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Understanding the mechanisms that generate shared morphologies across closely related taxa is important when identifying distinct evolutionary lineages using morphological characters. Desmognathus salamanders are an ideal group for testing hypotheses concerning the correlation between morphological similarity and genetic exchange within and among nominal species due to a pattern of high discordance between the two. Phylogeographic hypotheses are tested for populations of the D. quadramaculatus species complex throughout southern Appalachia by combining phylogenetic and population genetic methods with geographical information. Phylogenetic and phylogeographic inferences are then assessed in conjunction with morphological characteristics that have traditionally diagnosed taxonomic entities to understand the genetic basis of shared morphology in this complex, and to assess species boundaries. A history of fragmentation followed by range expansion is suggested as a recurrent pattern that has shaped the current population structure within this complex. The current taxonomy is found to unite populations that share similar morphologies due to parallel evolution rather than ancestry. We suggest revisions in taxonomy that will better reflect the evolutionary history of these lineages. Appreciation of the hidden genetic variation and homoplasious morphological variation often present in and among salamander species can foster the implementation of more appropriate methods for detecting and recognizing the complex history of these organisms.

Pantosteus, a subgenus of Catostomus, includes the mountain sucker (Catostomus playthyrnchus), whose speculated older origins in the Miocene/Pliocene can provide insight into the ancient geographical events of western North America. We believe that major geologic events influencing the diversification of mountain suckers include the rise of the Colorado Plateau, the connections between the ancient Snake River system and the Lahontan system and subsequently the connection of the Snake River system to the Columbia Basin, dispersal of mountain suckers across the continental divide, as well as the Pleistocene Bonneville flood. If this is true, we should see evidence of geologic separation and timing through studying the phylogenetics of the mountain sucker. In order to clarify relationships of the mountain sucker with respect to other Pantosteus species, we examined cytochrome b (cyt b) sequences for 144 mountain suckers, 24 other Pantosteus species, and ten outgroup species. Phylogenetic relationships among haplotypes were constructed based on maximum likelihood and Bayesian criterion. In an effort to provide better resolution at some nodes, we also sequenced additional mitochondrial genes (ND1, ND2, ATPase, ND4L, ND4, ND5, ND6, and cyt b) for a subset of 44 individuals taken from the major clades obtained from the cyt b phylogentic analyses. Trees from this data set were also constructed under maximum likelihood and Bayesian criterion. All phylogenetic analysis revealed that mountain sucker are paraphyletic, with two major clades of mountain suckers separated by other members of the subgenus Pantosteus. One clade included two sub-clades, one from the upper Snake River drainage/northern Bonneville/Green River drainage Basins and the other from the southern Bonneville Basin. The other major clade included sub-clades from the Lahontan Basin, Columbia River Basin, and Upper Missouri River Basin. Molecular clock analysis revealed that Pantosteus likely split from Catostomus during the Miocene and that major speciation events within Pantosteus occurred during the Pliocene and Pleistocene. Genetic structuring and gene flow estimates for mountain sucker populations, with groupings based on major drainage basins, were calculated with AMOVA and Fst estimates in Arlequin and revealed that most of the genetic structuring was explained by variation among drainage basins with limited gene flow occurring between drainage basins. Based on this study, the role of the Colorado Plateau's geologic history in the evolution of the mountain sucker remains unclear. However, all other geologic events as discussed in this study seem to have played a significant role in the evolution of the mountain sucker.

The understanding of diversification of intraspecific lineages can shed light on speciation processes and ultimately biogeographic patterns across multiple spatial and temporal scales. In this dissertation I investigated the geographical and ecological factors promoting diversification across the South American dry diagonal biomes (i.e. Cerrado, Chaco, and Seasonally Dry Tropical Forests - SDTFs), through a coupled approach between multilocus phylogeographic and geospatial methods, in the larger context of interpreting the consequences of the resulting patterns for the conservation of biodiversity and evolutionary processes. In Chapter 1 I evaluate biogeographic hypotheses previously proposed and emphasize that the dry diagonal biomes are particularly biodiverse and biogeographically complex, but poorly studied and under protected. I also propose testable predictions for the subsequent chapters and future diversification studies. In the subsequent chapters I adopt a biodiversity prediction approach based on estimating palaeodistributions and habitat stability surfaces to formulate and test spatially explicit diversification hypotheses based on squamate richness and phylogeography. In Chapter 2 I identify historically stable areas of SDTFs and in Chapter 3 I found that the historical climatic stability is a good predictor of Cerrado squamate richness. In Chapter 4 I use a multilocus dataset to estimate the phylogenetic relationships among described species of the lizard genus Phyllopezus (Phyllodactylidae), distributed across the ‘dry diagonal’ biomes. In Chapter 5 I used a dense sampling design focused in the species complex P. pollicaris (more individuals, localities, and markers), and coalescent phylogeographic methods to test the relative influences of Tertiary geomorphological vs. Quaternary climatic events on diversification in this lizard. I found unprecedented levels of cryptic genetic diversity, deep phylogeographic structure, and diversification dating back to at least the Neogene with persistence across Quaternary fluctuations. My dissertation emphasizes that patterns of diversification across the ‘dry diagonal’ biomes are much more complex than previously proposed and reflect the primary influence of geologically old processes. Evidence of allopatric and ecological speciation between lineages that coincide with genetic clusters associated with each of the biomes, contradicts early views that the biomes would have a shared diversification history. These patterns illustrate that low-vagility complexes, characterized by strong structure and pre-Pleistocene divergences, represent ideal radiations to investigate broad biogeography of associated biomes. Future studies should investigate patterns of temporal and spatial congruence across co-distributed taxa, and integrate morphological and further ecological data to refine species limits, taxonomy, and patterns of trait evolution across these radiations.

Chapter 1. The family Pteronarcyidae (Plecoptera) is a highly studied group of stoneflies and very important to a wide variety of aquatic studies. Several phylogenies have been proposed for this group recent decades, however there is little congruence between the various topologies. The present study revises the phylogeny of the group by combining molecular data from mitochondrial cytochrome oxidase subunit II, ribosomal subunit 12S, ribosomal subunit 16S, and nuclear loci ribosomal subinit 18S and Histone H3, with published morphological data in a parsimony-based total evidence analysis. The analysis produced a well-supported phylogeny with novel relationships within the genus Pteronarcys. Maximum Likelihood and Bayesian analyses produced topologies congruent with parsimony analysis. Character mapping revealed several homoplasious morphological characters that were previously thought to be homologous. Chapter 2. Phylogeographic studies in aquatic insects provide valuable insights into mechanisms that shape the genetic structure of aquatic communities. Yet studies that include broad geographic areas are uncommon for this group. We conducted a broad scale phylogeographic analysis of P. badia across western North America. In order to allow us to generate a larger mitochondrial data set, we used 454 seqeuncing to reconstruct the complete mitochondrial genome in the early stages of the project. Our analysis reveals what appears to be a complex history of isolation and multiple invasions among some lineages. The study provides evidence of multiple glacial refugia and suggests that historical climactic isolations have been important mechanisms in determining genetic structure of insects in western North America. Our ability to generate a large mitochondrial data set through mitochondrial genome reconstruction greatly improved nodal support of our mitochondrial gene tree, and allowed us to make stronger inference of relationships between lineages and timing of divergence events.

The Neotropical region is the most biologically diverse region on the planet. The region encompasses a variety of ecosystems and has long been the target of researchers interested in patterns of species diversity and distribution. More recently, molecular data have been incorporated into methods for reconstructing the historical relationships among geographical areas and their biotas. Molecular phylogenetics has provided insights into diversification patterns and the influence of Late Cenozoic events on the evolutionary history of the region. Nevertheless, considering the vast extent and complexity of the region, more studies are needed to fully appreciate the patterns of biogeography and the mechanisms that generate and maintain its biodiversity. Therefore, in Chapter 1 I employed molecular methods to reconstruct the phylogenetic relationships of the subfamily Sigmodontinae, which is the most diverse and widespread radiation of Neotropical rodents. I was able to evaluate controversial hypotheses about the paleogeographic scenarios implicated to explain the biogeography of sigmodontines. Advances in sequencing technology and analytical approaches have revolutionized the role of historical biogeography in elucidating the spatial and temporal context of diversification, and the integrative field of phylogeography was fundamental to the development of biogeography at the intraspecific level. However, the potential of phylogeography to unravel diverse historical scenarios in a tractable statistical framework has been largely unexplored for the Neotropics as a whole. In order to integrate more robust hypothesis testing to elucidate the evolutionary history of Amazonia's biota, I devoted Chapter 2 to a review of Amazonian phylogeography that I anticipate will improve the basis for interpreting the patterns and processes of diversification in Amazonia. Chapter 3 is a thorough species account of spiny rats of the genus Proechimys, which is poorly known taxonomically despite its diversity and widespread distribution in the Neotropics. This taxonomic revision will benefit researchers interested in using such information with coalescent-based methods of species delimitation aimed at an integrative and stable taxonomy. Lastly, Chapter 4 deals with the phylogeography of P. roberti. This species occurs in southeastern Amazonia and the Cerrado of central Brazil. I employed a dense taxon sampling and used coalescent-based methods to demonstrate that rivers and topography have a causal link to the geographic structure of P. roberti populations. In my dissertation, I used a combination of molecular genetics tools to provide a better understanding of the biogeography and evolution of some of the most diverse groups of Neotropical mammals. My dissertation interacts in many levels with my future research interests. These present and future efforts hold promise for unraveling the evolutionary history of the Neotropical region and its biota, and will assist in conservation decisions aiming at preserving its unparalleled biodiversity.

The conservation biology field seeks to preserve biodiversity and the processes shaping that variation. Conservation biology is intimately tied to evolutionary research, in order to identify evolutionary distinct lineages that may be in danger of disappearing. Interestingly, patterns and processes of lineage divergence and persistence change with respect to spatial and temporal scale. I seek to evaluate biodiversity, the factors that have shaped this heterogeneity, and how this variability persists. To accomplish this I used a phylogeographic approach as well as niche and population modeling on the Peromyscus maniculatus species group found widely distributed in North America. My emphasis was on the southeastern U.S. species P. polionotus and its distinct beach forms. At a continental scale, I found that environmental niches are likely involved in generating and/or maintaining genetic lineages within the P. maniculatus species group. These findings add to a growing number of studies that have identified lineages occupying different environmental spaces. At a regional scale, I supported the hypothesis that barrier islands on the Atlantic coast of Florida were colonized by an ancestral form of P. polionotus by a single colonization, from the central Florida area. Subsequently, at least two distinct lineages diverged (P. p. phasma and P. p. niveiventris). I also found evidence that suggests that the extinct form of beach mouse (P. p. decoloratus) is part of the P. p. phasma lineage. At the population level, I evaluated changes in genetic diversity in historical samples compared to those that experienced recent human encroachment on natural habitat I used tissue preserved in natural history collections to compare with live-trapped specimens, and found that P. p. niveiventris has maintained historical genetic diversity levels. I suggest that the continuation of historical levels of genetic diversity is due to the presence of a single large area iv of continuous habitat in the central portion of the species’ current distribution. Finally, I evaluated the importance of scrub and beach habitat to the population dynamics of beach mice. Beach mice have traditionally have been associated with beach dunes rather than with the scrub habitat found more inland on barrier islands. Using almost three years of capture-recapture data from Cape Canaveral Air Force Station (CCAFS), I created a stochastic matrix model to assess the relative contribution of populations from the two different habitats to a variety of demographic measures. Both field data and model results provided evidence that the population dynamics of beach mice may rely much more on scrub habitat than formerly documented. Overall, my research emphasized a hierarchical approach to evaluate biodiversity and the processes shaping differentiation at different spatial and temporal scales. The methods and findings give insight into speciation at different scales, and can be applied to a wide range of taxa for questions related to evolutionary and conservation biology.

Biogeography provides a window into the evolutionary history of populations, and helps explain the diversity and distribution of life through time. Viewed from a systematic perspective, biogeographic studies generate convincing arguments to explain the relationships among organisms and categorize them into useful taxonomies. When taxonomies do not reflect evolutionary histories, inaccurate representations of biodiversity confound future studies and conservation efforts. Two thamnophiine snakes, Nerodia clarkii and Nerodia fasciata, harbor unique morphological and ecological adaptations that obscured natural groupings, leading to controversial taxonomic delimitations. Additionally, population declines documented in N. clarkii compressicauda and N. clarkii taeniata led managers to list N. clarkii taeniata as threatened in 1977. I generated a baseline for continued biogeographic and systematic study of the Nerodia clarkii/fasciata clade. I used mitochondrial DNA to build a parsimony-based haplotype network, infer the phylogenetic relationships between the two species and their thamnophiine relatives, and estimate the divergence times of major N. clarkii/fasciata clades. With these data, I tested biogeographic and systematic hypotheses about the origin and distribution of diversity in this clade. I used principal components analyses to summarize morphological data and discuss ecological observations in search of characters that may unite genetic or taxonomic units. The analyses revealed a peninsular and a panhandle clade in Florida that appeared to iv diverge as a result of Pleistocene glacial fluctuations. I found no support genetically, morphologically, or ecologically for the current taxonomy, indicating a need for range-wide research to generate revised nomenclature. My results do not support the protection status of N. clarkii taeniata

The present thesis elucidates facts about a prominent steppe plant’s evolutionary history (i.e., geographic origin, migration route, distribution of genealogical lineages and polyploidization events) and contemporary population divergence (i.e., genetic diversity and differentiation, impacts of abiotic and biotic factors). Artemisia frigida has been chosen as the target species, with Mongolia as the focus study region. Because A. frigida is widely distributed in the both New and Old Worlds, it was a suitable candidate for the phylogeographic study. Moreover, because of its dominance in many communities in Mongolian steppes and tolerance for cold, drought and mechanical disturbances (grazing), evaluating the effect of environmental factors and grazing pressures on its population genetics was profitable. The overall goal of this thesis was to assess the effects of paleo- and current climate, and land use changes on the distribution of A. frigida’s genealogical lineages and genetic variations. The thesis is divided into two main parts: (i) Chapter 3 focuses on Phylogeography. Within this, section 3.3 depicts a study on the phylogeography of A. frigida, covering samples from its distributional range across the northern hemisphere. The study resulted in Asia being the species’ main origination and diversification center, and the species spread northwards to the Russian Far East and eventually crossed the Bering Strait to North America. Among four geographical regions sampled, seven genetic lineages were found, with Middle Asia having the most diverse populations. According to our phylogenetic analysis, two populations of Kazakhstan in Middle Asia represented the most likely ancestral diploids, and subsequent polyploidization events have occurred on several occasions independently. The observed phylogeographic patterns of the species showed that paleoclimate, especially glaciation events of the Quaternary has predominantly affected species’ current distribution, along with the expansion and contraction of the Eurasian steppe. The second part (Chapter 4) is dedicated to Population genetics to reveal the effects of the current climate and land use on population genetic variation. Three studies were conducted at local and regional levels, focusing on Mongolia. The first study (Section 4.1- review of local literature) was done to offer background information about Mongolian steppes, and the effects of climate and grazing on the steppe vegetation. As a result, steppe vegetation responded to grazing in different ways, depending on the interplay of local environmental factors. In particular, an overall negative effect of grazing was found in desert, dry and high mountain steppes, but no or even positive effects in meadow and mountain steppes. The study highlighted the importance of the interaction effect of local environmental conditions and grazing in Mongolian steppe vegetation. The second study (Section 4.2) employed large scale climatic gradient and local scale grazing gradients to assess the effects of grazing and environmental factors on the population genetics of A. frigida. Precipitation gradient covered 110 – 300mm difference of mean annual precipitation from central to southern Mongolia. While three levels of grazing gradient, such as heavy, moderate, and least grazed sites were examined. According to the study, grazing in overall, had no substantial effect on the genetic diversity of A. frigida, while environmental factors, i.e., summer precipitation and soil phosphorous content, promoted high genetic diversity. Genetic differentiation among populations across large climatic gradients was extremely low, suggesting the existence of considerable gene flow among populations across the steppes of Mongolia. The third study (Section 4.3) employed grazing exclosures to evaluate the genuine effects of grazing. Because Mongolia has a long-term nomadic pastoralism history, and grazing of large herbivores is already an integral part of the steppe vegetation. Thus, we utilized reference site fences along the Trans-Mongolian Railway (TMR), where fences have been built and maintained since 1955, resulting in over 60 years of grazing exclusion. In addition, we supplemented this with data from Hustai National Park (HNP), where three fences were established in 2003. As a result, we found a significant positive impact of grazing on the genetic diversity of A. frigida, implying that a certain level of grazing is beneficial for the species. While no grazing effect on the population genetic differentiation was detected, but climatic and soil variables strongly influenced population genetic structure. In summary, this thesis provided an in-depth investigation of the phylogeography and population genetics of the species A. frigida, which can stand as an exemplar for other Eurasian steppe species. Paleoclimate had largely shaped the current distribution pattern of the species, while contemporary climate and environmental heterogeneity promoted species’ polyploidization and genetic variation. Grazing by large herbivores showed no detrimental effect, or even a positive impact on the genetic diversity of A. frigida. Artemisia frigida populations in Mongolia are thus apparently genetically ‘healthy’, in spite of pervasive grazing in the region. Climate variables and environmental heterogeneity had a substantial impact on the species’ both genetic diversity and differentiation, indicating its higher sensibility to climate change than to land use change. The findings of the thesis could be valuable in understanding species genetic variation under global land use and climate changes.:Summary 4 List of Abbreviations 6 Chapter 1. Introduction 8 Chapter 2. Material and methods 10 2.1. Study region: Mongolian steppe 10 2.2. Focus species: Artemisia frigida Willd. 11 2.3. Molecular markers 12 Chapter 3. Phylogeography 13 3.1. Eurasian steppe and its evolutionary history 13 3.2. Artemisia L. (Asteraceae) as model plant for phylogeography 16 3.3. A case study: Phylogeography of Artemisia frigida Willd. 19 Chapter 4. Plant population genetics under changing climate and grazing……. 40 4.1. Climate – grazing interactions in Mongolian steppe 41 4.2. Climate – grazing interactions on plant population genetics 59 4.3. Effect of grazing exclusion on plant populations genetics 83 Chapter 5. Overarching synthesis and discussion 101 5.1. Molecular markers: pros and cons 102 5.2. A review of phylogeographical studies on Eurasian steppe plants 104 5.3. A review on plant population genetic studies in Mongolia 109 5.4. Outlook 113 Acknowledgements 114 References 115 Curriculum vitae 137 Confirmation 143

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24826号 / 理博第4975号 / 新制||理||1710(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 中野, 智之, 教授 朝倉, 彰, 教授 森, 哲 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM