ABSTRACT GENETIC VARIABILITY IN HYDRASTIS CANADENSIS L. USING RAPD ANALYSIS FEBRUARY 2009 KERRY J. KELLEY, B.A. MOUNT HOLYOKE COLLEGE M.A. UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Lyle Craker Hydrastis canadensis L. (goldenseal) is an endangered perennial wildflower species native to eastern North America. In this study, several populations of goldenseal, (both cultivated and wild type) were analyzed for genetic variability. The samples were collected from plant populations in North Carolina, Ohio, Pennsylvania and West Virginia and preserved using silica gel during collection. Random amplified polymorphic DNA (RAPD) analysis technique was used to generate DNA profiles from individual plants and to estimate genetic variability between groups (cultivated and wild type), among populations within groups and within populations using analysis of molecular variance (AMOVA) and a UPGMA clustering phenogram. Our results demonstrate that the bulk of genetic diversity may be within and among populations, but not between groups. This indicates the need for preservation and conservation efforts at the population level. The next step would be to study goldenseal populations more in depth for underlying causes of the genetic variability observed in this study. Further study of genetic variability with different molecular markers may be needed to clarify the level of diversity for the species at the group level. Increased knowledge of genetic variability and the identification of accessions of goldenseal would prove useful for reintroduction and cultivation strategies.

Sympatry is characterized by the existence of two or more species phylogenetically related occupying the same ecological niche. This event has already been recorded in species of crabs of the genus Aegla in Brazil between two species, and though morphological characteristics only. For Aegla, as in most crustaceans, there are few studies to verify if there is any relationship between sympatry and genetic diversity of populations living in this association. In the present study, besides a new record of sympatry between two species of Aegla for Brazil, Aegla platensis and Aegla spinipalma, occurring in Batú River, the occurrence of sympatry among three species, A. platensis, Aegla sp. (in preparation) and Aegla grisella, living in Cambará River, is also presented. To verify the existence of sympatry at the sampling points, besides the morphological characteristics used for the identification of individuals, the technique of DNA-Barcoding was applied using a fragment of 207 bp of the gene COI amplified in 22 individuals and from these sequences interspecific, intra- and interpopulation p-distance values were obtained. Sympatry between two species in Batú River was verified by both morphological and molecular data. For the species living in Cambará River mitochondrial and morphological data were not completely congruent, because some sequences of A. grisella clustered with individuals of other species, presenting low values of interspecific distance. However, these sequences probably are nuclear pseudogenes and when they were removed from the analysis, three species were identified in Cambará River. To verify if there is any relationship between sympatry and genetic diversity of A. platensis, two populations living in sympatry (Cambará and Batú rivers) and one allopatric population from Fiuza River were used. The technique of AFLP was applied to 120 individuals (20 from each population) revealing a high genetic variability for all populations and no relationship between sympatry and level of genetic diversity was found in A. platensis. A high population structuring was observed among populations, which is probably due to the distance among populations and to the low dispersion capability in aeglids. Results from the COI mitochondrial data and from the AFLP nuclear data presented some incongruence when compared, since using AFLP all the A. platensis populations and also the other species were separated through Bayesian analysis and UPGMA, but the same was not found for COI data. The incongruence between mitochondrial and nuclear data reinforces the idea that some A. grisella sequences are in fact nuclear pseudogenes co-amplified in PCR, due to the utilization of degenerated universal primers. / Simpatria é caracterizada pela existência de duas ou mais espécies filogeneticamente próximas ocupando um mesmo nicho ecológico. Este evento já foi registrado em espécies de caranguejos do gênero Aegla no Brasil, sendo estes registros realizados apenas entre duas espécies e utilizando para isto, apenas características morfológicas. Para Aegla, assim como na maioria dos crustáceos, existem poucos estudos destinados a avaliar se há alguma relação entre a simpatria e a diversidade genética das populações que vivem em tal associação. Neste estudo, além de ser apresentado mais um registro de simpatria entre duas espécies de eglídeos para o Brasil, sendo estas Aegla platensis e Aegla spinipalma ocorrendo no Rio Batú, também é apresentada ocorrência de simpatria entre três espécies, sendo estas A. platensis, Aegla sp. (em preparação) e Aegla grisella ocorrendo no Rio Cambará. Para verificar a existência de simpatria nos pontos de amostragem, além das características morfológicas utilizadas para a identificação dos indivíduos, foi realizada a técnica de DNA Barcoding com um fragmento de 207 pb do gene COI, amplificado em 22 indivíduos e a partir dessas sequências foram obtidos os valores de distâncias interespecífica, intra e interpopulacional através do modelo de distância p. A constatação da existência de simpatria entre duas espécies no Rio Batú foi verificada tanto pelos dados morfológicos quanto para os dados moleculares, porém para as espécies do rio Cambará os dados mitocondriais não foram totalmente congruentes com os morfológicos, pois algumas sequências de A. grisella agruparam com sequências de indivíduos de outras espécies e apresentaram baixos valores de distância interespecífica. Entretanto, estas sequências provavelmente são pseudo-genes nucleares e quando retiradas das análises permitiram a identificação de três espécies simpátricas no Rio Cambará. A fim de avaliar se existe alguma relação entre a simpatria e a diversidade genética de A. platensis foram utilizadas duas populações simpátricas desta espécie (Rios Cambará e Batú) e uma população alopátrica, oriunda do Rio Fiúza. A técnica de AFLP foi utilizada em 120 indivíduos (20 de cada população), onde constatou-se a existência de uma alta variabilidade genética em todas as populações, não tendo sido encontrada relação entre a simpatria e o nível de diversidade genética em A. platensis. Uma alta estruturação genética observada entre as populações, que provavelmente está associada à distância entre elas e à baixa capacidade de dispersão dos eglídeos. Os resultados do marcador mitocondrial COI e do marcador nuclear AFLP apresentaram algumas incongruências, quando comparados, visto que usando AFLP todas as populações de A. platensis e das demais espécies foram separadas por meio de análise Bayesiana e UPGMA, o mesmo não ocorrendo para o COI. A incongruência entre os dados nucleares e mitocondriais reforça a ideia de que algumas sequências de A. grisella são na verdade pseudo-genes nucleares coamplificados com o gene COI, devido à utilização de primers universais degenerados na PCR.

In the Western United States, the Great Basin is a geographic feature that is home to a variety of unique species, including Great Basin bristlecone pine (Pinus longaeva, PILO). P. longaeva is well known for the age of some of the trees, some of which are over 4000 years old. In this study 17 microsatellite markers (simple sequence repeat, SSR) are identified and used to genotype a selection of 480 individuals spread across 24 PILO populations and 60 individuals divided equally between 2 outgroup species, Pinus aristata and Pinus flexilis. One population of PILO, HMW, shows more genetic similarity with P. aristata, and is included with outgroup species in the analysis. The population structure of PILO is examined and found to be weakly related to the geographic distance between populations. The population statistics Fst, Gst, Rst, Dest and an AMOVA analysis suggest that there is a substantial amount of admixture at the individual level, similar to some other species of pines. Phylogenetic trees computed using the neighbor-joining method based on the average population genetic distance and based on individual genetic distance support the population structure results and show further evidence that most of the species variation is concentrated at the individual level, rather than inside of or between species. It is possible that gene flow is still occurring, or that gene flow has occurred recently enough that the separated populations have not yet diverged from each other in a measurable way.

<p>Music is greatly underappreciated in the scope of cross-cultural analysis. This is due in part to methodological problems plaguing recent comparative approaches, and modern ethnomusicology’s stance against cross-cultural analysis. Language, on the other hand, has a long history of cross-cultural study and recent advances in quantitative techniques, borrowed mostly from biology, have put language at the forefront of studying population prehistory from a cultural perspective. Chapter 2 of this thesis presents a novel quantitative approach to studying cross-cultural musical diversity based on the AMOVA methodology borrowed from population genetics. This method allows researchers to quantify the amount of variability found between as well as within populations, and gives us a measure of population-level divergence that accounts for intra-population variability. Our major finding is that the vast majority of musical variability (~98%) is found within populations rather than between. This approach solves many of the quantitative issues with the original Cantometrics approach, and is widely applicable to the analysis of many domains of culture. Aside from methodological issues a major open question is whether music has the requisite time-depth to answer questions about recent human pre-history. Chapter 3 focuses on addressing this question generally, and more specifically investigating which musical features trace population history most effectively. Using a corpus of songs from 9 Taiwanese aboriginal tribes and quantitative methods from chapter 2, we show that features related to song structure are correlated with mitochondrial DNA data from the same populations, while features of singing style are not. Both the quantitative methods and provisional support for music’s time depth presented here will hopefully usher in a new era of comparative musicology and provide scholars of pre-history with an additional tool to answer unresolved questions.</p> / Master of Science (MSc)

ABSTRACT COMPARATIVE SEQUENCE ANALYSIS OF THE INTERNAL TRANSCRIBED SPACER 2 REGION OF TURKISH RED PINE (Pinus brutia TEN.) AND NATURAL ALEPPO PINE (Pinus halepensis MILL.) POPULATIONS FROM TURKEY Tozkar, &Ouml / zge M.S., Department of Biology Supervisor: Prof. Dr. Zeki Kaya April, 2007, 107 pages Turkish red pine (Pinus brutia) is wide-spread and an important forest tree species in Turkey, occurring mainly in southern, western and north-western Turkey and as small isolated populations in the Black Sea region. Aleppo pine (Pinus halepensis) has naturally found only in Adana and Mugla provinces as small population in mixture with Turkish red pine. Although Turkish red pine and Aleppo pine are morphologically different, Turkish red pine has been regarded as subspecies of Aleppo pine by some taxonomists due to occurrence of natural hybridization between these two species. However, the phylogenic relationship between these species needs to be explored further. In the present study, by sampling overlapped populations of both species from Mugla and Adana provinces (4 populations of Turkish red pine and 3 populations of Aleppo pine), internal transcribed spacer (ITS) region of ribosomal DNA were comparatively studied with sequence analysis. Although ITS1, 5.8s and ITS2 regions of ribosomal DNA were studied with ITS primers, only ITS2 region was successfully amplified with polymerase chain reaction (PCR). The complete data set for this region was analysed using MEGA3.1 and Arlequin softwares. Analysis of molecular variance (AMOVA) demonstrated the highest genetic differentiation between Turkish red pine and Aleppo pine in Mugla with 100 percentage of variation. AMOVA analysis also indicated the possibility of low-level migration of genes between Turkish red pine and Aleppo pine populations in Adana with 50.65 percent of molecular variance. Haplotype comparison revealed that two major haplotypes were represented Based on the results of ITS2 region sequence analysis, Turkish populations of Aleppo pine and Turkish red pine populations could not be fully differentiated. In Mugla province Turkish red pine and Aleppo pine revealed more differentiation due to reproductive isolation. But in Adana province, two species shared more common genetic background due to possible hybridization. Since ITS2 region of nuclear ribosomal DNA revealed a few variable and parsimony informative sites for both species, thus, only ITS2 region of ribosomal DNA does not appear to be sufficient for fully resolving genetic relationships between Turkish red pine and Aleppo pine populations. Further studies including ITS1 and 5.8s regions of ribosomal DNA and populations included from major Aleppo pine distribution areas will be useful to understand the evolutionary relationship between Aleppo pine and Turkish red pine populations in Turkey.