Euryoryzomys, anteriormente reconhecido como Oryzomys do grupo nitidus, compreende um conjunto de sete espécies: E. emmonsae, E. lamia, E. legatus, E. macconnelli, E. nitidus, E. russatus e Euryoryzomys sp. SB (espécie reconhecida para o grupo com base em dados cariotípicos, porém ainda não descrita formalmente). Essas espécies estão distribuídas em regiões de elevada a baixa altitude, ocorrendo em zonas tropicais andinas, até as demais planícies. Com exceção de E. legatus, todas as outras espécies ocorrem no Brasil. Morfologicamente, os representantes desse gênero apresentam características semelhantes entre si, o que dificulta sua precisa identificação. Acredita-se que o número de espécies para o gênero esteja subestimado e, além disso, as relações entre as espécies permanecem pouco esclarecidas. Este trabalho teve como objetivo realizar estudos filogenéticos e filogeográficos em roedores do gênero Euryoryzomys, bem como avaliar o potencial do DNA barcoding na identificação de suas espécies. Para isso, foram utilizados os marcadores moleculares cyt-b, coxI e Interphotoreceptor Retinoid Binding Protein (IRBP), cujas análises enfocaram três perspectivas: sistemática molecular e as relações filogenéticas entre as espécies do gênero Euryoryzomys; teste de DNA barcoding na delimitação das espécies e filogeografia de E. russatus. O capítulo um traz uma revisão sobre a sistemática de roedores desde o nível de ordem até o gênero Euryoryzomys; o segundo capítulo abordou as relações filogenéticas entre as espécies de roedores do gênero Euryoryzomys e os padrões de diversificação, utilizando sequências dos genes mitocondriais (cyt-b, CoxI) e um nuclear IRBP. Os resultados evidenciaram que o gênero possui uma diversificação complexa devido à extensão geográfica que ocupa, e, além disso, está com sua diversidade subestimada - principalmente no bioma amazônico. É possível apontar para a ocorrência de pelo menos dois clados candidatos a novas espécies: Euryoryzomys sp. 1 (subclado de E. emmonsae); e Euryoryzomys sp. 2, originalmente descrita como espécimes pertencentes a E. nitidus. O terceiro capítulo investigou o potencial das sequências parciais do gene CoxI na identificação das espécies do gênero Euryoryzomys, por meio das estimativas de variabilidade intra e interespecíficas e também apontou diversidade subestimada do gênero, sendo possível observar, neste trabalho, a ocorrência de pelo menos oito espécies: Euryoryzomys sp. 3 (espécie irmã de E. macconnelli), E. macconnelli, Euryoryzomys sp. 1, E. emmonsae, Euryoryzomys sp. 2, E. legatus, Euryoryzomys sp. SB e E. russatus, além de E. lâmia e E. nitidus, que não compuseram a amostra do capítulo, totalizando 10 espécies para o gênero. Além disso, o método possibilitou a separação de espécies com números diplóides distintos, o que futuramente pode contribuir na identificação de amostras desconhecidas, provenientes principalmente da Amazônia. No quarto capítulo, foi realizado um estudo filogeográfico da espécie Euryoryzomys russatus e com o objetivo de investigar a sua diversidade genética ao longo da Mata Atlântica. Foi possível concluir que o principal processo responsável pela separação de linhagens de E. russatus pode ter sido isolamento por distância e que as linhagens estão separadas ao longo da MA principalmente em quatro grupos, sem entretanto, apresentam estruturação genética entre eles; o primeiro (grupo A) está restrito às regiões do RJ, ES e BA; o segundo (grupo B) entre PR, SC e norte do RS; o terceiro (grupo C) no RS; e o quarto (grupo D), ao longo de SP, RJ e PR. O intenso desmatamento na região compromete o entendimento da dinâmica do bioma em relação aos processos de diversificação das espécies, uma vez que a destruição de hábitats pode apagar assinaturas desses processos históricos. Dessa forma, medidas eficazes de conservação são necessárias / Euryoryzomys, previously recognized as Oryzomys nitidus group, heretofore comprises seven species: E. emmonsae, E. lamia, E. legatus, E. macconnelli, E. nitidus, E. russatus, and Euryoryzomys sp. (with 2n=76, recognized for the group based on karyotypic data, but it is not formally described until now). These species are widespread in Neotropics, occupying regions of high to low altitudes, including Andean areas and subtropical plains. Except for E. legatus, all the other species occur in Brazil. Morphologically, the representatives of this genus share similar traitsdifficulting accurate identification. The aim of this study is to conduct molecular studies based on molecular systematics and phylogenetic inferences, investigate the potencial of DNA barcoding in order to test the delimitation of the species of the genus Euryoryzomys, and also to develop phylogeographic studies in E. russatus, a species widespread in the Atlantic Forest. Based on this purpose, the present work encompasses four chapters: Chapter 1 contains a revision concerning the systematic position of the genus Euryoryzomys; Chapter 2 shows the investigation concerning phylogenetic relationships among species of the genus, and also inferences regarding patterns of diversification are discussed, using cyt-b, IRBP, and coxI sequences. According to these approaches the genus has a complex pattern of diversification due to its geographic distribution, moreover its diversity is underestimated, especially in the Amazon biome. We could point out at least two candidate clades as new species: Euryoryzomys sp. 1 (a subclade of E. emmonsae clade) and Euryoryzomys sp. 2, previously described as specimens belonging to E. nitidus. Chapter 3 investigates the potential of a partial sequence of the coxI for identification of the species of Euryoryzomys, and estimates intra- and interspecific variability, which revealed, once again, that the number of species is underestimated. At least eight species could be pointed out in this study: Euryoryzomys sp. 1, E. emmonsae, Euryoryzomys sp. 2, E. macconnelli, Euryoryzomys sp. 3 (sister species of E. macconnelli), E. legatus, Euryoryzomys sp. SB, and E. russatus (plus E. nitidus and E. lamia - not included in the analyses - and therefore resulting in at least 10 species for the genus). Moreover, the method was able to separatespecies with different diploid numbers, which can further contribute to the identification of unknown samples. Chapter 4 investigates the phylogeography of E. russatus, and also describes its genetic diversity over the Atlantic Forest (AF). The hypothesis of isolation by distance could be the main process responsible for the split of the lineages of E. russatus which were separated along the AF into four main groups: group A restricted to regions RJ, ES, and BA; group B encompasses PR, SC and northern of RS; group C composed of representatives from RS; and group D related to animals from SP, RJ, and PR. The massive deforestation in the Atlantic Forest undertakes the understanding concerning the dynamics of the biome and in relation to the processes of species diversification, since the destruction these historical processes, thus effective conservation measures are needed

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23041号 / 理博第4718号 / 新制||理||1676(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)教授 中野 伸一, 教授 曽田 貞滋, 教授 木庭 啓介 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The greenside darter, Etheostoma blennioides (Teleostomi: Percidae), is a wide-ranging polytypic taxon that occurs throughout eastern North America. A previous morphological study recognized four subspecies (blennioides, newmanii, gutselli, and pholidotum), several morphological races, and three zones of morphological intergradation. We generated complete cytochrome b (1140 bp) sequence data for 51 individuals from across the range of the greenside darter inclusive of all of the currently recognized taxa to assess genetic variation and taxonomic boundaries. Both maximum parsimony and mixed model Bayesian analyses resulted in two strongly supported deeply divergent clades including (1) a Tennessee River drainage clade, and (2) an Ohio River and Great Lakes basins, Interior Highlands, and Atlantic slope clade. Etheostoma blennius, a closely related congener, nested within the Tennessee River clade of E. blennioides, rendering the complex paraphyletic. Test of alternative topologies failed to support the current taxonomic designations. The inclusion of nuclear sequence data from intron 1 of the S7 ribosomal protein (523 bp) from a subset of the populations was included to independently test whether the currently recognized taxa conform to distinct evolutionary lineages and also to clarify potential issues associated with ancestral hybridization. Although the nuclear data was less variable than the mitochondrial data, the monophyly of several of the subspecies could not be rejected.

Northern Pike (Esox lucius) is an economically and ecologically valuable species with a circumpolar distribution across the Northern Hemisphere. Northern Pike have been shown to have low levels of genetic variation despite their great capacity to colonize new environments. Here, high-resolution resequencing data from 47 Northern Pike from across North America was used for SNP discovery and population analysis. Our analysis reveals an extraordinary lack of genetic variation among Northern Pike with observed heterozygosity (Ho) of just 0.0835. Our analyses suggest that two major groups of Northern Pike exist in North America that are separated by the North American Continental Divide. Genetic variation associated with the stratification of these two groups resides across the genome particularly in gene regions with multiple copy number variants and functions related to immunity, tissue permeability, and development. Northern Pike from Alaska and the Yukon River harbour about two times more heterozygosity than Northern Pike east of the Continental Divide with an average of one heterozygous SNP every 6,250 bases. Populations east of the Continental Divide possess a remarkable level of genetic homogenization with an average of just one heterozygous SNP every 16,500 bases. For comparison, an average of one heterozygous SNP per 309 bases was reported in herring (Martinez Barrio et al., 2016), one per 500 in Atlantic cod (Star et al., 2011), and one per 750 bases in Coho and chinook salmon (Koop, 2018). This is at least 5 – 10 fold less variation than is seen in humans (the 1000 Genomes Project Consortium, 2015). We observed a recently described master sex-determining gene, amhby, in three western North American populations but not in populations east of the Continental Divide. We could not resolve any signals indicating a genetic sex determination system was present in populations from southern Manitoba or the St. Lawrence River. This may indicate that environmental sex determination is at play in these populations. We found evidence of a possible female-heterozygous, male homozygous ZW-ZZ genetic sex-determination system in New Jersey Northern Pike. With the highest average of 181,268 heterozygous SNPs genome wide and the greatest Ho (0.3228) of all populations, as well as the presence of the sex-determining gene amhby indicate that Northern Pike from our Alaskan population are the oldest in North America. Fewer numbers of heterozygous SNPs (61,073), low Ho (0.0922), and the absence of amhby in Northern Pike east of the Continental Divide suggests that these are relatively young populations and are descended from a small founding population. These results imply that Northern Pike first came to North America through Beringia and colonized its North American range from there, possibly via pro-glacial lake formation and drainage. However, from the data herein it was not possible to trace how re-colonization occurred after the final retreat of glaciers at the end of the last ice age. This thesis provides a genetically high-resolution snapshot of Northern Pike population structure in North America. It demonstrates that organisms with largely homogenous genomes can be incredibly successful and resilient. Finally, it adds to the complex subject of sex determination in fish and provides insight into a sex determination system in transition. / Graduate / 2020-10-15

Lupinus lepidus (Fabaceae) contains many morphologically divergent varieties and was restricted in its range during the last period of glaciation. A combination of phylogenetic (with the trnDT and LEGCYC1A loci) and population genetics approaches (with microsatellites and LEGCYC1A are used here to characterize intervarietal relationships and examine hypotheses of recolonization of areas in the Pacific Northwest affected by glaciation. Sequenced loci are not found to form a clade exclusive to L. lepidus, nor are any of the varieties found to form clades. Population genetics analyses reveal only negligible genetic structure within L. lepidus, with the majority of variation being found within populations. Isolation-by-distance analysis reveals some correlation between population genetic distances and geographic distance. Microsatellite and sequence results are consistent with a scenario whereby the Oregon and Washington regions were rapidly colonized from the south, with independent invasions along the eastern and western sides of the Cascade Mountains. A predicted disjunction between northern and southern populations is found within the microsatellite data but not the sequence data, suggesting that northern populations were recolonized via a process involving the spread of novel microsatellite mutations, perhaps through the persistence of a glacial refuge isolated from southern populations. Varieties are not shown to be genetically isolated, and are interpreted as representing ecotypes, with local selection outpacing the effects of migration.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第17774号 / 理博第3897号 / 新制||理||1562(附属図書館) / 30581 / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 渡辺 勝敏, 教授 曽田 貞滋, 教授 疋田 努 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19756号 / 農博第2152号 / 新制||農||1038(附属図書館) / 学位論文||H28||N4972(農学部図書室) / 32792 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)准教授 田川 正朋, 教授 荒井 修亮, 教授 山下 洋 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The Eurasian steppe belt is the vastest grassland region worldwide, stretching approximately 8000 km from the Pannonian Basin in the west to the Amur river in the east, altogether covering more than 8 million km2. Due to its size and location, the vegetation of this biome was under strong influence of past climatic fluctuations that reached their peak in the Pleistocene. Fossil record of different steppe-associated flora and fauna places the onset of Eurasian open grasslands into Central Asia from which the grasslands spread westwards around 20 MYA and reached the East European Plain first in the Late Miocene around 8 MYA. However, while useful as a proxy to infer past vegetation patterns, fossils suffer from low-resolution power and thus fail to elucidate more detailed picture of the onset and the development of the Eurasian steppe belt flora. The working hypothesis driving the whole project was that molecular signals in typical steppe plant species reflect the climate-landscape history of the steppe and the biogeographic dynamics of steppe taxa and thus allow for a much finer resolution of the history of the steppe belt in comparison to floristic and fossil-based methods. By applying a plethora of different phylogenetic, phylogeographic and biogeographic methods, I first investigated the evolutionary history of four unrelated Brassicaceae taxa that can be nowadays found in the Eurasian steppe belt. Second, I tried to draw parallels with the climate-landscape history of the Eurasian steppe belt as inferred from the fossil record to test the above-mentioned working hypothesis. The first case study dealt with the economically important Brassicaceae genus Camelina, with an emphasis on C. microcarpa, which can be found across the whole Eurasian steppe belt. I uncovered that this taxon’s contemporary uninterrupted distribution was split along the north coast of the Caspian Sea approximately 1 MYA, dating back to the Apsheron and Baku transgression events. During this time period, a polyploidisation event took place giving rise to a new hexaploid taxon and subsequently preventing gene flow after the regression of the Caspian Sea. The second case study investigated the evolutionary history of Schivereckia, which exhibits a highly disjunct distribution along the East European Plain and the Balkans and the whole Schivereckia clade that can be nowadays found either at higher latitudes in the subarctic zone or mountain ranges of the northern hemisphere. My analyses placed the radiation of this clade at the beginning of Pleistocene, when low temperatures promoted speciation and radiation of cold-adapted flora and fauna. The study showed that the highly disjunct distribution of Schivereckia podolica mirrors the Pleistocene refugial areas of different ages and points towards a close evolutionary relationship between contemporary steppe and tundra flora. A third case study focussed on Sisymbrium. I uncovered that while Sisymbrium did not evolve in the Eurasian steppe belt, it invaded this area as well as the Mediterranean several times independently throughout the Pleistocene in the south-north and east-west trajectory, respectively. This study proved yet again how variable the Eurasian steppe flora is in terms of evolutionary onset and that many of steppe elements originated in its adjacent areas in the south. These then subsequently migrated to the Eurasian steppe belt only after it was already fully developed either towards the west into the Euro-Siberian steppe or to the east into the Mongol-Chinese steppe. The fourth case study investigated the evolutionary history of Capsella. A GBS-based approach was proven successful for inferring biological history of young taxa. I showed that contemporary steppe element Capsella orientalis invaded the Eurasian steppe belt long after it was already developed in the south-north trajectory. I acquired further insights in the evolutionary history of its cosmopolitan sister tetraploid C. bursa-pastoris and placed the origin of the whole genus into the late Pliocene continental Asia. All studies showed the importance of a taxon sample and offered an alternative nesting dating approach for reliable calibration in cases where no fossil record could be obtained. In conclusion, evolutionary history of all four investigated taxa were shaped by the same environmental changes that played a major role in the biological history of the Eurasian steppe belt. Overall, our working hypothesis that molecular signals in typical steppe plant species reflect the climate-landscape history of the steppe and the biogeographic dynamics of steppe taxa, turned out to be correct.

Lineage diversification in the Neotropics is an interesting topic in evolutionary biology and one of the least understood. The complexity of the region precludes generalizations regarding the historical and evolutionary processes responsible for the observed high diversity. Here, I use molecular data to infer evolutionary relationships and test hypotheses of current taxonomy, species boundaries, speciation and biogeographic history in several lineages of Neotropical snakes. I comprehensively sampled a widely distributed Neotropical colubrid snake and Middle American pitvipers and combined my data with published sequences. Within the colubrid genus Leptodeira, mitochondrial and nuclear markers revealed a phylogeograhic structure that disagrees with the taxonomy based only on morphology. Instead, the phylogenetic structure corresponds to specific biogeographic regions within the Neotropics. Molecular evidence combined with explicit divergence time estimates reject the hypothesis that highland pitvipers in Middle America originated during the climatic changes during the Pleistocene. My data, instead, shows that pitviper diversification occurred mainly during the Miocene, a period of active orogenic activity. Using multiple lineages of Neotropical snakes in a single phylogenetic tree, I describe how the closure of the Isthmus of Panama generated several episodes of diversification as opposed to the Motagua-Polochic fault in Guatemala where a single vicariant event may have led to diversification of snakes with different ecological requirements. This finding has implications for future biogeographic studies in the region as explicit temporal information can be readily incorporated in molecular clock analyses. Bridging the gap between the traditional goals of historical biogeography (i.e., area relationships) with robust statistical methods, my research can be applied to multiple levels of the biological hierarchy (i.e., above species level), other regional systems and other sub-disciplines in biology such as medical research, evolutionary ecology, taxonomy and conservation.

Marstonia lustrica is a poorly understood aquatic snail, relatively rare throughout its range and listed in the State of Massachusetts as Endangered (MNHESP 2010, Hershler et. al 1987). It is the northern-most cold temperate species of its genus, with other members of the genus occurring along the southern edge of its range and in the southeastern United States (Thompson 1977). The current range of M. lustrica appears to follow the maximum extent of the Laurentide Glacier (20–25 kya), extending from Minnesota to western Massachusetts. Research regarding the distribution, ecology, and phylogeny of M. lustrica in the State of Massachusetts and eastern New York raised the possible role of glaciers and pro-glacial lakes in the establishment and distribution of the snail, leading to the hypothesis that its distribution and evolution may be dependent upon glacial processes. A full range survey was completed in 2007 and 2008, with populations identified in 20 water bodies from Minnesota to Massachusetts, and Ohio to Ontario, Canada. Fifty-seven specimens from the 20 populations were sequenced for two mtDNA markers (COI and NDI), developing both phylogenetic trees and haplotype networks. Here I present those trees and networks, and correlate the distribution of these populations and their representative haplotypes with both glacial events and contemporary watersheds, using AMOVAs and Mantel tests to examine several v phylogeographic models. In addition to the results for M. lustrica, the unexpected occurrence of several other species of Marstonia spp. found across the range of M. lustrica are presented, including M. pachyta, M. comalensis, and M. hershleri.