The adaptive evolution of polar fishes: Structure, function and molecular phylogeny of hemoglobin

emperature affects all molecular processes and is the major determinant of habitat suitability. Whilst there is an increasing understanding of evolutionary adaptation to temperature in some processes, several key questions often remain open about the structure-function relationships associated with protein thermal adaptation. Proteins, such as hemoglobin, are highly sensitive to temperature and therefore, their structural and functional properties mirror the thermal conditions encountered by species during their evolutionary histories. The most stable thermal environments are aquatic; research on polar fishes has provided important insights into the details of thermal adaptation. In polar fishes, the evolution of hemoglobin includes adaptations with implications at the biochemical, physiological and structural levels. Although both are cold, the Northern and Southern polar oceans have very different oceanographic features. In comparison with Antarctic fish of the suborder Notothenioidei, Arctic fish are characterised by higher biodiversity and hemoglobin multiplicity. Within the study of the molecular bases of cold adaptation in fish inhabiting the polar habitats, and taking advantage of the information available on hemoglobin structure and function, the evolutionary history of the α and β globins of Arctic and Antarctic fish hemoglobins has been analysed, under the assumption of the molecular-clock hypothesis

Population genetic and phylogenetic insights into the adaptive radiation of Antarctic notothenioid fishes

Adaptive radiation is the evolution of ecological and phenotypic diversity within a rapidly multiplying lineage, a phenomenon that is considered responsible for a great part of Earthʼs biodiversity. It occurs as a response to ecological opportunity in the form of competitor-free habitat, extinction of antagonists, or the emergence of a key innovation. One of the most spectacular adaptive radiations in the marine realm is the diversification of notothenioid fishes in the freezing waters of Antarctica. This radiation has led to a unique dominance of the Antarctic marine habitat by notothenioids, and is often assumed to result from the key innovation of freeze resistance. Antifreeze glycoproteins are present in blood and tissue of Antarctic notothenioids and enable them to survive in their sub-zero environment. Notothenioids are further characterized by prolonged pelagic larval stages, that have been suggested to contribute to high levels of inter-population gene flow with oceanic currents, which seems to contradict the high speciation rates observed in the notothenioid adaptive radiation. This doctoral work uses molecular tools to investigate the character of gene flow in notothenioids as well as the origin of their diversification. It is demonstrated that larval dispersal is a common agent of long-distance gene flow in many notothenioid species. The key innovation hypothesis is corroborated by an extensive molecular dating of the divergence events of notothenioids and related acanthomorph fishes. New tools for the analysis of microsatellite markers and for Bayesian divergence date estimation are developed

Physiological and Evolutionary Implications of the Pattern of Expression of Oxygen-Binding Hemoproteins in Antarctic Notothenioid Fishes

Antarctic icefish do not express hemoglobin (Hb). Icefishes possess cardiovascular modifications including increased densities of blood vessels, larger ventricles and increased blood volume compared to red-blooded relatives. In addition to delivering oxygen to tissues, Hb degrades nitric oxide (NO), a small signaling molecule. To investigate the mechanism driving development of icefish cardiovascular characteristics, I present and test the hypothesis that loss of Hb results in increased steady-state levels of NO, triggering downstream signaling pathways such as angiogenesis. I measured NO breakdown products, as a proxy for NO, and found that icefish have higher steady-state levels of NO metabolites in their plasma compared to red-blooded notothenioids. Expression of angiogenesis genes (HIF-1α, PHD2, and VEGF) did not differ significantly between red- and white-blooded notothenioids indicating that, while NO levels are higher in adult icefish, angiogenesis is not active. To investigate whether loss of Hb directly can increase NO and stimulate angiogenesis, hematocrit of red-blooded N. coriiceps was severely reduced using the hemolytic agent, phenylhydrazine HCl. Anemic fish exhibited a significantly higher concentration of NO metabolites in the plasma than did control fish, indicating an increase in NO. Expression of HIF-1α, PHD2, and VEGF mRNA was higher in anemic animals compared to control N. coriiceps, suggesting a causative relationship between loss of Hb and induction of angiogenesis that likely is mediated via NO signaling. In addition to lacking Hb, several species of icefish have lost ability to express myoglobin (Mb), an oxygen-binding protein expressed in the ventricle of most notothenioids. Previous studies have indicated Mb expression was lost on four separate occasions during evolution of the icefish family. Sequencing of the Mb gene from D. hunteri identified a duplicated TATA box previously proposed as the mechanism responsible for loss of Mb in C. aceratus. Sequencing of Mb from all 16 species of icefish identified the duplicated TATA box is present in all but two icefish species. The presence of the duplicated TATA box in Mb-expressing icefish suggests that the loss of Mb in C. aceratus and D. hunteri may occur by a mechanism independent of the duplicated TATA box

Collaborative Research: Linkages among Mitochondrial Form, Function and Thermal Tolerance of Antarctic Notothenioid Fishes

Antarctic notothenioid fishes have evolved in the Southern Ocean for 10-14 MY under an unusual set of circumstances. Their characteristics include the complete absence of the circulating oxygen-binding protein, hemoglobin (Hb) within the Channichthyid (Icefish) family of notothenioids. Moreover, some species within the 16 members of this family have also lost the ability to express the oxygen-binding and storage protein, myoglobin (Mb) in cardiac muscle. Our previous work has determined that the loss of Hb and/or Mb is correlated with significant increases in densities of mitochondria within oxidative tissues, and extensive remodeling of these vital organelles. To date, nothing is known about how modifications in mitochondrial architecture of icefishes affect organelle function, or more importantly, how they affect organismal-level physiology. Most critical for Antarctic fishes is that mitochondrial characteristics have been linked to how well ectotherms can withstand increases in temperature. This collaborative research project will address the hypothesis that the unusual mitochondrial architecture of Antarctic Channichthyids has led to changes in function that impact their ability to withstand elevations in temperature. Specifically, the research will (1) determine if the unusual mitochondrial architecture of icefishes affects function and contributes to organismal thermal sensitivity, (2) identify differences in organismal thermal tolerance between red- and white- blooded notothenioids, (3) identify molecular mechanisms regulating changes in mitochondrial structure in icefishes. The results may establish channichthyid icefishes as a sentinel taxon for signaling the impact of global warming on the Southern Ocean. Broad impacts of this project will be realized by participation of high school biology teachers in field work through cooperation with the ARMADA project at the University of Rhode Island, as well as graduate education

Comparative population genetics, larval dispersal and evolutionary aspects of Antarctic fishes (Notothenioidei)

In this thesis, population genetic structures and evolutionary aspects of speciation in notothenioid fishes from the Southern Ocean have been investigated. Special emphasis was given to the role of pelagic larval dispersal on gene flow between geographically separated populations, since notothenioids are characterized by unusually long pelagic larval durations of up to one year, which is assumed to counteract genetic divergence of populations and ultimately allopatric speciation processes. By elucidating the population genetic structures of selected notothenioid species, inferences were made about the level of gene flow between populations. The major goal of this thesis was to compare the population genetic structures of sympatric species within the Atlantic sector of the Southern Ocean to determine factors responsible for regulating gene flow. Species-specific traits, such as larval durations, were expected to result in differences between the observed patterns, while environmental factors, such as oceanographic currents or frontal systems, were assumed to influence multiple species in a similar way. In addition, it has been tested whether the evolution of notothenioids fulfills the criteria of an adaptive radiation, of which only a few examples are known from the marine realm

Vergleichende Populationsgenetik, Larvenverdriftung und evolutionäre Aspekte von Antarktisfischen (Notothenioidei)

In this thesis, population genetic structures and evolutionary aspects of speciation in notothenioid fishes from the Southern Ocean have been investigated. Special emphasis was given to the role of pelagic larval dispersal on gene flow between geographically separated populations, since notothenioids are characterized by unusually long pelagic larval durations of up to one year, which is assumed to counteract genetic divergence of populations and ultimately allopatric speciation processes. By elucidating the population genetic structures of selected notothenioid species, inferences were made about the level of gene flow between populations. The major goal of this thesis was to compare the population genetic structures of sympatric species within the Atlantic sector of the Southern Ocean to determine factors responsible for regulating gene flow. Species-specific traits, such as larval durations, were expected to result in differences between the observed patterns, while environmental factors, such as oceanographic currents or frontal systems, were assumed to influence multiple species in a similar way. In addition, it has been tested whether the evolution of notothenioids fulfills the criteria of an adaptive radiation, of which only a few examples are known from the marine realm.In dieser Doktorarbeit wurden die populationsgenetischen Strukturen und weitere evolutionäre Aspekte von Artbildungsprozessen innerhalb von Antarktisfischen (Notothenioidei) aus dem Südpolarmeer untersucht. Ein besonderer Schwerpunkt war es, den Einfluss pelagischer Larvenverdriftung auf den Genfluss zwischen geografisch getrennten Populationen zu ermitteln, da notothenioide Fische durch ungewöhnlich lange, pelagische Larvenphasen von bis zu über einem Jahr charakterisiert sind, wodurch erhöhter Genfluss einer Auseinanderentwicklung von Populationen und damit auch allopatrischer Artbildung entgegen wirken kann. Die Untersuchung der populationsgenetischen Strukturen erlaubt es, Rückschlüsse auf das Ausmaß des genetischen Austauschs zwischen Populationen zu ziehen. Das Hauptziel dieser Arbeit war es, durch einen Vergleich der genetischen Populationsstrukturen sympatrischer Arten, Faktoren zu identifizieren, die maßgeblich den Genfluss beeinflussen. Dabei wurde angenommen, das artspezifische Merkmale, wie z. B. die Dauer der Larvenphase, in Unterschieden zwischen den beobachteten Populationsstrukturen zum Ausdruck kommen, während Umweltfaktoren, wie ozeanografische Strömungen und Fronten, verschiedene Arten gleichermaßen beeinflussen. Des weiteren wurde untersucht, ob die Evolution der Antarktisfische den Kriterien einer Adaptativen Radiation entspricht, für welche es im marinen Lebensraum bisher nur wenige bekannte Beispiele gibt

Evolution of immune genes in Antarctic fish

A superordem dos Notothenioidei inclui o maior número de representantes the peixes ósseos na plataforma continental da Antártida. As condicões abióticas e bióticas que dominam nesta região do mundo levaram à radiação e à especiação desta ordem. As baixas temperaturas que se deram durante o período do Eoceno Tardio levaram à flutuação da superfície ocupada pela calota glaciar no Oceano Antártico o que levou a uma redução do habitat disponível na plataforma continental. Por sua vez, a falta de habitat foi seguida por um declínio nas espécies de peixes ósseos e a uma alteração na relação predador-presa o que permitiu dispersão e diversificação das espécies que se adaptaram ao novo meio ambiente. Há 25 milhões de anos as condições ambientais tornaram-se mais estáveis criando um ambiente dominado por águas frias, ricas em oxygénio e nutrientes o que promoveu a adaptação radiativa dos Notothenoids. Os novos nichos ecológicos associados a condições ambientais estáveis providenciaram aos Notothenoids os requisitos para se tornarem a ordem de peixes ósseos domiante na plataforma continental da Antártida. Um dos pontos de interesse por esta ordem de peixes ósseos deve-se ao facto de ainda não se perceber qual será o impacto do aquecimento global nestas espécies. As diversas adaptações presentes nas várias espécies desta ordem também representam um fator de peso no que leva ao seu interesse científico. Estas vão desde elevada densidade de mitocôndrias e maior dimensão do miocárdio, à perda de resposta das proteínas de choque térmico, perda de hemoglobina, à evolução de proteínas anticongelantes - as adaptações observadas indicam quão bem se deu a adpatação desta ordem a um ambiente extremo. Entretanto, já foram desenvolvidos trabalhos que evidenciam que o sistema imune destas espécies também foi sujeito a adaptações promovidas pelo meio ambiente da Antártida. De modo geral, o sistema imune permite aos organismos superar perturbações que vão ao encontro da sua homeostase. O estudo do sistema imune em vertebrados, como os peixes, pode revelar aspetos importantes para o entendimento da evolução do sistema imune em vertebrados mais complexos. O objetivo deste trabalho é de estudar a evolução de genes que se enquadram no sistema imune de três espécies de peixes ósseos da região Antártida, Eleginops maclovinus que reside na região sub-polar da Antártida e duas espécies cuja a distribuição é limitada ao oceano da Antártida pela corrente circumpolar, Notothenia coriiceps e Dissostichus mawsoni. Para tal, foram escolhidas cinco famílias de genes que se relacionam com o sistema imune, estas foram os toll-like receptors (TLR), immunoglobulin superfamily (IgSf), phosphoinositide-3-kinase (PIK3), AKT/protein kinase B (AKT3) e as semaphorins (Sema). Os genomas e transcriptomas de 8 espécies de peixes foram obtidos de bases de dados de livre acesso enquanto os genomas e transcriptomas das espécies da Antártida foram proporcionados pelo laboratório do Professor LiangBiao Chen da Shanghai Ocean University. Depois de identificadas as sequências proteícas destas famílias no peixe modelo Danio rerio, procedeu-se a uma pesquisa de similaridade por BLAST entre estas últimas e os transcriptomas das restantes espécies de forma a identificar as sequências potencialmente homólogas. Estas sequências por sua vez foram filtradas de modo a somente reter, para cada família, as sequências que apresentavam um rácio de identidade desejado. Após um alinhamento múltiplo de sequências (MSA), foram escolhidos para cada uma das famílias o melhor modelo evolutivo. Com os MSA de amino ácidos foi possível construir para cada família uma árvore filogenética na qual foi possível identificar genes ortólogos. Com os genes ortólogos foi possível construir uma árvore filogenética de espécies. De seguida, os MSA de amino ácidos foram convertidos para alinhamentos de codões para permitir a estimação da taxa de substituição de nucleótidos nas árvores filogenéticas das espécies, que é dada por v= dN/dS onde dN equivale à taxa de substituição não-sinónima e dS a taxa de substituição sinónima. Com o valor de dS foi então possível resolver a equação T=Ks/2r, em que T representa o tempo de divergência a ser calculado, Ks é taxa de substituição sinónima e r é a taxa de substituição estimada obtida da bibliografia. Os resultados relativos ao número de sequências e a análise filogenética permitiram identificar variabilidade no número de genes encontrados em cada espécie tal como também foi possível observar que, quando presentes, os ortólogos das 11 espécies formavam uma árvore filogenética distincta. Estas observações levaram a estipular, tanto para os Notothenoids como para os restantes taxa analisados, que estas famílias de genes se enquadram num processo evolutivo denomiado de processo de nascimento e morte. As estimações dos tempos de divergência, obtidos nos nós para cada família de genes que representada num maior número de espécies resultaram em tempos de divergência similares ou superiores às estimativas dadas pelos registos fósseis. Por sua vez, os nós que apresentavam um menor número de epécies, indicaram tempos de divergência mais recentes do que os registos fósseis. As cinco famílias de genes nos Nototheniidae indicaram tempos de divergência recentes desde 7.1 milhões de anos (m.y.a) para Sema, 6.2 m.y.a para AKT3, 4.3 m.y.a para IgSf, 4 m.y.a para PIK3 e 2.5 m.y.a para TLR. As diferenças obtidas entre os tempos de divergência das cinco famílias de genes revelam uma possível relevância perante a adaptação dos Nototheniidae ao ambiente antártico, pois as famílias de genes que apresentam funções mais diversas também apresentam tempos de divergência mais antigos (Sema, AKT3, PIK3, IgSf) do que as famílias de genes com funções somente immunologicas (TLR). Finalmente, foi possível observar que estes tempos de divergência incluem-se dentro das estimativas cronológicas dadas para um fenómeno climatérico conhecido como transição climatérica do mioceno médio (MMCT) que LiangBiao Chen da Shanghai Ocean University. Depois de identificadas as sequências proteícas destas famílias no peixe modelo Danio rerio, procedeu-se a uma pesquisa de similaridade por BLAST entre estas últimas e os transcriptomas das restantes espécies de forma a identificar as sequências potencialmente homólogas. Estas sequências por sua vez foram filtradas de modo a somente reter, para cada família, as sequências que apresentavam um rácio de identidade desejado. Após um alinhamento múltiplo de sequências (MSA), foram escolhidos para cada uma das famílias o melhor modelo evolutivo. Com os MSA de amino ácidos foi possível construir para cada família uma árvore filogenética na qual foi possível identificar genes ortólogos. Com os genes ortólogos foi possível construir uma árvore filogenética de espécies. De seguida, os MSA de amino ácidos foram convertidos para alinhamentos de codões para permitir a estimação da taxa de substituição de nucleótidos nas árvores filogenéticas das espécies, que é dada por v= dN/dS onde dN equivale à taxa de substituição não-sinónima e dS a taxa de substituição sinónima. Com o valor de dS foi então possível resolver a equação T=Ks/2r, em que T representa o tempo de divergência a ser calculado, Ks é taxa de substituição sinónima e r é a taxa de substituição estimada obtida da bibliografia. Os resultados relativos ao número de sequências e a análise filogenética permitiram identificar variabilidade no número de genes encontrados em cada espécie tal como também foi possível observar que, quando presentes, os ortólogos das 11 espécies formavam uma árvore filogenética distincta. Estas observações levaram a estipular, tanto para os Notothenoids como para os restantes taxa analisados, que estas famílias de genes se enquadram num processo evolutivo denomiado de processo de nascimento e morte. As estimações dos tempos de divergência, obtidos nos nós para cada família de genes que representada num maior número de espécies resultaram em tempos de divergência similares ou superiores às estimativas dadas pelos registos fósseis. Por sua vez, os nós que apresentavam um menor número de espécies, indicaram tempos de divergência mais recentes do que os registos fósseis. As cinco famílias de genes nos Nototheniidae indicaram tempos de divergência recentes desde 7.1 milhões de anos (m.y.a) para Sema, 6.2 m.y.a para AKT3, 4.3 m.y.a para IgSf, 4 m.y.a para PIK3 e 2.5 m.y.a para TLR. As diferenças obtidas entre os tempos de divergência das cinco famílias de genes revelam uma possível relevância perante a adaptação dos Nototheniidae ao ambiente antártico, pois as famílias de genes que apresentam funções mais diversas também apresentam tempos de divergência mais antigos (Sema, AKT3, PIK3, IgSf) do que as famílias de genes com funções somente imunológicas (TLR). Finalmente, foi possível observar que estes tempos de divergência incluem-se dentro das estimativas cronológicas dadas para um fenómeno climatérico conhecido como transição climatérica do mioceno médio (MMCT) que ocorreu entre os 25-5 m.y.a. Esta correlação levou a considerar que as adaptações do sistema imune dos nototheniidae sejam subsequentes ao MMCT.The Notothenioidei suborder has the largest representation of teleost fish in the Antarctic continental shelf. Their speciation and adaptive radiation was the result of particular abiotic and biotic conditions in the Southern Ocean. During the Late Eocene the cooler temperatures enlarged the ice cover leading to the loss of shelf habitat. This loss of natural environment resulted in a decline of fish diversity followed by the radiation and diversification of those who could adapt to the new conditions. Since then, the stable environment that has governed the Southern Ocean for the last 25 million years promoted the adaption of Notothenoids to cold, oxygen rich waters, allowing them to become the main teleost suborder in the Antarctic shelf habitats. Furthermore, previous work has shown that in Notothenoids immune related genes have undergone adaptations due to their exposure to the environmental conditions of the Antarctic Ocean. Their immune related adaptations give us the opportunity to study the phylogenetic diversification among Notothenioids and other teleosts that adapted to different environments. We studied the evolution of five immune related gene families in eight non- Antarctic vertebrates and three notothenoids, Eleginops maclovinus, Notothenia coriiceps and Dissostichus mawsoni, through phylogenetic analysis and divergence times estimation using nucleic and protein sequences. Genes for five different gene families were obtained from the genome and transcriptome of the investigated species. A possible birth-and-death process may have been identified for the five immune gene families. Furthermore, the divergence times estimated for the nototheniidae indicate that after the Middle Miocene climatic transition, those species relied on the gene families that presented a broader range of functions for their adaption to the Antarctic environment

The adaptation of molecular temperature sensors to cold

The rates of all biological processes depend on temperature. Organisms rely on proteins that convert external temperature into biological signals to determine appropriate physiological or behavioral responses to temperature stimuli. Our understanding of the molecular basis of temperature sensation has grown rapidly for 25 years. However, our knowledge is primarily limited to a few organisms that live in similar thermal niches. To understand how thermosensors evolve, we require a broader knowledge of the function of these proteins at their biological limits. In this dissertation, we examine the evolution and function of putative molecular thermosensors in Antarctic notothenioid fishes, a group of vertebrates that live and diversify below the freezing point of water. We find these fishes express the same proteins that underlie thermosensation in model organisms: transient receptor potential (TRP) channel proteins. Surprisingly, when measured in the lab, these TRP proteins function at temperatures at least 20°C warmer than predicted by habitat temperature. We hypothesize that native cellular conditions shift the channel function, and find that lipid content, phosphorylation, and oxidation all have channel-specific effects on activation threshold. These data and other findings led us to posit that TRP channels may not function in temperature ranges relevant for these vertebrates at the extreme low end of their thermal habitats. Additionally, we examined the evolution of these same proteins on a local species, the Texas leaf cutter ant, Atta texana. A. texana are the most northern distributed Atta species. More recently established populations in north Texas have obligate fungal symbionts that are more cold tolerant compared to ancestral southern populations (Mueller et al., 2011). We found that, like the notothenioids, A. texana have a typical complement of TRP channels but only a subset are expressed in the sensory antennae. We found no difference in TRP channel expression or splicing along a temperature transect, but found these channels are under variable selective pressures in the coding sequences. We found expression of a putative heat sensor, HsTRPA, was correlated with temperature at the time of collection. These data support structural, rather than regulatory, evolution of TRP channels; however, regulatory changes in expression levels may be occurring over shorter timescales.Ecology, Evolution and Behavio