<p>The legally binding international declaration of the Convention on Biological Diversity (signed by over 180 countries) recently acknowledged the importance of conserving genetic diversity within livestock species. This study aimed to help Canada assess molecular diversity in its horse and pony (<i>Equus ferus caballus</i>) genetic resources. Here, 24 populations were examined, with special focus on the native Canadian, Mountain and Moorland, and Nordic pony populations, using two well accepted molecular tools. Additional horse breeds and feral populations were also included in this project as some may have influenced the development of the three equine groups of interest. Altogether, 821 individuals were genotyped at 38 microsatellite loci, and 280 individuals were sequenced using a 421 base pair portion of the mitochondrial displacement Hypervariable Region I.</p> <p>Results from the microsatellite analyses indicated that 13.33% of genetic diversity arose from breed differences, whereas 84.60% and 2.07% of diversity arose from within and among individuals respectively. The New Forest and Welsh breeds were found to be the most diverse while having the highest average effective number of alleles and allelic richness (4.31 and 6.01; 4.33 and 5.87 respectively). The Eriskay and Lac La Croix breeds were found to have the lowest average effective number of alleles and allelic richness (2.51 and 3.98; 2.83 and 4.01 respectively). Expected heterozygosities were lowest in the Lac La Croix (0.61) and highest in the Welsh and New Forest (0.74) breeds, whereas observed heterozygosities were highest in the Kerry Bog (0.77) and lowest in the Exmoor (0.57) breeds. The genetic structure and admixture analyses suggested that the most probable number of unique genetic clusters was 21 as opposed to the 24 predefined populations.</p> <p>Results from the mitochondrial sequence data revealed that there were 36 informative sites producing 62 haplotypes, 20 of which were previously unreported. The Connemara was found to have the highest haplotype diversity of the pony breeds (0.89); however, the Highland pony was found to have the highest nucleotide diversity and pairwise difference (0.16 and 6.73 respectively). In contrast, the Fell pony had the lowest haplotype diversity (0.22), and the feral Sable Island population had the lowest nucleotide diversity and pairwise difference (0.01 and 0.29 respectively). Multiple phylogenetic trees were reconstructed and produced similar topologies. In general, the Mountain and Moorland and Nordic breeds were spread among the clades, whereas native Canadian populations were most frequent in the D and E clades. Interestingly, a large portion of ponies were found within the rare E clade as opposed to horses.</p> <p>Information gathered from this project can be incorporated with other available data into pre-existing conservation/breeding programs currently managed by the various breed societies to ensure that the most optimal and sustainable strategies are in place.</p>

In this study, mtDNA was used to examine the phylogenetic and molecular relationships of the freshwater shrimp family Atyidae. The phylogeographical variations of two landlocked Caridina pseudodenticulata and Neocaridina denticulata were also discussed. Four genera and 32 species of the Atyidae were identified and divided into 6 groups. In addition to 14 previously recorded species, eight of them are new records, while another 10 species remain unconfirmed. The combination of morphological and molecular characteristics can effectively differentiate species within the family Atyidae. Analysis of demographic parameters indicates a recent population expansion of C. pseudodenticulata, but not for N. denticulata. The result of Bayesian skyline plot analysis implies that the expansion of C. pseudodenticulata began about 70,000 years ago, followed by a very recent rapid expansion approximately 4,000-6,000 years ago. Four major geographical regions of N. denticulata can be identified, including: (1) Southern Taiwan group; (2) Hualian group; (3) Northern Taiwan group; and (4) Ilan group. The study on the phylogenetic relationship of Atyidae in Taiwan could provide valuable information for future study of speciation and geological variation in tropical island.

Minnows of the genus Dionda (Cyprinidae, Teleostei) inhabit spring-fed streams in the southwestern United States and Mexico. Five nominal species of Dionda (D. argentosa, D. diaboli, D. episcopa, D. nigrotaeniata and D. serena) are found in streams and rivers in central and west Texas. Because Dionda require clean, flowing water, they serve as aquatic indicator species of biological impacts of drought and human water use. Consequently, the ecological and conservation status of species of Dionda are important relative to monitoring habitat deterioration. This study used genetic data from geographic samples of the five nominal species of Dionda in Texas waters to document the conservation-genetics status of populations in each species. Fish were collected in cooperation with the Texas Parks and Wildlife Department and the U.S. Fish and Wildlife Service. Data from 585 base pairs of the mitochondrially encoded, protein coding ND-5 gene and from 21 to 33 nuclear-encoded microsatellites were used to assess genetic variation, population structure, historical demography, and genetic effective size of samples of each of the five species. The sample from Independence Creek, initially assumed to be D. episcopa because of its location, was found to be D. argentosa. Results of genetic assays indicate that each geographic sample in each species should be treated as a separate population and managed in a way that preserves the natural diversity found within each species. Genetic data revealed that all of the populations evaluated may be compromised genetically and should be monitored further.

This dissertation has two major sections. In Chapter II, complete mitochondrial (mt DNA) genome sequences were used to construct a hypothesis for affinities of most major lineages of rodents that arose quickly in the Eocene and were well established by the end of the Oligocene. Determining the relationships among extant members of such old lineages can be difficult. Two traditional schemes on subordinal classification of rodents have persisted for over a century, dividing rodents into either two or three suborders, with relationships among families or superfamilies remaining problematic. The mtDNA sequences for four new rodent taxa (Aplodontia, Cratogeomys, Erethizon, and Hystrix), along with previously published Euarchontoglires taxa, were analyzed under parsimony, likelihood, and Bayesian criteria. Likelihood and Bayesian analyses of the protein-coding genes converged on a single topology that weakly supported rodent monophyly and was significantly better than the parsimony trees. Analysis of the tRNAs failed to recover a monophyletic Rodentia and did not reach convergence on a stationary distribution after fifty million generations. Most relationships hypothesized in the likelihood topology have support from previous data. Mt tRNAs have been largely ignored with respect to molecular evolution or phylogenetic utility. In Chapter III, the mt tRNAs from 141 mammals were used to refine secondary structure models and examine their molecular evolution. Both H- and L-encoded tRNAs are AT-rich with different %G and GC-skew and a difference in skew between H- and L-strand stems. Proportion of W-C pairs is higher in the H-strand and GU/UG pairs are higher in the L-strand, suggesting increased mismatch compensation in L-strand tRNAs. Among rodents, the number of variable stem base-pairs was nearly 75% of that observed across all mammals combined. Compensatory base changes were present only at divergences of 4% or greater. Neither loop reduction nor an accumulation of deleterious mutations, both suggestive of mutational meltdown (Muller's ratchet), was observed. Mutations associated with human pathologies are correlated only with the coding strand, with H-strand tRNAs being linked to substantially more of these mutations.

The goal of this study was to examine the impact of Africanization on the genetic structure of the Welder Wildlife Refuge feral honey bee population by scoring mtDNA and microsatellite polymorphisms. Adult honey bee workers, collected between 1991 and 2001, were screened for mtDNA using the cytochrome b/BglII, ls rRNA/EcoRI, and COI/HinfI PCR-based assays. The procedure allowed identification of four mitotypes: eastern European, western European, A. m. lamarckii, and A. m. scutellata. The relative frequencies of the four mitotypes changed radically during the 11-year period. Prior to immigration of Africanized honey bees, the resident population was essentially of eastern European maternal ancestry. The first colony of A. m. scutellata mitotype was detected in 1993. Between 1995 and 1996 there was a mitotype turnover in the population from predominantly eastern European to predominantly A. m. scutellata. From 1997 onward, most colonies (69 %) were of A. m. scutellata mitotype. The temporal change in mtDNA was paralleled by nuclear DNA. The 12 microsatellite loci analyzed indicated (1) the mechanism of Africanization of the Welder population involved both maternal and paternal bi-directional gene flow (hybridization) between European and Africanized honey bees; and (2) the resident panmitic European population was replaced by panmitic asymmetrical admixtures of A. m. scutellata and European genes. The steepest increase in the proportion of introgressed A. m. scutellata nuclear alleles occurred between 1994 and 1997. The post-Africanization gene pool was composed of a diverse array of recombinant classes with a substantial European genetic contribution (mean proportion of European-derived alleles was 37 % as given by mR estimator or 25 % as given by mY estimator, for 1998-2001). If European genes continue to be retained at moderate frequencies, then the Africanized population is best viewed as a "hybrid swarm" instead of "pure African". The most radical change in the genetic structure of the Welder Wildlife Refuge feral honey bee population (observed between 1995 and 1997) coincided with arrival of the parasitic Varroa mite. We suggest that Varroa likely hastened the demise of European honey bees and had a major role in restructuring the Welder Wildlife Refuge feral honey bee population.

Doctor of Philosophy / A number of types of molecular polymorphisms can be used for studying genetic relationship and evolutionary history. Microsatellites are hypervariable and can be very useful tools to determine population structure, distinguish sibling species, as well as verifying parental relationships and pedigrees. However, while microsatellite polymorphisms are useful for solving relationships between populations within a species, relations among species or genera will probably be obscured due to a high degree of homoplasy —identity arising from evolutionary convergence not by descent. For long range evolutionary history, such as phylogeny from old world monkey to human, mtDNA markers may be better candidates. The aim of this thesis is to assess molecular polymorphisms of different types and their optimal use in different situations. Two widely separated taxa were used for testing –the green monkey Chlorocebus sabaeus, and the sibling dipteran flies Bactrocera tryoni and B. neohumeralis, known collectively as the Queensland fruit fly. In the present study a complete 16,550 bp mtDNA sequence of the green monkey Chlorocebus sabaeus is reported for the fist time and has been annotated (Chapter 2). Knowledge of the mtDNA genome contributes not only to identification of large scale single nucleotide polymorphisms (SNPs) (Chapter 4) or other mtDNA polymorphisms development, but also to primate phylogenetic and evolutionary study (Chapter 3). Microsatellites used for the green monkey paternity and pedigree studies were developed by cross-amplification using human primers (Chapter 5). For studies of population structure and species discrimination in Queensland fruit fly (Chapter 7), microsatellites were isolated from a genomic library of Bactrocera tryoni (Chapter 6) The total length of 16550 bp of complete mtDNA of the green monkey C. sabaeus, which has been sequenced and annotated here, adds a new node to the primate phylogenetic tree, and creates great opportunity for SNP marker development. The heteroplasmic region was cloned and five different sequences from a single individual were obtained; the implication of this are discussed. The phylogenetic tree reconstructed using the complete mtDNA sequence of C. sabaeus and other primates was used to solve controversial taxonomic status of C. sabaeus. Phylogenies of primate evolution using different genes from mtDNA are discussed. Primate evolutionary trees using different substitution types are compared and the phylogenetic trees constructed using transversions for the complete mtDNA were found close to preconceived expectations than those with transversions + transitions. The sequence of C. sabaeus 12SrRNA reported here agrees with the one published by ven der Kuyl et al. (1996), but additional SNPs were identified. SNPs for other regions of mtDNA were explored using dHPLC. Twenty two PCR segments for 96 individuals were tested by dHPLC. Fifty five SNPs were found and 10 haplogroups were established. Microsatellite markers were used to construct a genealogy for a colony of green monkeys (C. sabaeus) in the UCLA Vervet Monkey Research Colony. Sixteen microsatellites cross-amplified from human primers were used to conduct paternity analysis and pedigree construction. Seventy-eight out of 417 offspring were assigned paternity successfully. The low success rate is attributed to a certain proportion of mismatches between mothers and offspring; the fact that not all candidate fathers were sampled, the limitations of microsatellite polymorphisms; and weakness of the exclusion method for paternity assessment. Due to the low success rate, the pedigree is split into a few small ones. In a complicated pedigree composed of 75 animals and up to four generations with multiple links a power male mated with 8 females and contributed 10 offspring to the pedigree. Close inbreeding was avoided. Population structure within two species of Queensland fruit fly Bactrocera tryoni and Bactrocera neohumeralis (Tephritidae: Diptera) is examined using microsatellite polymorphisms. Queensland fruit flies B. tryoni and B. neohumeralis are sympatric sibling species that have similar morphological and ecological features. They even share polymorphism at the molecular level. Mating time difference is the main mechanism by which they maintain separate species. In the present study, 22 polymorphic and scorable microsatellites were isolated from B. tryoni and tested in the two species sampled from sympatric distribution areas. Pairwise genetic distance analysis showed explicit differentiation in allele frequencies between the two species, but very weak differences between conspecific populations. Gene flow is higher within B. tryoni than within B. neohumeralis, and gene exchange between the two species exists. An averaging linkage clustering tree constructed by UPGMA showed two major clusters distinguishing the two species, and it appears that population structure is highly correlated with geographic distance. The relationship between molecular markers, evolution, and selection are discussed using comparative studies within two large taxa: primate and insect. The degree of conservation and polymorphism in microsatellites varies between taxa, over evolutionary time.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22480号 / 農博第2384号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5260(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 吉村 剛, 教授 井鷺 裕司, 講師 YANG Chin-Cheng / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Agriculture, with different Neolithic cultures, starts in the Near East more than 10,000 years ago. This new way of life has very different archaeological manifestations that previous Mesolithic. After its Near Eastern emergence, the farming practices rapidly penetrated into southeastern Europe and the first signs of Neolithic in Central Europe are already 7,000 years old. It is being considered that the cultural innovations influenced demographic growth of the populations that have taken part in the Neolithic spread. In such situation, new mutations would have to fix and could form new specific haplogroups for Europe with ancestral ties to the Near East. Phylogeographic studies such as founder analysis of European and Near Eastern mtDNA sequences found that the European Neolithic component was enriched mainly by haplogroups J and T1, and that the genetic contribution of farming economy in European gene pool is about 10 - 20%. However, studies like these have not been yet realized in particular parts of Europe. The aim of this thesis is to disentangle the internal variability of Central European haplogroups J and T1 thought to be involved in the Neolithic demic diffusion. We classified these haplogroups from the HVS-I mtDNA sequences of 281 samples of the recent population of the Czech Republic. We...

We determined genetic variance and gene flow across multiple scales (reaches, tributaries, and catchments) to examine the dispersal ability of the caddisfly, Neothremma alicia in streams along the Wasatch Range in the Rocky Mountains of Utah. Neothremma alicia is one of the most abundant caddisflies in this region. We generated DNA sequence data (mitochondrial COI) from 34 reaches, nested in 15 tributaries distributed across 3 adjacent catchments. We identified 47 haplotypes from a total of 486 individuals. The most abundant haplotype (H1) was found at all sites/reaches and comprised 44% of the total number of individuals sequenced. The remaining rare haplotypes (46) were recently derived from the dominant, H1 haplotype. All of the rare haplotypes were restricted to a single catchment with 81 % restricted to either a single tributary or to two adjacent tributaries. We found the largest FST values among tributaries and the smallest FST values between reaches within tributaries suggesting that dispersal and gene flow is largely confined to within tributaries. This result supports the observation that aerial adults commonly crawl and fly along the stream corridor, especially in deeply incised valleys of mountainous regions. Our analyses show that this population has experienced a bottleneck that may have reduced population genetic variance from many haplotypes to one single dominant haplotype, H1. The rare haplotypes may have diverged since the bottleneck from the H1 haplotype and thus, have not had time to disperse outside their catchment and in most cases outside their specific tributary. Our analyses indicated that the bottleneck took place between 1,000 and 10,000 years ago. Thus, it appears that most rare haplotypes have been unable to colonize outside of the tributary they originated in for around 1,000 years.

No / The 15th century Inuit mummies excavated at Qilakitsoq in Greenland in 1978 were exceptionally well preserved and represent the largest find of naturally mummified specimens from the Arctic. The estimated ages of the individuals, their distribution between two adjacent graves, the results of tissue typing, and incomplete STR results led researchers to conclude that the eight mummies formed two distinct family groups: A grandmother (I/5), two daughters (I/3, I/4), and their two children (I/1, I/2) in one grave, and two sisters (II/6, II/8) and a daughter (II/7) of one of them in the other. Using mtDNA from hair and nail, we have reanalyzed the mummies. The results allowed the unambiguous assignment of each of the mummies to one of three mtDNA haplogroups: A2b (I/5); A2a (I/2, I/3, II/6, II/8); A2a-311 (I/1, I/4, II/7), excluded some of the previous relations, and pointed to new ones. I/5 is not the grandmother/mother of the individuals in Grave I, and she is not maternally related to any of the seven other mummies; I/3 and I/4 are not sisters and II/7 is neither the daughter of II/6 nor of II/8. However, I/1 may be the child of either I/4 or II/7 and these two may be sisters. I/2 may be the son of I/3, who may be the daughter of either II/6 or II/8, and these two may be sisters. The observation of haplogroups A2a and A2b amongst the 550-year-old Inuit puts a lower limit on the age of the two lineages in Greenland.