abstract: The greatest barrier to understanding how life interacts with its environment is the complexity in which biology operates. In this work, I present experimental designs, analysis methods, and visualization techniques to overcome the challenges of deciphering complex biological datasets. First, I examine an iron limitation transcriptome of Synechocystis sp. PCC 6803 using a new methodology. Until now, iron limitation in experiments of Synechocystis sp. PCC 6803 gene expression has been achieved through media chelation. Notably, chelation also reduces the bioavailability of other metals, whereas naturally occurring low iron settings likely result from a lack of iron influx and not as a result of chelation. The overall metabolic trends of previous studies are well-characterized but within those trends is significant variability in single gene expression responses. I compare previous transcriptomics analyses with our protocol that limits the addition of bioavailable iron to growth media to identify consistent gene expression signals resulting from iron limitation. Second, I describe a novel method of improving the reliability of centroid-linkage clustering results. The size and complexity of modern sequencing datasets often prohibit constructing distance matrices, which prevents the use of many common clustering algorithms. Centroid-linkage circumvents the need for a distance matrix, but has the adverse effect of producing input-order dependent results. In this chapter, I describe a method of cluster edge counting across iterated centroid-linkage results and reconstructing aggregate clusters from a ranked edge list without a distance matrix and input-order dependence. Finally, I introduce dendritic heat maps, a new figure type that visualizes heat map responses through expanding and contracting sequence clustering specificities. Heat maps are useful for comparing data across a range of possible states. However, data binning is sensitive to clustering cutoffs which are often arbitrarily introduced by researchers and can substantially change the heat map response of any single data point. With an understanding of how the architectural elements of dendrograms and heat maps affect data visualization, I have integrated their salient features to create a figure type aimed at viewing multiple levels of clustering cutoffs, allowing researchers to better understand the effects of environment on metabolism or phylogenetic lineages. / Dissertation/Thesis / Chapter 2 Excel file of transcriptome responses / Chapter 2 Perl scripts / Chapter 3 Cluster Aggregation Perl script / Chapter 4 Example of the top-down clustering method used to construct dendritic heat maps / Chapter 4Perl scripts and dendritic heat map images / Chapter 4 Perl scripts and dendritic heat map images / Doctoral Dissertation Geological Sciences 2017

Orientador: Claudio de Oliveira / Banca: Guaracy Tadeu Rocha / Banca: Daniela Calcagnotto / Banca: Anderson Luis Alves / Banca: Ricardo Cardoso Benine / Resumo: Os Characiformes são peixes exclusivamente de água doce e encontram-se distribuídos nas Américas e na África, atingindo maior diversidade nas principais drenagens neotropicais. A família Characidae é o grupo mais especioso entre os Characiformes, porém, a relação dessa família com outras famílias é ainda incerta. São conhecidas cerca de 1000 espécies de Characidae das quais cerca de um terço estão distribuídas em 14 subfamílias, e as demais não tem uma posição filogenética clara, sendo incluídas em um grande grupo considerado incertae sedis em Characidae. A subfamília Cheirodontinae compreende cerca de 60 espécies, sendo um grupo de characídeos amplamente distribuídos nas bacias hidrográficas da América do Sul e Central, incluindo espécies trans-andinas. Os gêneros de Cheirodontinae atualmente estão divididos e, três tribos: Cheirodontini, Compsurini e Odontostilbini. No presente estudo, o principal objetivo foi investigar as relações de Cheirodontinae com as subfamílias de Characidae e as relações internas dos membros de Cheirodontinae através da análise de sequências de DNA mitocondrial (16S e Citocromo b) e nuclear (RAG1, RAG2 e Myh6). As análises mostraram que Spintherobolus não pertence à subfamília e Cheirodon stenodon, que era considerado incertae sedis em Characidae, deve fazer parte da mesma. Diversos gêneros apareceram polifiléticos, principalmente Odontostilbe. As espécies trans-andinas e andinas, são as espécies mais antigas da subfamília. As relações observadas nas análises são bastante diferentes das correntemente aceitas para Cheirodontinae e assim é proposta uma nova classificação para o grupo. O gênero Holoshesthes é considerado válido e pertencente a um clado juntamente com o gênero Aphyocheirodon e Acinocheirodon. Odontostilbe forma um clado monofilético com as espécies antes pertencentes à Serrapinnus... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Characiformes are exclusively freshwater fishes and they are found distributed in Americas and Africa, reaching more diversity in the major Neotropical drainages. The family Characidae is the most specious group among characiforms, but the relationships among this family and other families remains unclear. It is known about 1,000 species belonging to Characidae, one third distributed in 14 subfamilies, and the remaining does not have a clear phylogenetic position, and currently are included in a large group considered incertae sedis in Characidae. The subfamily Cheirodontinae comprises about 60 species, being a characid group widely distributed in the South and Central America hydrographic basins, including trans-Andean species. The genera of Cheirodontinae are currently divided in three tribes: Cheirodontini, Compsurini, and Odontostilbini. In the present work, the main goal was investigate the internal relationships of the members of Cheirodontinae through sequencing and analysis of mitochondrial (16S rRNA and Cytochrome b) and nuclear (RAG1, RAG2, and Myh6) genes. These analyses shown that Spintherobolus does not belong to the subfamily and Cheirodon stenodon, which was considered incertae sedis in Characidae, belongs to the same. Several genera are polyphyletic, mainly Odontostilbe. The trans-Andean and Andean species are the older species of the subfamily. The relationships observed in the analyses are very different of the currently accepted to Cheirodontinae and thereby it is suggested a new classification to the group. The genus Holoshesthes is considered valid and belonging to a clade jointly with the genus Aphyocheirodon and Acinocheirodon. Odontostilbe form a monophyletic clade with the species currently belonging to Serrapinnus, a new species, and Compsura heterura. The tribes Cheirodontini, Compsurini and Odontostilbini are preserved, with different compositions and a new tribe is suggested (Pseudocheirodontini) / Doutor

Esta tese está dividida em três partes: I Filogenética Básica, II Filogenia de Pilocarpinae e III Novidades Taxonômicas em Esenbeckiinae. Parte I Filogenética Básica - fornece uma rápida revisão de alguns dos métodos básicos atualmente em uso na filogenética, envolvendo aspectos teóricos e operacionais, assim como algumas de suas possíveis implicações. O Capítulo 1 discute dois conceitos importantes (grupo e caráter) e interrelacionados, mostra como pode ser construído um modelo estocástico para o tratamento de caractres, enfatizando sua adequação e demonstranto como homologia e, por conseqüência grupos, podem ser adequadamente tratados sob ótica probabilística. O Capítulo 2 apresenta os métodos de construção e otimização de árvores uzando máxima verossimilhnaça e análise bayesiana. Parte II Filogenia de Pilocarpinae - apresenta a filogenia de Pilocarpinae baseada em dados moleculares (espaçadores ITS1, ITS2 e gene 5.8 S do DNA nuclear e espaçador trnG-S do DNA platidial) e a filogenia de Pilocarpus Vahl baseada em dados moleculares (mesmas regiões usadas anteriormente) e morfológicos. O Capítulo 3 apresenta a filogenia em nível genérico da subtribo Pilocarpinae e de gêneros relacionados (Helietta e Balfourodendron ), mostrando que, exceto Esenbeckia , os gêneros tradicionalmente reconhecidos (Metrodorea , Pilocarpus e Raulinoa - monoespecífico) emergem como monofiléticos (embora a subtribo não) e que Balfourodendron e Helietta (ambos da subtribo Pteleinae) possuem relações mais estreitas com parte dos gêneros de Pilocarpinae do que com o gênero-tipo de sua própria subtribo (Pteleinae) e reúnem-se (junto com Esenbeckia , Metrodorea e Raulinoa ) em um clado caracterizado pela presença de inflorescências ramificadas, para o qual foi criada uma subtribo, ficando Pilocarpinae monogenérica; além disso, este capítulo apresenta um protocolo para detecção de burn-in em análises filogenéticas bayesia- nas usando métodos já bem estabelecidos em estudos de convergência de MCMC. Por sua vez, o Capítulo 4 apresenta a filogenia das espécies de Pilocarpus baseada em dados morfológicos e moleculares; essa filogenia, associada a simulações computacionais, é utiliza como base para traçar hipóteses evolutivas sobre os padrões foliares e de estivação da corola no gênero, mostrando como os estados desses caracteres se comportam nas árvores obtidas e quão apropriado é utilizar os diferentes estados como sinapomorfias/homoplasias usando o método MCMC como base e contrastando com o mapeamento com parcimônia, deixando claro que sina- pomorfia/homplasia é mais adequadamente tratada como uma questão de probabilidade. Parte III Novidades Taxonômicas em Esenbeckiinae representa um reflexo das atividades de campo e de análise de material de herbário. No Capítulo 5 é apresentada uma redescrição de E. cowanii Kaastra, espécie anteriormente conhecida apenas da Guiana Francesa e apenas pelo material tipo, cuja morfologia floral era desconhecida e foi encontrada nos Estados do Acre, Mato Grosso, Pará e Rondônia durante as expedições de campo que fiz para a Amazônia; além disso, é proposto um epítipo para o táxon. O Capítulo 6 apresenta a descrição de uma nova espécie de Esenbeckia (embora ainda sem diagnose latina), coletada nos estados do Acre e Rondônia e caracterizada pela posse de brácteas persistentes. / This dissertation is composed of three major parts: I Basic Phylogenetics, II Phylogeny of Pilocarpinae, and III Taxonomic Novelties in Esenbecki- inae. Part I Basic Phylogenetics - provides a mini-review of some basic methods currently used in phylogenetics, covering theroretical and operational issues, and some of their implications as well. In the Chapter 1 I discuss two major concepts in phylogenetics, namely groups and characters, demonstrate how to build an evolutionary model and emphasize the importance of models in phylogenetics. As an outcome, the meanings of character evolution and groups are reviewed and improved under a probabilistic view. Chapter 2 presents an introduction to the very basic methods of tree construction and optimization using maximum likelihood and bayesian methods. Part II Phylogenetics of Pilocarpinae - presents a phylogeny of Pilocarpinae based on molecular data (internal trancribed spacers ITS1, ITS2, gene 5.8 S - from the nuclear DNA , and spacer trnG-S - from the plastidial DNA), and a phylogeny of Pilocarpus based on morphological and molecular (same DNA regions used before) evidence. Chapter 3 presents a generic-level phylogeny of the Pilocarpinae and allied genera (Balfourodendron and Helietta), which supports the monophyly of the traditionally recognized genera (Metrodorea and Pilocarpus , Raulinoa - monospecific), except Esenbeckia (which is included in a polytomy), whe- reas the subtribe itself is not monophyletic; it is also shown that Balfourodendron and Helietta (both from subtribe Pteleinae) are more closely related to some genera of Pilocarpinae than to the type genus of their own subtribe (Pteleinae), and emerge (together with Esenbeckia , Metrodorea , and Raulinoa ) nested within a clade that has multi-axis inflorescences, for which I created a new subtribe, leaving the Pilocarpinae monogeneric; moreover, this Chapter presents a new2 protocol to be used in MCMC diagnosis in phylogenetic studies. In the Chapter 4 , in turn, the phylo- geny of Pilocarpus is investigated based on morphological and molecular data; that phylogeny, combined to computer simulations, is then used to propose evolutionary hypotheses of leaf blade and corolla aestivation patterns, and show how appropriate the use of character states as synapomorphy/homoplasy can be using the MCMC method; additionally the MCMC and parsimony character mapping procedures are compared, and it is shown that synapomorphy/homoplasy is just a matter of probability. Part III Taxonomic Novelties in Esenbeckiinae is clearly a direct result of my field expeditions to the Amazon, and herbarium work. In the Chapter 5 I present a re-description and epitypification of E. cowanii Kaastra, previously known only from the type locality (and by the type specimens) and whose floral morphology was unknown, which I collected in Acre, Mato Grosso, Pará, and Rondônia States during my collecting trips in the Amazon; further, given the poor type material, I propose an epitype for the species. In the Chapter 6 I describe a new species of Esenbeckia (still without latin diagnose), which I collected in Acre and Rondonia states, whose diagnostic feature is the presence of persistent bracts.

HIV in the UK in the 1980s was concentrated within men who have sex with men (MSM) and people who inject drugs (PWID) but heterosexual sex is now the most frequently reported risk behaviour. As these risk groups are associated with different virus populations, this is reflected in the subtype diversification of the UK epidemic, which was historically dominated by subtype B. I have made use of a national database of HIV sequences collected during routine clinical care, which also contains data on age, sex, route of exposure & ethnicity. The 2014 release of the UK HIV Drug Resistance Database contained data from over 60,000 patients. In this thesis, I first describe the development of novel tools that rapidly and automatically identify HIV clusters within phylogenetic trees containing tens of thousands of sequences because they represent transmission chains within the larger infected population. I use these tools to compare the HIV subtype B epidemics in the UK and Switzerland, which had both been described separately but using different approaches. Working with Swiss colleagues, I was able to analyse the epidemics in exactly the same way without having to share sensitive data. I found clustering in the UK to be much higher at relaxed thresholds than in Switzerland (34% vs 16%) indicating that the UK database is more likely to capture transmission chains. Down sampling revealed that this pattern is driven by the larger size of the UK epidemic. At tighter cluster thresholds, the epidemics were very similar. I next use these tools to analyse the spread of emerging subtypes A1, C, D and G in the UK. I found both risk group and cluster size to be predictive of cluster growth, which I tested using simulations and a GLM. Growth of MSM and crossover clusters was significantly higher than expected for subtypes A1 and C, indicating that crossover from heterosexuals to MSM has contributed to their expansion within the UK. Numbers were small for subtypes D and G but the proportion of new diagnoses linking to MSM and crossover clusters was similar to A1 and C, suggesting that the same pattern may be emerging for D and G. I conclude by evaluating the accuracy of a method previously described by our group to generate transmission networks from HIV sequences. The interpretation of clustering patterns from phylogenetic trees is difficult because of the absence of a standardised statistical framework. In contrast, a body of work exists that relates disease transmission to networks. Using large simulated datasets, I developed algorithms which eliminate improbable links. I then reconstructed improved UK transmission networks for subtypes A1, B and C and compare network metrics (such as the degree distribution) between risk groups. Together with other evidence, this thesis demonstrates that the UK HIV epidemic continues to be driven by transmission among MSM. The UK epidemic is no longer compartmentalised and the crossing over of subtypes across risk groups has been facilitated by MSM also having sex with women.

Pseudoscorpions are a diverse lineage of arachnids with a rich history of taxonomic study. However, they remain one of the lesser-known groups of arachnids and many questions about these enigmatic arthropods remain. The present work revises the taxonomy and systematics of the family Neobisiidae in the Southwest, documenting the existence of several new species and a hitherto unknown clade from the Sky Island region of southeastern Arizona. It also describes the venom of a pseudoscorpion for the first time, through comparative transcriptomic studies. Seven new species are described and assigned to the genus Globocreagris, extending the known range of this genus from California into Arizona, Oregon, and Washington. The monophyly of the subfamily Neobisiinae was tested using two genes (COI and 28S). Molecular phylogenetic analysis of both genes and the pattern of trichobothrial placement on the chelae supports removing Parobisium from the subfamily Neobisiinae, and placing it within the subfamily Microcreagrinae, a reassignment here made. Although it has long been known that most pseudoscorpions possess venom glands in their pincers which they use to subdue their arthropod prey, the components of the venom have never been identified. Using comparative transcriptomics from the pedipalps of Globocreagris the first putative venom proteins in pseudoscorpions were identified. Putative venom proteins include astacin-like metalloproteases, chitinases, cysteine-rich secretory proteins, Kunitz-type serine protease inhibitors, phospholipase A2, and scorpion La1-like peptides.

Geometric morphometrics, the science about the study of shape, has developed much in the last twenty years. In this thesis I first study the reliability of the phylogenies built using geometric morphometrics. The effect of different evolutionary models, branch-length combinations, dimensionality and degrees of integration is explored using computer simulations. Unfortunately in the most common situations (presence of stabilizing selection, short distance between internal nodes and presence of integration) the reliability of the phylogenies is very low. Different empirical studies are analysed to estimate the degree of evolutionary integration usually found in nature. This gives an idea about how powerful the effect of integration is over the reliability of the phylogenies in empirical studies. Evolutionary integration is studied looking at the decrease of variance in the principal components of the tangent shape space using the independent contrasts of shape. The results suggest that empirical data usually show strong degrees of integration in most of the organisms and structures analysed. These are bad news, since strong degree of integration has devastating effects over the phylogenetic reliability, as suggested by our simulations. However, we also propose the existence of other theoretical situations in which strong integration may not translate into convergence between species, like perpendicular orientation of the integration patterns or big total variance relative to the distance between species in the shape space. Finally, geometric morphometrics is applied to the study of the evolution of shape in proteins. There are reasons to think that, because of their modular nature and huge dimensionality, proteins may show different patterns of evolutionary integration. Unfortunately, proteins also show strong functional demands, which influence their evolution and that cause strong integration patterns. Integration is then confirmed as a widespread property in the evolution of shape, which causes poor phylogenetic estimates.

Temnospondyls are the most species-rich group of early amphibians, but species-level phylogenetic analyses of this large clade have so far only incompletely sampled the group. This study represents the largest and most comprehensive species-level phylogenetic study of Temnospondyli, sampling 99 taxa for 297 morphological characters from all seven continents through nearly 170 million years of their evolutionary history. Results of this analysis support the monophyly of several clades. Phylogenetic definitions are updated and three new clades names are proposed: Eutemnospondyli, Neostereospondyli, and Latipalata. Major splits within temnospondyl evolution are recovered at the base of Eutemnospondyli (Euskelia and Limnarchia) and Neostereospondyli (Capitosauria and Trematosauria). Archegosauriodea is recovered within Euskelia. Dendrerpeton is recovered as the immediate sister taxon of Dissorophoidea, not Eryopoidea. This arrangement suggests that for subclade-level analyses of dissorophoids, which bear on the `Temnospondyl Hypothesis' for a putative origin of Lissamphibia within dissorophoids, the convention of rooting on Dendrerpeton and including eyropoids in the ingroup should be re-evaluated in light of the new temnospondyl topology. Study of the tempo and mode of evolution within temnospondyl amphibians has been limited in the past by the availability of a clade-wide, species-level phylogenetic analysis. The phylogenetic dataset generated by this study has allowed for investigation into rates of origination and extinction amongst this long-lived group at a scale not previously available for exploration. Extinction rate and origination rate, when calculated strictly from stratigraphic data, showed a high correlation with the number of sampled localities, indicating a strong influence on this evolutionary signal by sampling and rock record biases. But when rates were augmented with phylogenetic data, four periods of increased lineage origination are discernible from the Pennsylvanian to the Early Triassic. The largest of these origination events coincides with the Permo-Triassic mass extinction, indicating that amphibians were taking advantage of favorable conditions during the largest biological crisis in the Phanerozoic record. Temnospondyl amphibians are the second most abundant fossil vertebrates in the Permo-Triassic Karoo Basin of South Africa. Paleohistological investigation of these amphibians was hampered by small sample size and taxa available for sampling. Incorporation of paleohistologic data from other analyses helped to alleviate this problem; however, Temnospondyli remains under sampled in paleohistological analyses. Results show cyclic growth and a lifespan of thirty years or more in basal stereospondyls, convergence to sustained, non-cyclic growth in terrestrial temnospondyls, support findings based on gross morphology that Lydekkerina is a terrestrial stereospondyl, and suggest that ribs are a viable source of skeletochronologic information in temnospondyls and should serve as preferred material when proximal limb diaphyses are not available. Sustained, azonal growth in Micropholis is unlike that of Apateon or extant caudatans, suggesting a possible adaptation to local conditions in the earliest Triassic of Gondwana.

Soil fungi release up to three-fold more carbon dioxide (CO2) to the atmosphere than human activity through the decomposition of dead organic matter (i.e. litter) in soil. Ecologists have frequently observed a pattern of fungal community assembly during litter decomposition, wherein different fungal taxa dominate different stages of decay in individual ecosystems. However, we still lack a complete understanding of how these diverse fungal communities generate decomposition. My dissertation helps to fill this knowledge gap by elucidating the cross-ecosystem taxonomic patterns of fungal decomposer succession and identifying biological features that correlate with these lineage-specific patterns. I conducted a meta-analysis of fungal decomposer succession in 22 ecosystems to identify the taxonomic patterns that were consistent across ecosystems and test environmental correlates of those patterns. I found a phylogenetic signal to succession and observed that the relative abundance of Ascomycetes was negatively correlated with observed peak decay stage, the relative abundance of Zygomycetes was positively correlated with peak decay stage, and the relative abundance of Basidiomycetes remained relatively constant throughout decay. I also found that plant litter type and climate factors were correlates of peak decay stage. Next, I performed laboratory culture experiments to test two longstanding hypotheses: that innate potential activity of plant cell wall-degrading enzymes and intrinsic growth rate underlie organisms’ peak decay stage. I found that potential cellulase activity and growth rate correlated negatively with peak decay stage and that growth rate explained more than a third of the phylogenetic signal to peak decay stage. Finally, I completed a comparative genomics study of fungi that have different observed decay stages. I found that protein domains related to plant C-degrading enzymes and growth-related biochemical pathways are part of the biology that underlies the succession of fungi and that transcriptional regulation and stress response may also underlie succession. This dissertation generates novel insights into the biology that underlies fungal decomposer community turnover during plant litter decay. This work provides direction for future research into the drivers of the assembly of decomposer communities and has important implications for efforts to incorporate fungal biology into predictive models of decomposition and terrestrial C-cycle dynamics.

Almost all organisms have approximately a 24-hour circadian rhythm that enables them to anticipate their environment’s daily rhythmicity. Anticipation increases their likelihood of success in foraging, reproduction, predation, and other life events. Therefore, a disruption of their endogenous clock results in detrimental physiological consequences that significantly impact organisms’ fitness. Surprisingly, we have found numerous spider species with free-running periods that deviate greatly from 24 hours. Free-running period (FRP) is a standard measurement of the period of an organism’s circadian rhythm found by measuring periodicity of behaviors or physiology under constant conditions (e.g., constant darkness and temperature). So far, these extreme spider FRPs have only been observed in the superfamily Araneoidea, but we have only limited sampling of species outside this clade. Therefore, we want to fill this data gap of non-araneoid spiders to deepen our understanding of the evolution of circadian clocks in spiders. Also, we will observe if significant deviation from 24 hours and wide variation in FRP are common to all spider species or are only characteristics of araneoid spiders. Here, we describe the FRPs of four non-araneoid spider species belonging to the RTA clade: Schizocosa avida, Phidippus audax, Agelenopsis pennsylvanica, and Mecaphesa celer. We detected significant free runs (mean + SD) at p<0.001 using Lomb-Scargle periodograms in three out of the four species: S. avida (23.84 ± 1.03 h); P. audax (22.67 ± 0.36 h); and A. pennsylvanica (23.97 ± 0.32 h). However, M. celer was found to be arrhythmic under constant conditions. These findings of near 24-hour FRPs with low deviation among the RTA species, along with previous data, strongly suggest that extreme FRPs are confined to the Araneoidea clade. Thus, we have phylogenetically localized a major evolutionary change in the circadian system of spiders occurring in the Araneoidea clade, approximately 170 million years ago.

The North American Astereae clade (Asteraceae: Astereae) represents an ecologically important lineage whose species frequently comprise early to mid-successional ecosystems primarily throughout the United States. In the eastern U.S., most species are perennial suffrutescent herbs whereas many in the western U.S. are shrubs or subshrubs, particularly in the Solidagininae s.l. The delimitation of this subtribe, however, has remained unclear as molecular phylogenetics have not resolved whether the Solidagininae s.s. and another clade, the Gutierrezia lineage, collectively form the Solidagininae s.l. To evaluate the relationships among and within these lineages, high-throughput sequencing was employed across the North American Astereae. Highthroughput sequencing was also used to clarify relationships of one taxonomically contentious genus within the Gutierrezia lineage, Euthamia. Additional Euthamia specimens were amplified via polymerase chain reactions for sequencing two loci to increase phylogenetic sampling within the genus. Subsequent species delimitations based on molecular phylogenetics and morphological evaluations from literature were used to model species distributions through ecological niche modeling. Niche comparisons via the R package ‘Humboldt’ further assessed whether the most closely related species differed considerably in their environmental niche occupation. These collectively outlined the distributions of all nine Euthamia species and indicated that hypothetical sister taxa have diverged environmentally for both allopatric and sympatric species.