TCT TCG TGT GTA GAT 333 333 333 333 TCT TCG TGT GTA GAT 333 556 666 666 AAC TCT ?GT AAT TCG TGC GTA GAT GTG AAT AAT TCG TGC GTG GAT GTG GT? AAC AAT TCA TG?AAC GTA TCT GAT TGT GTG AAT AAT TCA TGT AAC TCA TGT GTA AAC AAT TCA AAT TCT TGC GTA GAT GTT AAT TCA TGC GTA AAT TCG TGT GTG GAT GCA AAC 555 567 890 123 456 789 012 345 678 901 GCT AAC TCTGCT TGT GTA GAT GCA GCT GCA AAC TCA TGT GTG GAC GCT GCT GCT TTG GCA AAT TCT TGT GTG CTG TTG GCA TTG GCT TTA TTA GTG TTG GCA GTA TTG 333 333 333 901 234 GTT TTG GTT TTG GCA GTA GTT TTG GCA GTG CTG GCA GTT GTC TTG GCA AAT GTT GTA TTA GCT GTC GTT TCG GCT GTT TTG TTA 444 455 555 TTA TTA TTA 333 333 GGT TTG GGT TTA GGC CTA GGT GGC TTA 345 678 GGT GGT GGG TTA GGT GGT TTG GGA CTG GTT TTA 9 GGT TTA GTT TTG GCA — 13 GGT TTG GTT TTG — 15 GGG TTA — 12 GGA TTG 17 GGC TTG — 10 — 7 — 4 GGT TTG GTA — 11 — 1 3— 5 GGG TTA GTT GGT TTG — 19 — 20 — 6 Position: 444 thomomyus thaeleri--2 GGA TTA GTT TTA idahoensis w e lle r i- - crovelloi--18 w e lle r i c e n tr a lis c r a ig i warmanae— 16 w e lle r i aurei--8 musculi— 14 G. G.egonus or G. shastensis-- G. G. G. G. idahoensis G. G. G. chihuahuae G. G. G. G. c r a ig i- - G. th a e le r i G. G. G. th a e le r i G. G. thomomyus

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix G ACT ACA ACG ACC 567 000 ACC ACT ACA GTC ACT GTA GTT GTT 234 GTC ATC GTC ACT ATT ATT GTT ACT ATT GTC ACT ATT GTA ATC GTA ACC ATT GTT ATC GTC ACT ATC GTA ACC ATC GTA ACC 000 000 455 555 555 ATT GTT ACT ATT ATC GTT ACT ATC GTC ACT CAT CAT CAT CAT CAT CAT CAT ATT GTA ACA 678 901 CAT CAT CAT CAT ATT (Appendix G con'd) G (Appendix TCA TCA TCA TCA TCC CAT TCC 000 000 444 444 345 TCT CAT TCC CAT TCC ATT ATT 000 444 012 ATC TCC CAT ATT GTG ATT individuals. Locality ATA ATC 333 ATA ATT TCC CAT 000 ATA ATC TCA ATA ATA ATC TCA GGC GGT ATA ATT TCT CAT GGT ATG ATT GGC ATG ATC TCA GGC ATA ATT TCC CAT ATC GGT GGT Thomomys TTT TTT 333 333 TTT TTT TTC 000 000 TTT GGT ATA ATT TTT GGT ATA ATT TTT GGC ATA TTT TTT GGA ATA ATT GGG GGA TTC GGC GGA TTT GGA 890 123 456 789 GGG GGA TTT GGT ATG ATC 000 223 GGT CCA CCT CCA CCA CCT GGC CCC GGT CCC GGT CTC 234 567 TTA CCC GGT TTT GGT ATA ATT TCA ATT CTG ATT CTC CCA GGA TTT GGC ATA ATT CTT CC? G?? ATC CTA ATT CTC CCA GGA TTT GGG ATC ATC TTA TTA ATT CTT CCA GGA TTT GGC ATG 111 122 222 222 CTA ATT CTC CCA TTA CTA ATT CTC CCA GGA TTT GGA ATA ATT TCC TTA ATC CTA 111 345 678 901 ATT ATT TTA ATC ATT TTA ATT TTA ATT ATT ATC TTA ATC TTG ATT TAC ATT TTA ATT CTT CCA GGA TTT GGC ATG ATT TCA CAT ATC GTC ACT TAC TAC 111 TAT ATC CTA ATC CTC CCA GGC TAC ATT TTA TAC TAC ATT TTA ATT CTT CCA TAT TAC ATC TAC TAC ATC CTA ATT CTT TAC TAC ATT TTA ATT CTA CCA TAC TAC TAT TAT ATC TTA ATT TTA CCT GGG TTT GGT ATA ATT TCC CAT ATT GTC 000 GTT GTT GTT 789 012 GTT GTT GTT GTT GTA TAT ATT TTA ATC TTG CCT GGG GTT GTT GTT GTT GAA GTC GAG GTT GAA GAA GAA GTT GAA GTT GAA GTT TAT ATT TTA GAG GTC GAA GAA ??T ??T ??T GAG ??T ??T ??T GAA ??T ??A GAA ??A GAA ??A GAA ??G GAA ??A 000 000 123 456 000 000 000 000 000 000 000 000 000 13 ??T GAA GTT TAC ATT TTA ATT CTC — 11 ??T GAG — 5 ??G — 7 — 3 — 4 — 6 — 19 — 17 — 15 — 16 — 8 — 2 ??A — 1 Position: a lp o id es a lp o id a s umbrinus--20 ??T townsendii— 12 ??T t ta lp o id e s umbrinus ta lp o id e s umbrinus umbrinus umbrinus— 18 ??T GAA t b o tta e b o tta e--1 0 Jbofctae--9 ??T GAA mazama m onticola T. T. bulbivorus T. umbrinus T. umbrinus--14 T. T. T. T. T. townsendii-- T. T. T. T. mazama T. T. T. T. T. T. T. Nucleotide sequences of a 379 base-pair region of the COI gene of 20 numbers correspond with Appendix D.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 168 ATG ATG ATA ATA ATA ATA 234 ATA ATA ATA ATA ATA ATA ATA ATG ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA 011 111 ATA ATA ATA ATA ATA ATA 111 111 ATA ATA ATA ATA ATA GCT GCC GCT GCT GCT GCT 678 901 GCC 111 GCC GCT GCC TGA GCC TGA TGA GCT TGA 345 TGA TGA TGA TG? TGG GCT (Appendix G con'd) G (Appendix GTA GTA TGA GCT ATG ATA GTA TGA GCT 012 GTA TGA GTA TGA GCC GTA GTA GTA ATA ATA GTG TGA GCC ATA GTT ATA GTG ATA GTG ATA GTA TGA ATA GGT ATA GGA GGA GGA ATA GTA TGA GGT ATA GTA TGA GCT GGT ATA GTA TGA GCT ATA ATA GGA ATA GTA TGA 456 789 GGT ATA 999 999 000 000 000 GGA ATA GGA ATG GTA GGT ATA GGT ATA GTA GGG ATA ATA ATA ATA GGA ATA ATA ATA ATA GGA 123 999 ATG GGA ATA ATA GGA ATA GTA TGA GCC ATA ATA ATA ATA ATA ATA ,ATA TAC ATG TAT ATG GGA TAT TAT ATA GGA TAT TAC TAT TAC TAT 890 TAT TAT TAC GGT GGA GGT GGC TAT GGT TAT GGG TAT GGT TAT TTC GGA TTC GGC TTT GGG TTC GGT TAT TTT TTC TTC GGC TTC GGT TAT ATA GGT TTT TTT TTC GGT TTC TTT GGG TTC GGG TTT GGT TAT CCC CCT CCT 901 234 567 CCT CCT CCT CCT 788 888 888 889 GAA CCC TTC GAA CCT GAG CCC GAA CCT 000 000 000 000 000 000 000 000 111 111 AAG GAG CCA TTC AAG GAA CCC AAG GAA CCT AAG GAA AAA AAA GAA AAA GAA CCTAAA TTC GGT AAG GAA CCA AAA GAA AAA AAA AAA GAG 000 AAG GAG CCT TTT GGC AAA GAA AAA AAA AAG GAA CCA AAA AAG 777 777 777 AAG AAA AAA GAG GGA GGG AAG 666 GGT AAA GGT AAG GGT GGC AAA 000 000 GGT AAA AAA GAG CCT TTT GGTGGC TAC AAA GGT AAA TCA GGA AAA AAA GAA CCT TCA GGA AAG TCA GGA TCA TCA GGA TCA GGA TCA TCA GGA TCA TCA GGC AAG TCA TCA 666 TCT GGT AAATCG AAA GAG TCA TCA 000 TCT TAC TAC TAT TAT 666 TAT TAC TAT 000 TAT TAC TAC TAC TCG GGA AAA TAT TAT TAC TAC TAT TAT TAC TAT TAC 890 123 456 789 012 345 678 TAT TAT TAT TAC TAC TAT TAT 556 TAC TAT TAC TAC 000 TAT TAT — 11 TAC — 12 --13 --5 — 6 TAC --18 — 15 TAC — 10 — 1 Position: townsendii umJbrinus--14 umbrinus— 17 TAT TAC TCA GGA AAA talpoides--7 talpoides— 4 umbrinus— 16umbrinus--19 TAC TAC TCA GGA AAG mazama--2 mazama T. T. bulbivorus T. umbrinus T. T. umbrinus T. T. townsendii T. T. T. umbrinus— 20 T. bottae--8 T. b o tta e T. T. monticola--3 T. talpoidesT. talpoidesT. T. bottae--9 T. T.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. '■D o\ ATA ATA ATA ATA ATA ATA ATA ATA AT? ATA ATA ATA 677 ATA ATA 111 GGA ATA GGA GGG GGA ATA GGA GGA GGA GGG GGA GGA ATA GGC GGT GTC GTT GTT GGG GTT GTC GTT GTA GT? GTG GGT ACA ACA ACA GTC GGA ACA ACA ACA GTC ACA GTA GGA ATA ACA ACA GTA ACA GTT GGA ATA ACA 111 111 111 012 345 678 901 ACA GTA ACA GTT GGA ATA ACA GTT ACA ACA ACA GTA GGA ACA ACA GTA (Appendix G con’d) G (Appendix TTT TTT TTT TTT TTT TTT 555 666 666 666 111 TTT TTT TTT TTT TTC TTT TTT ATA 111 555 ATA TTT 456 789 ATA ATA ATA TTC ATA ATA CAC CAT CAT ATA CAC AT? TTT 111 CAT CAT CAC CAT ATGCAT TTT CAT CAT CAT CAT CAT CAT ATA CAT CAC GCG CAT CATGCA ATA CAT CAC ATA TTT GCA CAT CAT ATA GCA CAT GCA CAT CAT ATA TTT GCA CAT GCA CAT CAC ATA GCA CAT CAT ATA GCT CAC CAT GCA GCA CAT CAC ATA TGA TGA TGA TGA GCA CAT CAC ATA TTT ACA GTA GGA 111 111 111 444 444 445 555 TGA GCA 234 567 890 123 TGA GCA TGA TGA GTA TGA GTA GTA TGA GTA TGA GCA GTA TGA GCA CA? GTA TGA GCA GTA 111 GTA TGA 901 GTT TGG GCT ATT GTA TGA ATC GTA ATT ATT GTA TGA ATT TTT ATT GTT TTT ATT GGC TTT ATT GGA TTT GGG TTT ATC GGA GG? TTT ATC GGC TTT ATT 012 345 678 CTA GGT TTT CTA GGA TTT ATT CTA GGC TTC ATC CTA GGC TTT ATT GTA CTA CTA GGC TTT CTA GGC TTT ATT GTA TGA GCA TTA CTA CTA GGA TTT ATT GTA TTA TTA GGA TTT 789 TTA GGA TTT TTT 222 222 333 333 333 344 GGA TTC CTA GGT TTT GGT TTC GGA 222 GGA TTT GGA TTC CTA GGG TTC ATT GTA TGA ATT GGA TTC 890 123 456 112 ATT GGA TTC ATC GGG TTT TTA GGG TTT ATC GTA TCT ATT GGATCT TTC ATT 111 111 111 111 111 111 111 111 111 TCC ATC GGA TTT TTA GGA TTT ATT GTA 567 TCC ATT TCA ATT TCA AT? GGG TTT TCA TCT ATT GGG TTT TTA TCT ATT TCC ATT GGG TTT -12 TCA ATC GGA TTC 5 TCT ATT 14 — 13 TCA — 7 TCA ATT GGA — 3 — 16 TCT ATT GGA TTC 8 TCT ATT GGA TTT 1 Position: talpoides--6 umbrinus— 17 TCA townsendii- umbrinus--20 TCC ATT GGA TTC TTA GGC TTT ATT GTG TGA GCA CAT CAC ATA umbrinus--18 TCA ATT GGA TTT umbrinus-- Jbottae--9 b o tta e -- mazama--2 m onticola T. T. umbrinus T. T. T. umbrinus— 19 TCC ATT GGA TTC TTA T. talpoidesT. T. T. bulbivorus--llT. T. T. umbrinus--15 T. townsendii T. talpoides— 4 T. T. talpoides-- T. mazama-- T. T. bottae--10 T.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACT ACC ACT ACT ACT 678 ACA ACT ACT CCT ACT CCT CCT CCC CCT ACT CCA ACA CCA ACA CCA CCA ATC CCC ATT CCT ACC ATT ATC CCC ACT ATC CCT ATT CCT ACC 012 345 ATT CCT ATT 222 222 222 GCT ATC CCC ACT GCT GCT ATT CCT ACC GCT 111 222 222 222 GCT ATC GCT ATC CCT ACT GCT 222 GCT GCT ATT (Appendix G con'd) G (Appendix ATT GCT ATT GCC ATT ATT GCT ATC CCT ACT ATT 222 ATT ATT ATT ATT ATT ATT GCT ATT ATT ATT ATT ATT GCT ATT ATT ATT GCT ATT ATT ATT GCT ATT ATT ATT ATT ATC ATT ATT GCC 123 456 789 ATT ATT ATT ATT ATT ATT ATA ATG ATA ATA ATT ATT 222 222 ATA ACT ACA 222 ACA ATA GCT ACT ATA 000 000 001 111 111 GCT ACA ATA GCT TCT GCT ACT ATA TCT GCT ACC ATA TCA GCT ACT ATA TCC 900 TCC 122 222 TCC ACA TCT GCT ACT ATA ACA TCT GCT ACA TCT GCT ACT ACA TCT GCT ACT ATA ACA TCT GCCACA ACC TCT GCT ACT ATA ACA ACA TCT GCT ACT ATA ACA TCT GCT ACT ATA 999 111 ACA TCT GCT TTT TTT TTT TTT ACA 111 TTT ACA TCT GCT ACA ATA ATT TTT ACA TTT TTC ACA TCT GCT ACT ATA ATT ATT GCT ATC CCT ACG TAC TTC TAT 999 999 TAC TAC GCT TAC TTT GCT TAT TTT GCA GCC GCT TAT TTT GCC TAC 111 111 GCC TAT TTT GCT TAC CGA CGA CGA CGA CGA GCC TAT TTT ACA TCT GCT ACT ATA ATT ATT GCC CGA CGA GCT TAT TTT ACA CGA CGG GCT TAC CGA GCA ACT CGA GCC TAT TTT ACA ACT ACT CGA GCC TAC ACA ACC GAT ACT GAT ACT 890 123 456 789 012 345 678 901 234 567 890 GAT ACT CGA GAT GAT ACT 778 888 888 888 GAT ACC GAT ACC GTA GTA GAT GTA GAT ACT CGA GCT TAT TTT ACA 567 GTA GAC GAT 111 111 111 111 111 GAC GTT GAT GTA GAT ACT GAC GTA 234 GAT GTA 13 — 11 GAC — 4 GAT GTA GAT ACC CGG GC? — 5 GAT GTA GAC — 6 — 7 GAT GTA GAT — 15 GAT — 19 — 9 GAC GTA GAT ACT CGA GCC TAT TTT — 8 — 2 GAT GTA GAT ACC CGA GCC TAC TTT ACA TCA GCT ACT ATA ATT ATT GCT ATT — 1 GAT GTC GAT ACC CGA GCT TAC TTT ACA TCA GCT ACT Position: 777 777 umbrinus— 20 GAC GTA GAC ACT townsendii— 12 GAC GTA umbrinus— 18 umbrinus umbrinus— 16 GAT GTA GAC ACT CGA GCC TAC ta lp o id es umbrinus--17 umbrinus— 14 GAT GTG GAT ACT CGA bottae— 10 GAT GTA GAT b o tta e mazama mazama T. T. T. T. T. umbrinus T. T. T. bulbivorus T. townsendii-- T. talpoides T. T. T. T. b o tta e T. T. T. T. talpoides T. talpoides T. monticola--3 GAC GTC

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TCA TCC TCG TCA TCA TCT TCT TCT TCT TCC TCC 222 TGA TGA TCA TGA TCG TGA TCA TGA TGA TCA TGA TGA TCA TGA TCA TGA TGA TCC TGA TGA 888 888 TGA TGA TGA AAA TGA AAA AAA AAA AAA AAA AAA TGA TCA AAA TGA TCA AAA 789 012 345 AAA TGA 222 222 AAA AAA AAA ATT ATT AAG ATT (Appendix G con'd) G (Appendix AAT ATT AAA AAT ATT 123 456 777 777 777 AAT ATT AAA AAT AAC ATT AAG AAT ATT AAT GGA AAT ATT GGA AAT ATT GGA AAT ATT GGA GGA AAC ATT GGA AAT ATT AAA 890 GGA AAT ATC 667 GGT AAC GGT GGT GGA AAT ATT GGA GGG GGA GGA GGA AAT ATT GGA 567 CAC GGT CAC GGA CAC CAC GGT CAC CAC CAC CAT GGT GGG AAT ATT CAC GGA GGA AAT ATT AAA 666 666 CAT GGT GGG CAC GGA CAC GGT CAT GG? GGT CTT CTT 566 CTA CTT 222 222 222 222 222 222 CTT ACT CTT ACT CTT ACT CTT CAT GGA GGG ACT CTT 222 ACC CTT GCT ACT CTT CAC GGT GGA AAT ATT AAA GCT ACT CTT GCC GCT ACT GCT ACT CTA GCC GCT ACT CTT CAC 222 GCT ACC CTT CAT GGT 345 678 901 234 GCT GCT ACT GCT ACC GCT ACC CTT CAT GGT GGG GCT ACT CTT CAT GGT GGT AAT ATT TTA 555 555 555 012 TTG TTG TTG CTA 222 TTA CTA TTA GCT TGA CTG GCT ACC CTT TGA CTG 222 TGA TGA CTT GCT ACT CTT TGA AGC AGC TGA CTG AGC TGA AGC TGA TTA AGC AGC TGA CTG GCC AGC TGA TTA AGC TGA AGC TGA AGC TGG 444 444 AGC AGC TTT 222 222 TTT AGC TGA TTT 444 TTT AGC TGA TTA TTT AGC TGA TTA TTT AGC TGA TTT AGC TGA CTA GCT ACT TTT AGC GTT TTC GTT TTC 890 123 456 789 GTT GTT TTC 222 GTC GTA GTA GTT AAA GTC TTC 567 AAA GTT AAA GTT TTC AAA GTC TTC AAA GTT TTC AAG GTC AAA AAA GTT AAA AAA GTT TTT AAA GTC TTC AAA AAA GTA AAA GTA GTA AAA GTA AAA GTA GTA AAA GTT TTT AGT TGA TTA GCT ACT CTT CAC GGG 333 333 334 234 GTA GTA GTA GTA AAA GTA GTA GTA GTA GTA GTA GTA GTA GGT 222 222 222 GGC GTA AAA GTT TTC 901 GGT GGT 233 GGA GGT GGA GGT GGA — 13 — 11 GGC — 3 GGA — 4— 7 GGA GGA — 5 GGA GTG AAG GTT TTT AGT TGA CTA GCT ACC — 9 — 10 — 2 — 1 Position: Cownsendii--12 GGC umbrinus— 16 GGT umbrinus--19 GGC umbrinus— 20 ta lp o id e s umbrinus— 17 GGT umbrinus--14 umbrinus— 15 GGT umbrinus— 18 GGT b o tta e m onticola b o tta e --8 mazama mazama T. T. townsendii T. T. T. T. talpoides--6 T. T. b o tta e T. T. T. T. T. talpoides T. T. bulbivorusT. T. T. T. talpoides T.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IO ACG ACA ACA ACA ACA ACT ACT ACT ACT CTA ACA TTA ACA CTA ACA CTG ACA CTT CTA 333 444 CTC ACT GGT CTA ACA GGC CTG GGG CTA ACA GGC GGT CTA GGT GGT CTA 333 333 333 333 456 789 012 GGT GGT CTC GGC GGT CTC ACT GGG GGA GGT CTA ACA GGA GGA GGA GGC CTA ACA GGA GGA GGA GGA 333 GGA GGA GGT CTT ACT GGA GGA (Appendix G G con'd) (Appendix GTA GTT 333 333 223 ATT ACG GTA ACA ACA ACA GTT 222 ACA GTT GGG GGT ACA GTT GGA GGA CTG TTC TTT TTT TTC 333 333 TTT ACA GTT GGC 222 TTT ACA CTT TTT ACA GTA GGG GGT CTT CTC TTC ACA GTA CTT CTT CTT CTT CTT TTC ACA GTT TTT TTT TTT CTT TTC ACA GTA TTT TTC CTA TTC CTT TTC ACA GTT ATT TTT CTT TTC ATT TTT CTT TTC ACAATT GTA ATT TTT CTC TTC ACA GTA GGA GGC ATT TTT CTC TTC ACA GTA ATT ATT ATT TTT ATC TTT CTT TTC ACA GTA GGA GGT CTG ATC ATC TTT CTT TTT ACA GTA ATT TTT ATT TTC CTT 333 333 333 ATT TTC CTT TTT ACA GTA GGG ATT TTC ATT TTC ATT TTT TTT TTT TTC TTT 333 TTT 111 111 111 122 GGA GGT TTC GGT TTC GGC TTT GGT TTT GGC TTT GGT TTC GGT TTT GGT TTC TTA GGC CTA TTA GGT TTT ATT TTA GGT TTT ATT TTC CTT TTT GCA TTA GCA GCT TTA GGT GCC TTA GCA TTA GCG TTG GGT TTT GCA TGA GCC TTA GGT TTC TGA GCT TTA GGC TTC 890 123 456 789 012 345 678 901 234 567 890 123 TGA 990 000 000 000 TGA TGA GCA CTA CTC TTA 567 222 223 333 333 333 999 ATA ATA CTA TGA GCA TTA ATA CTA TGA GCA CTA GGA 234 ATG TTA TGA GCC TTA GGT ATA CTA TGA ATA CTA TGA GCA ATA CTA TGA GCT TTA GGC TTT ATT TTT CTT TTT ACA GTT GGG ATA TTA TGA ATA CTA ATA TTA TGA 999 222 ATA TTA TGA ATA CTA GCT 901 GCT GCT GCT GCT 899 GCT GCA ATA CTA TGA GCA CCA GCT ATG CCA GCT ATA TTA TGA CCA GCT CCA GCT CCA 678 222 222 CCA CCA GCT CCA CCC GCA CCA GCA ATA CTA TGA GCA CTA GGC CCA GCA ATA CTA TGA GCA CTA CCA GCA ATA CTA TGA GCA TTA -13 — 11 -20 CCA — 4 CC? — 6 CCC GCA Position; 888 townsendii--12 umbrinus--15 CCC GCT talpoides— 7 umbrinus--l€ CCC umbrinus— 14 CCA b o tta e --10 b o tta e --9 bottae--8 mazama--1 CCA GCA ATA CTA monticola--! T. T. T. umbrinus- T. umbrinus--19 T. T. townsendii- T. T. bulbivorus T. T. umbrinus— 17 T. umbrinus— 18 CCA GCC ATA TTA TGA GCC TTA T. T. T. T, talpoides--5T. talpoides T. mazama--2 T. talpoides T. T.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CTT CTT CTC CTT CTT CTT CTT CTG TTA CTT TTA 788 CTG GTA GTT GTA CTC GTA CTC GTA GTA GTA GTA GTA GTA 333 333 678 901 GTA GTT GTA TTA ATT ATT ATT ATT GTA ATT GTA CTA ATT GTA ATC 333 ATT GTA CTC GAT GAT ATT GAC ATT GAT GAT GAT GAT ATT GAC GAT 777 777 777 GAT ATT GTTGAC TAC ATT GAT ATT GTTGAC CTC GAC ATT GAT ATT TTG GAC ATT TTA GAC CTA GAC ATT TTA TTA TTA 789 012 345 CTA TCA TCA TTA TCA TCA CTA 666 666 TCT TCA 456 TCC TCA TCA TCA TTA TCA TCA TCA TTA TCA TCA TTA GAT ATT,GTA CTC TCA TCA TTA 333 333 333 333 TCA TCA TCA CTA AAT 333 556 666 AAC AAT TCA TCA TTG AAT TCA TCA TTA AAC TCA TCG AAT TCA TCC CTA GAT ATT 555 TCT AAT TCC TCT AAT TCA TCT AAC TCA TCA AAC TCA TTA TCA AAC CTA TCA AAC TTA TCA AAC TCA TCA TTA TTA TCA AAC TCA TTA TCA TTA TCG CTA TCA AACCTA TCA TCA TCA AAT TTA TTG 555 TTA TCA AATCTA TCA TCA CTA 234 567 890 123 TTA CTA TTA TCA AAC TCA CTA TCA AAC TCA TTA TCA AAT TCA TCTTTA TTA CTA GTA GTA GTT GTT GTA GTG 455 901 GTT GTT ATT ATT 444 ATC GTT GGA ATT GTA GGA GGA ATT GGA GGA ATT GTG 444 333 333 333 333 333 345 678 GGG ATT GTT GGG ATT GTT GGA ATC GTT GGG ATC GTC GGA ATT GTT CTA TCT GGA ATT GTT GG? 11 GGG ATT — 12 — 5— 6 GGG ATT GTT — 3— 4 GGA ATT — 19 GGA ATT GTA — 15 GGA ATT — 9 — 8 GGA ATT Position: townsendii— 13 talpoides--7 umbrinus— 20 umbrinus--18 umbrinus— 14 umbrinus— 16 ta lp o id e s townsendii umbrinus— 17 GGA ATC bottae--10 GGA ATT GTT mazama— 1 T. T. T. umbrinus T. b o ttaT. e T. T. T. umbrinus T. T. T. bulbivorus-- T. T. mazama--2 T. talpoides T. T. b o tta e T. T. monticolaT. talpoidesT.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Vita

Theresa Ann Spradling was bom in Hugoton, Kansas, on 12 July 1966. She lived the majority of her childhood in El Dorado, Kansas, and graduated from El Dorado High School in 1984. She attended Emporia State University, where she received her bachelor of science degree in biology, with a minor in chemistry, in May, 1988. She then attended the University of North Texas and earned a master of science degree in biology in August, 1991. She is currently finishing her doctoral work in zoology at Louisiana State University and anticipates graduation in August, 1997.

174

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Theresa Aim Spradling

Major Fields Zoology

Title of Piiftrtitiffli* Relative Rates of Molecular Evolution In Rodents and Their Symbionts

Approved*

Major Profease

EXAMINING COMMITTEE:

^Lu-U

-£ /C'hjrx (xj

Date of «»—

June 24 1997

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.