A cultura da batata (Solanum tuberosum L) está se tornando cada vez mais importante, tanto do ponto de vista dos produtores, dos pesquisadores e dos consumidores, por ser um dos alimentos protéicos mais consumidos em todo o mundo, entretanto, o Brasil depende de variedades importadas, originárias de clima temperado o que não condiz com as nossas condições, refletindo assim em valores inferiores de produtividade e de qualidade. Apesar da grande evolução que esta cultura apresentou em todos estes anos de cultivo se faz necessário a busca por materiais mais produtivos, adaptados e resistentes. Os programas de melhoramento convencionais são extremamente importantes para a seleção de novos progenitores, entretanto o tempo gasto para desenvolver e lançar uma variedade é bastante longo. Diante deste cenário, novas metodologias estão sendo cada vez mais utilizadas no processo de identificação de bancos de germoplasma e cultivares mais promissoras. O objetivo deste trabalho foi avaliar, por meio de marcadores microssatélites, 108 acessos de batata de cinco coleções contendo variedades comerciais, clones para programas de melhoramento e cultivo orgânico, visando a caracterização genética, a identificação de duplicatas e de possíveis parentais para uso nos programas de melhoramento. Para a caracterização molecular foram utilizados 10 iniciadores específicos (primers), gerando-se um total de 50 alelos (bandas) os quais foram analisados como dados binários, sendo que a partir destes dados foi obtida uma matriz de similaridade utilizando o coeficiente de similaridade de Jaccard. Com este coeficiente e com o método aglomerativo UPGMA, foram realizadas análises de agrupamento utilizando o software NTSYSpc e um método de reamostragens (bootstraps), gerando dendrogramas que permitiram a distinção genética entre os acessos. O conteúdo de informação de polimorfismo (PIC) e a heterogozidade esperada (He) foram significativos, sendo que os maiores valores foram 0,8594 e 0,8725, respectivamente, para o primer STM0019a. Em média, o número de alelos por loco foi cinco, variando de dois alelos para os primers STM 1053 e STM 1104 até 13 alelos por loco para o primer STM0019a. Para facilitar a visualização dos resultados, além de serem avaliados como um todo os 108 acessos foram divididos em grupos de acordo com as coleções (variedades comerciais, clones e cultivo orgânico), sendo que a maior variação pelo coeficiente de Jaccard foi de 0,39 a 0,93 para 57 acessos das coleções de cultivares orgânicos e comerciais. Ao se avaliar os 108 acessos juntos, o coeficiente de Jaccard variou de 0,42 a 0,93, mostrando a grande variabilidade genética entre os acessos das cinco coleções. Foram observadas seis possíveis duplicatas [ATLANTIC (Canadá) e ATLANTIC (Chile); 67-2 e 17-10 (clones CNPH1); COLORADO e ÁGATA (EPAMIG); 253 E 266 (clones CNPH2); MELODY e APTA 21-54 (cultivo orgânico); e 387-1 (E1) e VOYAGER], identificando-se também os acessos mais distantes geneticamente [clone 383-19 (EmbrapaCNPH1) e a cultivar comercial HPC-7B], permitindo desta forma a identificação de possíveis parentais para os programas de melhoramento. Os altos níveis de polimorfismo observados paraSolanum tuberosum sugerem que os marcadores microssatélites podem ser uma ferramenta útil para detectar as diferenças genéticas entre cultivares de batata. / The cultivation of potato (Solanum tuberosum L) is becoming increasingly important from the point of view of producers, researchers and consumers, for representing one of the food protein mostly consumed in the world. However, Brazil depends on imported varieties, originated from temperate climate which does not complies with our conditions, thus reflecting in lower productivity and quality. Despite the great progress that this crop presented in all these years of cultivation, it is necessary to search for more productive, adapted and resistant materials. The conventional breeding programs are important for the selection of new parents, but the time spent to develop and launch a new variety is quite long. In this scenario, new approaches are being increasingly used in the identification of germplasm banks and most promising cultivars. The objective of this study was to evaluate, using microsatellite markers, 108 accessions of five potato collections containing commercial varieties, clones for breeding programs and organic farming varieties, aiming at the genetic characterization, identification of duplicates and possible parents to be used in potato breeding programs. For the molecular characterization, 10 specific primers were used, generating a total of 50 alleles (bands) which were analyzed as binary data, and from this data a similarity matrix was obtained using the Jaccard coefficient of similarity. With this coefficient and the UPGMA method, cluster analysis were carried out using the NTSYSpc software and bootstraps analyses, generating dendrograms which allowed the genetic distinction between accessions. The polymorphism information content (PIC) and expected heterozygosity (He) were both significant, with the highest values (0.8594 and 0.8725, respectively) obtained for primer STM0019a. On average, the number of alleles per locus was five, ranging from two alleles for primers STM 1053 and STM 1104 to 13 alleles per locus for primer STM0019a. To facilitate the visualization of the results, in addition to being evaluated as a whole, the 108 accessions were divided into groups according to the collections (commercial varieties, clones and organic farming), where the highest variation for the Jaccard coefficient (0.39 - 0.93) was found for the 57 accessions of organic and commercial cultivars collections. When assessing the 108 accessions together, the Jaccard coefficient ranged from 0.42 to 0.93, showing a high genetic variability between accessions of the five collections. Six possible duplicates were found [\'ATLANTIC (Canada) and ATLANTIC (Chile); 67-2 and 17-10 (clones CNPH1); Color and AGATE (EPAMIG); 253 E 266 (clones CNPH2); MELODY and APTA 21-54 (organic farming); and 387-1 (E1) and VOYAGER], and also the more genetically distant accessions [clone 383-19 (EmbrapaCNPH1) and the commercial cultivar HPC- 7B] were identified, thereby enabling the identification of potential parents for breeding programs. High levels of polymorphism observed for Solanum tuberosum suggest that microsatellite markers can be a useful tool to detect the genetic differences between potato cultivars.

Heliothis virescens (Fabricius) é uma das pragas-alvo do algodão geneticamente modificado que expressa a proteína Cry1Ac de Bacillus thuringiensis Berliner (Bt). Estudos sobre a suscetibilidade de H. virescens à proteína Cry1Ac e sobre a estrutura genética e padrões de fluxo gênico nas escalas locais e regionais desse inseto são fundamentais para a implementação de programas de Manejo da Resistência de Insetos (MRI) no Brasil. Dessa forma, os principais objetivos do trabalho foram: (a) avaliar a suscetibilidade à proteína Cry1Ac em populações de H. virescens coletadas nas principais regiões produtoras de algodão no Brasil (Bahia, Goiás, Mato Grosso e Mato Grosso do Sul) durante as safras agrícolas 2007/08 e 2008/09; e (b) avaliar a variabilidade genética e fluxo gênico de populações de H. virescens provenientes das culturas de algodão (safras 2007/08, 2008/09 e 2009/10) e de soja (safra 2009/10) utilizando sequências de DNA mitocondrial (DNAmit). As linhas-básica de suscetibilidade à proteína Cry1Ac foram estabelecidas mediante o uso de lagartas neonatas, por meio de bioensaios de incorporação das diferentes concentrações da proteína em dieta artificial. Para avaliar a variabilidade genética e o fluxo gênico entre populações de H. virescens utilizou-se sequências de DNAmit das subunidades I e II da citocromo oxidase COI e COII e a subunidade 6 da desidrogenase dinucleotídica da adenina nicotinamida nad6. As CLs50 estimadas variaram de 0,18 a 0,66 µg de Cry1Ac/mL de dieta para as populações coletadas na safra 2007/08 (variação de 3,7 vezes). Da mesma forma, as concentrações efetivas médias para a inibição do desenvolvimento larval (CE50) variaram de 0,0053 a 0,0161 µg de Cry1Ac/mL dieta (variação de 3,0 vezes). A partir da análise conjunta dos dados de concentração-mortalidade de todas as populações avaliadas, foram definidas e validadas as concentrações diagnósticas de 3,1 e 5,6 µg de Cry1Ac/mL de dieta para programas de monitoramento da resistência de H. virescens à proteína Cry1Ac no Brasil. Baseadas em análises de agrupamento (Neighbor-Joining e Análise de Componentes Principais) e Bayesiana (Structure) foram verificadas uma baixa estruturação entre as populações de H. virescens de diferentes regiões, bem como para aquelas coletadas em plantas hospedeiras diferentes. As análises de AMOVA também indicaram baixa estruturação genética entre as populações de H. virescens estudadas independente da cultura (Fst= 0,019) ou escala geográfica (Fst= 0,012), sugerindo um nível significativo de fluxo gênico. Em média, a distância genética entre as amostras foi de 0,1%. Uma rede de haplótipos obtida com os dados combinados resultou em 35 haplótipos, com quatro haplótipos únicos presentes somente nas amostras coletadas em soja. A principal característica dessa rede é a forma de estrela na distribuição dos haplótipos, bem como a ocorrência de muitos alelos em baixa frequência. Esse tipo de rede é característico de populações que passaram por uma recente expansão populacional e, de fato, a história demográfica de H. virescens, baseada no teste de distribuição da diferença genética par-a-par entre haplótipos (distribuição de Mismatch) e nos resultados negativos nos testes de neutralidade seletiva indicam também um episódio de expansão populacional recente. As informações obtidas no presente trabalho serão fundamentais para o acompanhamento da efetividade das estratégias de manejo da resistência de H. virescens à proteína Cry1Ac no Brasil. / The tobacco budworm, Heliothis virescens (Fabricius), is one of target pests of genetically modified cotton expressing Cry1Ac insecticidal protein derived from Bacillus thuringiensis Berliner. Studies on susceptibility of H. virescens to Cry1Ac and the genetic structure and gene flow patterns at local and regional levels are crucial for establishing an Insect Resistance Management (IRM) program for Bt cotton in Brazil. Thus, the objectives of this study were (a) to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac from major cotton-growing regions in Brazil (Bahia, Goiás, Mato Grosso and Mato Grosso do Sul) in the cropping seasons of 2007/08 and 2008/09; and (b) to evaluate the genetic variability and gene flow among H. virescens populations from cotton (2007/08, 2008/09 and 2009/10 cropping seasons) and soybean (2009/10 cropping season) with mitochondrial DNA markers. Baseline susceptibility data to Cry1Ac protein were estimated with neonate larvae thereby using diet incorporation bioassays. Genetic variation and gene flow among H. virescens populations were evaluated by using mitDNA sequences of cytochrome oxidase subunities I and II COI e COII and the subunity 6 of dinucleotide dehydrogenase of adenine nicotinamide nad6. The estimated LC50 values varied from 0.18 to 0.66 µg of Cry1Ac/mL of diet among the 2007/08 populations (3.7 fold variation). Similarly, the EC50 values based on growth inhibition ranged from 0.0053 to 0.0161 µg of Cry1Ac/mL of diet for the 2007/08 populations (3.0 fold variation). A joint analysis of the mortality data across all tested populations was used to develop candidate diagnostic concentrations for future monitoring programs. The proposed diagnostic concentrations of 3.1 and 5.6 µg of Cry1Ac/mL of diet were validated against field-collected populations from 2008/09 and will form the basis for future resistance monitoring programs with H. virescens. Based on cluster analysis (Neighbor-Joining and Principal Coordinate Analysis) and Bayesian analysis (Structure), a low structure was detected among H. virescens populations either by regions or host plants. AMOVA analysis also indicated low genetic structure among H. virescens populations across crops (Fst= 0.019) or geographic scale (Fst= 0.012), suggesting a significant gene flow. The mean genetic distance among samples was 0.1%. The haplotype network obtained with joint data resulted in 35 haplotypes, with four unique haplotypes present only in samples collected from soybean crop. The major characteristics of the haplotype network were the star-like pattern and the occurrence of many alleles at low frequencies. This type of network is typical for populations that passed through a recent population expansion and, in fact, the demographic history of H. virescens, based on distribution test of pair-wise genetic difference among haplotypes (Mismatch distribution) and negative results from tests of selective neutrality also indicate an episode of a recent population expansion.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-04-26T19:53:52Z No. of bitstreams: 1 2012 - Simone Quinelato Bezerra.pdf: 1703088 bytes, checksum: c39ad4f0bc5fb8b05085a7e4684060b9 (MD5) / Made available in DSpace on 2017-04-26T19:53:52Z (GMT). No. of bitstreams: 1 2012 - Simone Quinelato Bezerra.pdf: 1703088 bytes, checksum: c39ad4f0bc5fb8b05085a7e4684060b9 (MD5) Previous issue date: 2012-03-30 / FAPERJ / Aiming to decrease the chemicals acaricide use and their damages, new alternatives for ticks control has been studied. Metarhizium anisopliae s.l. is one of the most studied fungi in agricultural pest management programs, since it has great acaricide potential. Therefore, this study aimed to characterize molecular and morphologically, as well as evaluate the virulent potential of 30 M. anisopliae s.l. isolates from different geographical regions, hosts or substrates allowing the selection of virulent isolates in order to be further investigated for field programs of microbial control of pests. Initially, the analyses of morphological characterizations of the isolates were made to confirm their identification. Each isolate had its conidial potential production evaluated. The colonies studied showed morphological characteristics consistent with those described in the literature. The colonies diameter varied between 29.66 mm and 51.33 mm among isolates. There was both length and width variation in the conidia and phialides in the same isolate, as well as the presence of grouped and solitary phialides. The conidial production potential was variable among isolates, but both conidial size and colonies diameter did not influence the conidial production; isolates with low conidial production showed similar colony size in comparison to isolates with high potential. In a second stage of the study, the virulence of these isolates was evaluated to Rhipicephalus microplus larvae treated with one of the four different conidial concentrations (105, 106, 107 or 108 conidia.mL-1). The lethal action of Brazilian M. anisopliae s.l isolates to R. microplus larvae were confirmaded with high mortality among the isolates, which in general was proportional to the conidia concentration of the treatments. Most isolates killed larvae population with 107 conidia.mL-1 concentration, however the most virulent isolates presented lethal concentration of 106 conidia.mL-1 with main percentages of mortality nearly 100% at day 20 after treatment. In addition, the genetic variability of these isolates was performed to evaluate their relationship with other species of Metarhizium sp. through RFLP-PCR analysis and ITS1-5.8S-ITS2 rDNA sequencing. No specificity pattern was observed when isolates from the same region, host or substrate were grouped. Low genetic variability was observed among isolates, which were basically grouped into two groups. The CG 344 isolate was shown to be genetically distant from the remaining Brazilian isolates studied, but according to the ?GenBank? sequences comparison, it was related to the Metarhizium genus. It is suggested that this variation occured owing the lack of procedures that could generated morphological and molecular changes, which probably contribute to this low genetic variability. The present study allowed the detection of M. anisopliae s.l. isolates with highly virulence to R. microplus larvae, that may be considered potential biocontrol agents for this tick species, emphasizing the importance of molecular tools for identification and characterization of fungal isolates, ensuring the product quality, their success implement and the environmental track of the fungi at field biological control programs. / Na tentativa de diminuir a utiliza??o de produtos qu?micos e os danos por eles causados, novas alternativas para o controle de carrapatos vem sendo estudadas. O fungo Metarhizium anisopliae ? um dos mais estudados em programas agropecu?rios de manejo de pragas, pois apresenta grande potencial acaricida. Baseado nisso, o presente estudo objetivou a caracteriza??o morfol?gica, molecular e a avalia??o da virul?ncia de 30 isolados brasileiros de M. anisopliae s.l. provenientes de diferentes regi?es geogr?ficas, hospedeiros ou substratos, com a finalidade de selecionar isolados mais virulentos para utiliza??o em futuros programas de biocontrole de carrapatos. Inicialmente os isolados foram caracterizados morfologicamente para confirma??o de sua identifica??o, tamb?m sendo avaliado o potencial de produ??o de con?dios de cada isolado. As col?nias estudadas apresentaram caracter?sticas morfol?gicas compat?veis com as descritas na literatura. O tamanho das col?nias variou entre 29,66 mm e 51,33 mm de di?metro. Houve varia??o no comprimento e na largura de con?dios e fi?lides num mesmo isolado, assim como a presen?a de fi?lides agrupadas e solit?rias. O potencial de produ??o de con?dios foi vari?vel entre os isolados, por?m tanto o tamanho dos con?dios quanto o di?metro das col?nias n?o influenciaram a produ??o de con?dios. Numa segunda etapa do estudo, foi avaliada a virul?ncia destes isolados sobre larvas de Rhipicephalus microplus tratadas com uma das quatro diferentes concentra??es de con?dios (105, 106, 107 ou 108 con?dios/mL). Foi confirmada a a??o letal dos isolados brasileiros de M. anisopliae s.l. sobre larvas de R. microplus, geralmente ocorrendo de forma diretamente proporcional a concentra??o conidial dos tratamentos. A maioria dos isolados ocasionou a morte de metade da popula??o de larvas com a concentra??o de 107 con?dios/mL; os isolados mais virulentos apresentaram esta concentra??o letal com 106 con?dios/mL, com percentuais m?dios de mortalidade de larvas pr?ximos de 100% ao 20? dia ap?s tratamento. Al?m disso, buscou-se avaliar a variabilidade gen?tica destes isolados e sua rela??o com outras esp?cies do g?nero Metarhizium atrav?s da an?lise de RFLP-PCR e do sequenciamento da regi?o ITS1-5.8S-ITS2 do rDNA. N?o foi observado um padr?o de especificidade para o agrupamento entre isolados oriundos de mesma regi?o, hospedeiro ou substrato. Foi observada variabilidade gen?tica entre os isolados que basicamente se agruparam em dois grupos. O isolado CG 344 mostrou-se geneticamente distante de todos os outros, mas de acordo com a compara??o com sequ?ncias obtidas do ?GenBank? mostrou-se relacionado ao g?nero Metarhizium. Esta varia??o pode ser devido ao fato deste isolado ter sido poupado de processos que gerassem altera??es morfol?gicas e moleculares, o que possivelmente contribuiu para a pequena variabilidade gen?tica obsevada. O presente estudo possibilitou a detec??o de isolados brasileiros de M. anisopliae s.l. com elevada virul?ncia para larvas de R. microplus, podendo ser considerados potenciais agentes no biocontrole desta esp?cie de carrapato, ressaltando a import?ncia da utiliza??o de ferramentas moleculares para identifica??o e caracteriza??o destes isolados, contribuindo para a qualidade do produto, o sucesso de sua aplica??o e o monitoramento de um isolado introduzido no ambiente com finalidade de controle biol?gico.

Heliothis virescens (Fabricius) é uma das pragas-alvo do algodão geneticamente modificado que expressa a proteína Cry1Ac de Bacillus thuringiensis Berliner (Bt). Estudos sobre a suscetibilidade de H. virescens à proteína Cry1Ac e sobre a estrutura genética e padrões de fluxo gênico nas escalas locais e regionais desse inseto são fundamentais para a implementação de programas de Manejo da Resistência de Insetos (MRI) no Brasil. Dessa forma, os principais objetivos do trabalho foram: (a) avaliar a suscetibilidade à proteína Cry1Ac em populações de H. virescens coletadas nas principais regiões produtoras de algodão no Brasil (Bahia, Goiás, Mato Grosso e Mato Grosso do Sul) durante as safras agrícolas 2007/08 e 2008/09; e (b) avaliar a variabilidade genética e fluxo gênico de populações de H. virescens provenientes das culturas de algodão (safras 2007/08, 2008/09 e 2009/10) e de soja (safra 2009/10) utilizando sequências de DNA mitocondrial (DNAmit). As linhas-básica de suscetibilidade à proteína Cry1Ac foram estabelecidas mediante o uso de lagartas neonatas, por meio de bioensaios de incorporação das diferentes concentrações da proteína em dieta artificial. Para avaliar a variabilidade genética e o fluxo gênico entre populações de H. virescens utilizou-se sequências de DNAmit das subunidades I e II da citocromo oxidase COI e COII e a subunidade 6 da desidrogenase dinucleotídica da adenina nicotinamida nad6. As CLs50 estimadas variaram de 0,18 a 0,66 µg de Cry1Ac/mL de dieta para as populações coletadas na safra 2007/08 (variação de 3,7 vezes). Da mesma forma, as concentrações efetivas médias para a inibição do desenvolvimento larval (CE50) variaram de 0,0053 a 0,0161 µg de Cry1Ac/mL dieta (variação de 3,0 vezes). A partir da análise conjunta dos dados de concentração-mortalidade de todas as populações avaliadas, foram definidas e validadas as concentrações diagnósticas de 3,1 e 5,6 µg de Cry1Ac/mL de dieta para programas de monitoramento da resistência de H. virescens à proteína Cry1Ac no Brasil. Baseadas em análises de agrupamento (Neighbor-Joining e Análise de Componentes Principais) e Bayesiana (Structure) foram verificadas uma baixa estruturação entre as populações de H. virescens de diferentes regiões, bem como para aquelas coletadas em plantas hospedeiras diferentes. As análises de AMOVA também indicaram baixa estruturação genética entre as populações de H. virescens estudadas independente da cultura (Fst= 0,019) ou escala geográfica (Fst= 0,012), sugerindo um nível significativo de fluxo gênico. Em média, a distância genética entre as amostras foi de 0,1%. Uma rede de haplótipos obtida com os dados combinados resultou em 35 haplótipos, com quatro haplótipos únicos presentes somente nas amostras coletadas em soja. A principal característica dessa rede é a forma de estrela na distribuição dos haplótipos, bem como a ocorrência de muitos alelos em baixa frequência. Esse tipo de rede é característico de populações que passaram por uma recente expansão populacional e, de fato, a história demográfica de H. virescens, baseada no teste de distribuição da diferença genética par-a-par entre haplótipos (distribuição de Mismatch) e nos resultados negativos nos testes de neutralidade seletiva indicam também um episódio de expansão populacional recente. As informações obtidas no presente trabalho serão fundamentais para o acompanhamento da efetividade das estratégias de manejo da resistência de H. virescens à proteína Cry1Ac no Brasil. / The tobacco budworm, Heliothis virescens (Fabricius), is one of target pests of genetically modified cotton expressing Cry1Ac insecticidal protein derived from Bacillus thuringiensis Berliner. Studies on susceptibility of H. virescens to Cry1Ac and the genetic structure and gene flow patterns at local and regional levels are crucial for establishing an Insect Resistance Management (IRM) program for Bt cotton in Brazil. Thus, the objectives of this study were (a) to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac from major cotton-growing regions in Brazil (Bahia, Goiás, Mato Grosso and Mato Grosso do Sul) in the cropping seasons of 2007/08 and 2008/09; and (b) to evaluate the genetic variability and gene flow among H. virescens populations from cotton (2007/08, 2008/09 and 2009/10 cropping seasons) and soybean (2009/10 cropping season) with mitochondrial DNA markers. Baseline susceptibility data to Cry1Ac protein were estimated with neonate larvae thereby using diet incorporation bioassays. Genetic variation and gene flow among H. virescens populations were evaluated by using mitDNA sequences of cytochrome oxidase subunities I and II COI e COII and the subunity 6 of dinucleotide dehydrogenase of adenine nicotinamide nad6. The estimated LC50 values varied from 0.18 to 0.66 µg of Cry1Ac/mL of diet among the 2007/08 populations (3.7 fold variation). Similarly, the EC50 values based on growth inhibition ranged from 0.0053 to 0.0161 µg of Cry1Ac/mL of diet for the 2007/08 populations (3.0 fold variation). A joint analysis of the mortality data across all tested populations was used to develop candidate diagnostic concentrations for future monitoring programs. The proposed diagnostic concentrations of 3.1 and 5.6 µg of Cry1Ac/mL of diet were validated against field-collected populations from 2008/09 and will form the basis for future resistance monitoring programs with H. virescens. Based on cluster analysis (Neighbor-Joining and Principal Coordinate Analysis) and Bayesian analysis (Structure), a low structure was detected among H. virescens populations either by regions or host plants. AMOVA analysis also indicated low genetic structure among H. virescens populations across crops (Fst= 0.019) or geographic scale (Fst= 0.012), suggesting a significant gene flow. The mean genetic distance among samples was 0.1%. The haplotype network obtained with joint data resulted in 35 haplotypes, with four unique haplotypes present only in samples collected from soybean crop. The major characteristics of the haplotype network were the star-like pattern and the occurrence of many alleles at low frequencies. This type of network is typical for populations that passed through a recent population expansion and, in fact, the demographic history of H. virescens, based on distribution test of pair-wise genetic difference among haplotypes (Mismatch distribution) and negative results from tests of selective neutrality also indicate an episode of a recent population expansion.

Species are one of the fundamental components of biology and the accurate delimitation of species is important in evolutionary, systematic and ecological studies, yet there is still confusion over how species can be recognised. Examining different characters allows multiple lines of evidence for successful and accurate species delimitation and identification. In this thesis, morphological, genetic and behavioural variation is investigated within an endemic species of ground weta, Hemiandrus pallitarsis, in the North Island, New Zealand. Twelve morphological characters were measured, and mitochondrial cytochrome oxidase I DNA sequences were analysed from populations across the distributional range of H. pallitarsis. Both methods provide no evidence of a species complex within H. pallitarsis. Instead, the morphometric results suggest females are significantly larger than males, and ground weta in Palmerston North are significantly smaller than weta further north. Additionally, genetic analyses found substantial population structuring, large genetic distances, and an historical south to north pattern of movement in the North Island. The pattern of vibratory drumming behaviour followed that predicted by morphology and geographic proximity – drumming signals were more similar between geographically close populations and did not match the patterns of genetic isolation. Overall, this thesis was able to show that H. pallitarsis is morphologically, genetically and behaviourally variable across the North Island.

The research field of genetics provides useful tools to investigate the biology of species that are difficult to observe and study and are especially valuable in guiding the conservation of endangered species. The Chatham Island Tāiko (Tchāik, Pterodroma magentae) is the world’s most endangered seabird with an estimated population size of just 120-150 birds, including only 8-15 breeding pairs. This thesis used genetic techniques to investigate aspects of Tāiko biology and relationships in order to aid Tāiko conservation. The mitochondrial cytochrome b gene and duplicated regions of domain I of the mitochondrial control region were DNA sequenced in almost the entire known Tāiko population. The level of genetic variation revealed in Tāiko was unexpectedly high considering endangered species typically exhibit low genetic diversity. Sequencing of ancient DNA from subfossil Tāiko bones allowed an investigation of the past level of genetic variation and the species’ previous geographic distribution. A large proportion of the genetic diversity of the extinct Tāiko populations was retained in the remnant population. However, genetic variation in Tāiko chicks was low, thus genetic diversity in the population could be lost in just a few generations. There are many nonbreeding Tāiko so DNA sexing was used to examine sex ratios in the population. Almost all unpaired birds were male, which signified a potential Allee effect (i.e. that a reduced density of potential mates is decreasing population productivity). Further understanding of the Tāiko mating system and behaviour was obtained by parentage, sibship and pairwise relatedness analyses of genotypes at eight microsatellite DNA loci. It is important that Tāiko are found so they can be protected from introduced predators. The results of mitochondrial DNA sequencing and microsatellite DNA genotyping indicated that there are likely to be more Tāiko breeding in undiscovered areas. Analysis of philopatry using both mitochondrial and nuclear markers can assist conservation by the identification of areas to search for these undiscovered individuals. Tāiko may have once and could still be found on islands near South America since DNA sequencing showed the Magenta Petrel type specimen (collected in 1867 in the South Pacific Ocean) is a Tāiko.

<p>In this thesis the genetic variation of two grouse species, the Chinese grouse (<i>Bonasa sewersowi</i>) and the Black grouse (<i>Tetrao tetrix</i>) was examined with neutral genetic markers: microsatellites. Habitat fragmentation and isolation leads to structuring among and loss of genetic variation within populations.</p><p>The Chinese grouse in a small population in Lianhuasan nature reserve was found to have undergone a population bottleneck and as a result of isolation and possible inbreeding showed genetic impoverishment hereof.</p><p>The Black grouse populations in Europe face various different conditions from widely distributed areas of suitable habitat in the northern and eastern parts of its range to highly naturally and anthropogenically fragmented habitat landscapes in the west.</p><p>Structure among populations was found in Great Britain where Wales, Scotland and England showed characteristics of three different genetic entities, indicating very little or no geneflow between these populations. </p><p>The Dutch population showed signs of loss of genetic variation as to be expected from a population that has historically decreased in population size from several thousands to tens of individuals in a matter of decades. However the possibility to spot signs of a bottleneck was impaired due to the short time-window in which this can be observed in a population with such a low effective population size (N_E).</p><p>The sampled populations in Europe clustered into five different groups of genetic identities. The different clusters were: Great Britain-, the Netherlands-, Fenno-Scandian-, Alpine- and lowland German-Austrian populations. The level of genetic variation when compared over all these different populations decreased as a sign of isolation and small N_E. However it was not feasible to separate the impact of these two factors.</p>

In this thesis the genetic variation of two grouse species, the Chinese grouse (Bonasa sewersowi) and the Black grouse (Tetrao tetrix) was examined with neutral genetic markers: microsatellites. Habitat fragmentation and isolation leads to structuring among and loss of genetic variation within populations. The Chinese grouse in a small population in Lianhuasan nature reserve was found to have undergone a population bottleneck and as a result of isolation and possible inbreeding showed genetic impoverishment hereof. The Black grouse populations in Europe face various different conditions from widely distributed areas of suitable habitat in the northern and eastern parts of its range to highly naturally and anthropogenically fragmented habitat landscapes in the west. Structure among populations was found in Great Britain where Wales, Scotland and England showed characteristics of three different genetic entities, indicating very little or no geneflow between these populations. The Dutch population showed signs of loss of genetic variation as to be expected from a population that has historically decreased in population size from several thousands to tens of individuals in a matter of decades. However the possibility to spot signs of a bottleneck was impaired due to the short time-window in which this can be observed in a population with such a low effective population size (NE). The sampled populations in Europe clustered into five different groups of genetic identities. The different clusters were: Great Britain-, the Netherlands-, Fenno-Scandian-, Alpine- and lowland German-Austrian populations. The level of genetic variation when compared over all these different populations decreased as a sign of isolation and small NE. However it was not feasible to separate the impact of these two factors.

Genes are the foundation of evolution and biodiversity. The genetic structure of natural populations needs to be understood to maintain exploited resources rationally. This thesis focuses on genetic variability and methods to determine spatial and temporal genetic heterogeneities. Intense human exploitation generates particular challenges to conserve genetic diversity of fishes since it has genetic effects. My research concerns one of our most valuable fish species: the Atlantic herring (Clupea harengus). I analyzed Atlantic herring samples from the North and Baltic Seas. The objectives were to determine: 1) spatial genetic structure, 2) whether allozymes and microsatellites provide similar descriptions of the differentiation pattern, or 3) if they are influenced by selection, 4) factors affecting statistical power when testing for genetic differentiation, and 5) the temporal stability of the genetic structure. The results show: 1) very low levels of spatial genetic differentiation in Atlantic herring; a major component is a difference between the Baltic and North Seas, 2) a concordant pattern with allozymes and microsatellites, 3) that selection influences a microsatellite locus, which can be a low salinity adaptation, 4) that statistical power is substantial for frequently used sample sizes and markers; the difference in power between organelle and nuclear loci is partly dependent on the populations’ stage of divergence, and 5) no changes in amount of genetic variation or spatial genetic structure over a 24-year period; the selection pattern in one microsatellite locus remained. The notion that the large population sizes make herring unlikely to lose genetic diversity may be disputed. I found small local effective population sizes, and the evidence of selection hints of a distinct evolutionary lineage in the Baltic. When Atlantic herring is managed as very large units, there can be detrimental genetic effects if certain population segments are excessively harvested.

Plants have spread to almost everywhere in the world. As they disperse, they meet many different environments to which they may be able to adapt. For a plant species to adapt to a new environment, genetic variation is needed. The individuals differ from each other in their genetic composition, which often means differences in phenotypes. Those individuals that manage to reproduce will form the next generation. With different conditions in different environments, it will not be the same phenotypes that reproduce everywhere. In that way, plant species will form into a mosaic of locally adapted populations varying genetically as the species disperses. After the last ice age plants have started to disperse away from the equators. With increasing latitudes come increasing challenges to migrating plants. As plant species disperse northwards along this gradient of varying conditions individuals are selected for cold adaptive traits like flowering time and freezing tolerance, acquired by cold acclimation. In this way, genetic variation from the original populations for these traits becomes sorted out along a latitudinal cline. The aim of this thesis was to understand how selection along a latitudinal gradient has shaped natural variation in cold adaptive traits in plants dispersing northwards, and specifically, to investigate what variation can be observed in phenotypes for these traits and how these traits correlate with genetic variation in genes known to be involved in cold acclimation. In this study significant variation was found in a sample of the model plan Arabidopsis thaliana accessions in cold adaptive traits flowering time and freezing tolerance. A clear latitudinal cline in the cold adaptive traits freezing tolerance for A. thaliana was observed. Analysis of nucleotide polymorphism for the cold responsive ICE1 (inducer of CBF expression 1) transcription factor revealed a haplotype structure with two allelic clades as well as unusually high levels of synonymous polymorphism. Nucleotide polymorphism analysis for the transcription factors CBF1, CBF2 and CBF3 (C-repeat binding factors) that play a key role in regulating the expression of a group of target genes known as the “CBF regulon” showed a distinct geographical haplotype structure. One haplotype was dominant in southern accessions while in the other northern accessions overrepresented. There was a significant effect of CBF haplotype on both freezing tolerance and flowering time even after correcting for latitude. Significant differences in CBF expression levels were found between the different CBF genes as well as between different accessions. Sequence variation at CBF was shown to have a significant effect on expression levels of CBF2. No clear correlations were found between CBF gene expression and freezing tolerance or temperature sensitivity for any of the accessions used in the study. This highlights the complex relationship between sequence variation in candidate genes and gene expression, and the problems associated with unraveling the genetic basis of ecologically important traits.