Biogeographers have long searched for common processes responsible for driving diversification in the Holarctic region. Although terrestrial flora and fauna have been well studied, much of the marine biogeographic work addresses patterns and processes occurring over a relatively recent timescale. A prerequisite to comparative biogeographic analysis requires well-resolved phylogenies of similarly distributed taxa that diverged over a similar timeframe. The overall aim of my Ph.D. thesis was to address fundamental questions in the systematics and biogeography of a family of Holarctic fish (Osmeridae) and place these results in a broad comparative biogeographic framework. With eight conflicting morphological hypotheses, the northern hemisphere smelts have long been the subjects of systematic disagreement. In addition to the uncertainty in the interrelationships within this family, the relationship of the Osmeridae to several other families remains unclear. Using DNA sequence data from three mitochondrial and three nuclear genes from multiple individuals per species, I reconstructed the phylogenetic relationships among the 6 genera and 15 osmerid species. Phylogenetic reconstruction and divergence dating yielded a well-resolved phylogeny of the osmerid genera and revealed several interesting evolutionary patterns within the family: (1) Hypomesus chishimaensis and H. nipponensis individuals are not reciprocally monophyletic, suggesting that they are conspecific and H. chishimaensis is a recently evolved freshwater ecotype that invaded the Kuril Islands following the last glaciation, (2) The trans-Pacific sister relationships in Hypomesus based on lateral line scale counts are not supported, implying that this phenotype evolved in parallel on each side of the North Pacific Ocean, (3) The Plecoglossidae are the Osmeridae sister group, (4) Over half of the characters from previous studies show evidence of parallel evolution; however, 27 traits reflect ancestral relationships, (5) Multiple divergences within the Osmeridae date to both the mid-Miocene cooling period and the Pliocene Bering Seaway opening, suggesting these events were important in the evolution of these fishes, and (6) Divergences in many marine taxa for which dated phylogenies are available are also correlated with these time periods. Future research should target additional Holarctic marine taxa for further comparative analysis. / Science, Faculty of / Zoology, Department of / Graduate

no / Degradation of nuclear DNA is a hallmark of programmed cell death. Epidermal keratinocytes die in the course of cornification to function as the dead building blocks of the cornified layer of the epidermis, nails, and hair. Here, we investigated the mechanism and physiological function of DNA degradation during cornification in vivo. Targeted deletion of the keratinocyte-specific endonuclease DNase1-like 2 (DNase1L2) in the mouse resulted in the aberrant retention of DNA in hair and nails, as well as in epithelia of the tongue and the esophagus. In contrast to our previous studies in human keratinocytes, ablation of DNase1L2 did not compromise the cornified layer of the epidermis. Quantitative PCRs showed that the amount of nuclear DNA was dramatically increased in both hair and nails, and that mitochondrial DNA was increased in the nails of DNase1L2-deficient mice. The presence of nuclear DNA disturbed the normal arrangement of structural proteins in hair corneocytes and caused a significant decrease in the resistance of hair to mechanical stress. These data identify DNase1L2 as an essential and specific regulator of programmed cell death in skin appendages, and demonstrate that the breakdown of nuclear DNA is crucial for establishing the full mechanical stability of hair.

No / Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear and mitochondrial DNA appear to be present in almost all hair and nail samples, the quality of DNA, both in quantity and length of amplifiable DNA fragments, vary considerably not just by species, but by individual, and even within individual between hair types.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-11T12:09:07Z No. of bitstreams: 1 2016 - Joao Bosco Varela.pdf: 1890246 bytes, checksum: 359c15e2f52f2ea873d394cbbfa5479c (MD5) / Made available in DSpace on 2017-01-11T12:09:07Z (GMT). No. of bitstreams: 1 2016 - Joao Bosco Varela.pdf: 1890246 bytes, checksum: 359c15e2f52f2ea873d394cbbfa5479c (MD5) Previous issue date: 2016-02-24 / The study of ticks and tick-borne disease is increasingly dependent upon the use of molecular biological techniques that are employed in pathogen detection and for the accurate identification of ticks, particularly immature stages. The successful application of molecular methods, principally the polymerase chain reaction (PCR), can only be achieved if the DNA present in the tick was effectively preserved and could be extracted efficiently. The current study compared three fixatives (RNAlater, zinc salts (ZN) and isopropanol) for the ability to preserve the nuclear and mitochondrial (mt) DNA of larvae and nymphs of Amblyomma parvum and larvae of Amblyomma scultptum. DNA was extracted from ticks at times between 72h to 12 months post fixation using a phenol-chloroform procedure and examined using PCR assays for nuclear (internal transcribed spacer 2; ITS2) and mitochondrial (12S rDNA, subunit 1 of cytochrome c oxidase (COI) and D-loop) sequences. The efficiency of amplification was analyzed quantitatively (number of samples which produced amplicon) and qualitatively (relative intensity of bands observed on agarose gels). It was observed that the ITS2 sequence could be amplified in the majority (93,39%, n= 283/303) of the samples, in each of the three fixatives, although qualitative differences were observed, particularly with A. sculptum preserved in ZN. In contrast, fixation in isopropanol effectively abolished the ability to amplify the mitochondrial marker sequences of A. parvum and also resulted in inferior amplification (qualitative), of the D-loop target with A. sculptum. Those effects were observed in samples fixed for as little as 72h. The detrimental effects of isopropanol were also observed in samples extracted using an alkaline lysis method (Hotshot). Samples of A. parvum larvae preserved in RNAlater for 30 months showed an amplification efficacy of 100% in the ITS2 and COI assays, irrespective of the extraction method. Mitochondrial sequences are a central component of the majority of molecular studies of ticks. The findings of this study indicate that isopropanol should be avoided as a fixative for immature stages of ticks. Instead, the use of RNAlater is recommended in order to permit the consistent recovery of amplifiable mtDNA / O estudo de carrapatos e doen?as transmitidas por eles ? cada vez mais dependente da utiliza??o de t?cnicas de biologia molecular que s?o empregadas na detec??o de pat?genos, e a acurada identifica??o desses artr?podes, em particular os est?gios imaturos. A aplica??o bem-sucedida dos m?todos moleculares, principalmente, a rea??o em cadeia da polimerase (PCR), s? pode ser alcan?ada se o DNA presente no carrapato foi eficazmente preservado e extra?do de forma eficiente. O estudo comparou tr?s fixadores (RNAlater, sais de zinco (ZN) e isopropanol) avaliando a sua capacidade de preservar DNA mitocondrial (mtDNA) e nuclear de larvas e ninfas de Amblyomma parvum e larvas de Amblyomma scultptum. O DNA foi extra?do dos carrapatos, em tempos entre 72h e 12 meses ap?s a fixa??o por meio da t?cnica de fenol-clorof?rmio e lise alcalina (?Hot Shot?) e examinadas usando ensaios de PCR para sequ?ncias nucleares (espa?ador interno transcrito 2; ITS2) e mitocondriais (12S rDNA, subunidade 1 do citocromo c oxidase (COI) e D-loop). A efici?ncia de amplifica??o foi analisada quantitativamente (n?mero de amostras que produziram ?amplicon?) e qualitativamente (intensidade relativa das bandas observadas em g?is de agarose). Foi observado que a sequ?ncia ITS2 foi amplificada na maioria (93,39%, n= 283/303) das amostras em cada um dos tr?s fixadores, embora tenham sido observadas diferen?as qualitativas, particularmente com A. sculptum preservado em ZN. Em contraste, a fixa??o em isopropanol afetou negativamente a capacidade de amplificar as sequ?ncias dos marcadores mitocondriais de A. parvum e tamb?m resultou na amplifica??o inferior (qualitativa), do alvo D-loop com A. sculptum. Esses efeitos foram observados em amostras fixadas por apenas 72 horas. Os efeitos prejudiciais de isopropanol tamb?m foram observados igualmente em amostras extra?das usando um m?todo de lise alcalina (?HotShot?). As amostras de larvas de A. parvum preservadas em RNAlater durante 30 meses, mostrou uma efic?cia de amplifica??o de 100% nos ensaios para ITS2 e COI, independentemente do m?todo de extra??o usado. Sequ?ncias mitocondriais s?o um componente central da maioria das pesquisas moleculares com carrapatos. Os resultados deste estudo indicam que isopropanol deve ser evitado como um fixador para fases imaturas de carrapatos. Em vez disso, o uso de RNAlater ? recomendado, a fim de permitir a recupera??o consistente de mtDNA amplific?ve

Submitted by ANALORENA CIFUENTES RINCÓN null (lorenacifuentesmvz@gmail.com) on 2016-12-05T19:02:32Z No. of bitstreams: 2 Dissertação Lorena (Versão final)2.pdf: 4872200 bytes, checksum: 6e585a9162115d6ddb21dc9ffd88b0ee (MD5) Dissertação Lorena (Versão final)2.pdf: 4872200 bytes, checksum: 6e585a9162115d6ddb21dc9ffd88b0ee (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-06T15:42:33Z (GMT) No. of bitstreams: 1 cifuentesrincon_a_me_jabo.pdf: 4872200 bytes, checksum: 6e585a9162115d6ddb21dc9ffd88b0ee (MD5) / Made available in DSpace on 2016-12-06T15:42:33Z (GMT). No. of bitstreams: 1 cifuentesrincon_a_me_jabo.pdf: 4872200 bytes, checksum: 6e585a9162115d6ddb21dc9ffd88b0ee (MD5) Previous issue date: 2016-08-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A espécie Mazama americana (veado-mateiro) é conhecida como a maior espécie pertencente ao gênero Mazama, e também um dos cervídeos mais abundantes e amplamente distribuídos na floresta Neotropical. A espécie já passou por diferentes classificações taxonômicas ao longo do tempo devido a potencial existência de diversas espécies dentro do que hoje é conhecido como M. americana. É considerada um complexo de espécies crípticas, pois apresenta alta taxa de paralelismo morfológico entre indivíduos com uma variação cromossômica coerente em termos geográficos, sugerindo a existência de unidades evolutivamente distintas. Assim, o objetivo do trabalho foi propor um neótipo para M. americana a partir da caracterização morfológica, citogenética e molecular de um topótipo, no sentido de fazer uma descrição emendada da espécie a partir de uma visão integrativa, permitindo assim a comparação com outros padrões já descritos na literatura considerados M. americana. Para tanto, um indivíduo foi coletado na localidade tipo da espécie (Guiana Francesa), e caracterizado por técnicas de morfologia tradicional (medidas cranianas, coloração da pele, biometria corporal) e morfometria geométrica, assim como por análises citogenéticas (banda C, banda G, coloração Ag-NOR, coloração convencional de Giemsa) e moleculares (análises filogenéticas de genes mitocondriais e nucleares). Os resultados corroboram evidências da existência de um complexo de espécies dentro do que hoje se considera M. americana, já que, segundo os seus padrões citogenéticos, o topótipo não se enquadra em nenhuma variante da espécie até agora conhecida. Molecularmente, as análises indicam a existência de pelo menos duas espécies diferentes dentro deste táxon. A similaridade morfológica é clara dentro de todas as variantes, comprovando mais uma vez que as unidades taxonômicas do veado-mateiro são muito difíceis de se diferenciar só pelos caracteres morfológicos. Deste modo, no presente trabalho, é proposto um neótipo o qual é o ponto de partida para a descrição de novas espécies e possível mudança completa na nomenclatura do gênero Mazama. / The red brocket deer species, Mazama americana, is known as the largest species of the genus Mazama, and also one of the most abundant deer and widely distributed in Neotropical forests. This species has undergone different taxonomic classifications over time due to the potential existence of several species within what is currently known as M. americana, which is considered as a complex of cryptic species. Individuals included in this species show a high morphological convergence between them with a consistent chromosomal variation in geographical terms, suggesting the existence of different evolutionary units. The objective of this study, therefore, was to obtain the morphological, cytogenetic and molecular profile of a M. americana topotype to gain an exact description of the species and be able to compare them with other standards already described in the literature for M. americana. For this purpose, an individual was collected at the type locality of this species (French Guiana) and characterized by traditional morphological techniques (cranial measurements, skin color, body biometrics), geometric morphometry, cytogenetic analysis (Band C, Band G Ag-NOR staining, conventional Giemsa staining) as well as molecular analysis (phylogenetic analyzes of mitochondrial and nuclear genes). Among highlights of the results, cytogenetic analysis showed a pattern that does not fit into any of the Mazama americana variants until now known. Similarly, the molecular analysis revealed the existence of, at least, two different species within this taxon, being clear the morphological similarity in all variants, proving once again that the red brocket variants are impossible to differentiate only by morphological characters. All these results, therefore, corroborate the existence of several species within M. americana species, contributing largely to the taxonomy of this species, as well as to the description of new species within the genus Mazama and the generation of a new holotype.

The aim of this thesis is to investigate how discussions in archaeology and genetics influence the consensus on human origins and migrations in the South Pacific. By analyzing the genetic research on chicken- and sweet potato-DNA, I present a general overview of how genetics and archaeology shape the understanding of how humans have colonized the Pacific. By deconstructing a review on how the Pacific was settled based on aDNA, I analyze a geneticist’s perspective on archaeological problems. Through this analysis I suggest how archaeology should be approached on a theoretical level in order to be relevant in understanding human migrations in the Pacific. I propose that archaeology’s strength lie in interpreting material culture through an agency perspective in order to reach a dimension not obtainable by biological perspectives / Syftet med denna uppsats är att undersöka hur diskussioner i arkeologi och genetik påverkar hur vi ser på mänskliga migrationer i Oceanien. Genom att analysera den genetiska forskning som gjorts på kyckling och sötpotatis ges en övergripande bild av hur genetik och arkeologi formar den förståelse som finns för hur människan koloniserat Söderhavet. Genom att dekonstruera en sammanställning av den genetiska forskning som gjorts på mänskligt DNA i Oceanien analyseras en genetikers synsätt på arkeologiska problemställningar. Genom analysen i denna uppsats föreslår jag hur arkeologi borde arbeta på ett teoretiskt plan för att vara relevant i hur vi förstår Oceaniens migrationsmönster. Jag föreslår att arkeologins styrka ligger i att tolka den materiella kulturen genom ett agency-perspektiv för att komma åt en dimension av migrationsproblematiken som inte går att nås genom biologiska perspektiv.

Two of the most pervasive threats to species biodiversity are invasive species and habitat loss and degradation. Invasive species are often relatively insensitive to disturbance and many expand their range into disturbed and fragmented habitats. This dissertation uses an interdisciplinary approach to investigate how anthropogenic habitat disturbance is precipitating a range expansion in an invasive toad species, Bufo nebulifer, which is driving a decline in its native congener, B. fowleri. I employed a remote sensing and GIS study using historical data to compare changes in the two species distributions and habitat changes, a molecular genetic study to identify interspecific hybrids and their potential effects on the parental species, and an experimental ecology study to look at the effects of competition and predation on the two species. The results of the landscape level analyses of species' distributional changes in different disturbance levels showed that both species' distributions have changed significantly. The distributions of the two species are inversely affected by habitat disturbance; the distribution of B. fowleri in highly degraded habitat has contracted while the expansion of B. nebulifer increased substantially. The molecular genetic study successfully demonstrated the use of nuclear and mitochondrial markers to identify cryptic hybrids and their maternal lineage. Three hybrids were detected using nuclear introns and a morphologically cryptic hybrid was identified using mitochondrial DNA as the progeny of a cross that was previously thought to be inviable. Although relatively few hybrids were currently found, the identification of a cryptic hybrid implies that the rate of historical hybridization may have been drastically underestimated. Ecological studies showed that competition with B. nebulifer tadpoles had a negative effect on both body size measures and survival to metamorphosis for B. fowleri tadpoles. The addition of predators to experiment did not favor the survival of B. fowleri over B. nebulifer. Bufo fowleri's inability to compete with its invasive congener could be a driving mechanism for the decline of B. fowleri and the expansion of B. nebulifer. The methods discussed in this dissertation offer promising and practical new approaches for evaluating and managing changes in the distribution of species of conservation concern.

Resumo: Com os objetivos de elucidar a origem genética do suíno casco-de-burro e de contribuir para sua conservação, realizou-se uma caracterização genética em 110 animais, oriundos das regiões Nordeste (NE), Centro-Oeste (CO) e Sudeste (SE), usando-se duas classes de marcador molecular e análises citogenéticas. Foram encontrados 13 haplótipos mitocondriais entre os cascos-de-burro, sendo que apenas um foi comum às três subpopulações (NE, CO e SE). O valor médio da diversidade haplotípica e o da nucleotídica na população total foram 0,61 e 0,05 respectivamente. Por meio do DNA mitocondrial, as subpopulações de casco-de-burro apresentaram menor distância genética da população da raça portuguesa bísara. No entanto o haplótipo mais frequente nos cascos-de-burro e o único comum a todas as subpopulações pertence à raça ibérica. A variabilidade genética média obtida por meio dos 25 microssatélites na população total foi: número de alelo = 9,8; conteúdo de informação polimórfica = 0,73; heterozigose esperada = 0,69; heterozigose observada = 0,58; consaguinidade (Fis) = 0,15; e apenas seis loci apresentaram-se em equilíbrio de Hardy-Weinberg. Considerando-se a divisão da população nas três subpopulações – que, por meio do DNA nuclear, estiveram mais próximas da população duroc e da bísara –, os valores observados para os índices de fixação foram: 0,10 para Fis, 0,09 para Fst e 0,18 para Fit. Os cascos-de-burro possuem o número diploide 2n = 38, não sendo verificado miscigenação com o javali. Os resultados demonstram origem genética ibérica para os cascos-de-burro, com posterior introgressão alélica das raças internacionais importadas no século passado / Abstract: With the purpose of elucidating the genetic origin of Brazilian Mulefoot pigs and to contribute to their conservation, 110 animals from Northeast (NE), Central- West (CW), and Southeast (SE) Brazil were characterized using two molecular marker classes and cytogenetic analysis. A total of 13 mitochondrial haplotypes was found, but only one was common to the three subpopulations (NE, CW, SE) of Brazilian Mulefoot pigs. The total population presented mean haplotype and nucleotide diversity values of 0.61 and 0.05, respectively. Mitochondrial DNA analysis showed that the Brazilian Mulefoot pig subpopulations presented the shortest genetic distance from the Portuguese Bísara breed. However, the most frequent haplotype found in the Brazilian Mulefoot population, and the only one common to all subpopulations belongs to the Ibérica breed. The mean genetic variability of the total population, obtained using 25 microsatellites, was: allele number = 9.8; polymorphic information content = 0.73; expected heterozygosity = 0.69; observed heterozygosity = 0.58; inbreeding = 0.15; and only six loci displayed Hardy-Weinberg equilibrium. Considering the three studied subpopulations – which were closer to the Bísara and Duroc populations, based on nuclear DNA – the values observed for the fixation indexes were: 0.09 for Fis, 0.10 for Fst, and 0.18 for Fit. Brazilian Mulefoot pigs have a diploid number of 2n = 38, which indicates that there is no interbreeding with wild boars. The results demonstrate that the genetic origin of Brazilian Mulefoot pigs is Iberian, with later allele introgression from foreign breeds imported during the 20th century / Orientador: Jeffrey Frederico Lui / Coorientador:Carlos Manuel M. Santos Fonseca / Coorientador: Maria Aparecida Cassiano Lara / Banca: Sandra Aidar de Queiroz / Banca: Vera Fernanda Martins Hossepian de Lima / Banca: Samuel Rezende Paiva / Banca: Eucleia Primo Betioli Contel / Doutor

<p>I use morphological, vocal, molecular, behavioural, ecological and distributional data to re-evaluate the systematics of three passerine bird groups, the <i>Mirafraassamica </i>complex (bush-larks), the genus <i>Seicercus</i> ("spectacled-warblers"; with emphasis on the the <i>S. burkii</i> complex) and the genus <i>Motacilla</i> (wagtails). Two new species are described: <i>Seicercus soror</i> and <i>Motacilla samveasnae</i>. I propose that the polytypic species <i>M. assamica</i> should be treated as four separate species: <i>M. assamica</i>, <i>M. affinis</i>, <i>M. microptera</i> and <i>M. marionae</i> (it is also remarked that the proper name of the latter is <i>M. erythrocephala</i>). That is primarily supported by vocalisations and mitochondrial DNA. The latter data set also suggests that <i>M. assamica</i> sensu lato is paraphyletic, since <i>M. erythroptera</i>, which is always treated as a separate species, is nested within the <i>M. assamica</i> complex. I propose that the polytypic species <i>S. burkii</i> comprises six sibling species. Some of these are found to breed sympatrically, although mainly or entirely segregated altitudinally. Mitochondrial DNA suggests that the <i>S. burkii</i> complex is non-monophyletic, and also that the divergence of the different taxa is much older than indicated by morphological and vocal data. According to the molecular phylogeny, both the genera <i>Seicercus</i> and its assumed sister genus <i>Phylloscopus</i> are paraphyletic. That is corroborated by independent data. The phylogenetic study of the genus <i>Motacilla</i> reveals incongruence between mitochondrial DNA, nuclear DNA and non-molecular data. I conclude that the nuclear gene tree reflects the organismal phylogeny more faithfully than the mitochondrial gene tree. The latter is likely to have been affected by introgressive hybridisation, possibly also stochastic lineage sorting. The most remarkable result that is strongly supported by both mitochondrial and nuclear DNA is that <i>M. flava</i> is non-monophyletic.</p>

<p>DNA sequences from the <i>rps16</i> intron and the <i>psbE-petL</i> spacer from the chloroplast genome, the ribosomal nuclear ITS region, and introns from the low copy nuclear genes <i>RPA2</i>, <i>RPB2</i>, <i>RPD2a</i> and <i>RPD2b</i>, are in different combinations used to infer phylogenetic relationships in <i>Sileneae</i> (<i>Caryophyllaceae</i>). Used in concert, the biparentally inherited nuclear regions are useful to distinguish between paralogy due to allopolyploidy and single gene duplications, respectively, because the latter are not expected to give rise to repeated phylogenetic patterns in potentially unlinked sequence regions. In addition, the sequences resolve previously poorly known relationships in the tribe <i>Sileneae</i>. Several independent losses and incomplete concerted evolution are inferred between the two <i>RPD2</i> paralogues in a subgroup of <i>Silene</i>.</p><p>An allopolyploid origin is suggested for the tetraploid <i>S. aegaea</i>, with the maternal ancestor from the diploid <i>S. pentelica</i> lineage, and the paternal contributor from the diploid <i>S. sedoides</i> lineage.</p><p><i>Silene involucrata</i> originated as an allotetraploid with the diploid lineage of Arctic <i>S. uralensis</i> as cytoplasmic donor and the diploid Siberian/Northeast Asian <i>S. ajanensis</i> lineage as pollen donor. A subsequent allopolyploidization with the <i>S. ajanensis</i> lineage as pollen donor and the tetraploid <i>S. involucrata</i> lineage as cytoplasmic donor resulted in the hexaploid lineage of <i>S. sorensenis sensu lato</i>.</p><p>A monophyletic origin of the North American polyploids is rejected. One lineage consists of tetraploid <i>S. menziesii</i> and its diploid allies. A separate lineage leads to a clade consisting of both diploid and polyploid Arctic, European and Asian taxa in addition to the majority of the North American polyploids. The tetraploid <i>S. californica</i> and the hexaploid <i>S. hookeri</i> are derived from separate allopolyploidization events between these two lineages.</p>