Developmental, morphological and physiological effects of chronic low doses of ionising radiation on plants on earth and in space

Conclusions in the current literature are essentially unanimous regarding what is known about the effects of ionising radiation on plants. It is agreed that acute high-dose effects (primarily from laboratory tests) are well-documented and understood but that the same cannot be said for lower doses. ‘Low-dose’ research is itself contentious until there is sufficient understanding to define what constitutes a ‘low’ dose. Studies using lower doses typically (but with some notable exceptions) feature dose-effect relationships at doses to plants orders of magnitude lower than high-dose studies (i.e. they examine doses measured in μGy-mGy as opposed to kGy-Gy). A compilation of data reported here emphasises not only the lack of studies that utilise low and environmentally-realistic doses of ionising radiation (μGy-mGy) but also the particular lack of such experiments that used a controlled environment. Additionally, the compilation revealed a lack of transgenerational studies. In research reported here five generations of Arabidopsis thaliana were grown in soil contaminated with Cs-137 at low field-relevant dose rates (35 μGy/h). The developmental stages defined by a long-established phenotypic model (Boyes et al., 2001) were charted over entire life cycles. A detailed analysis of leaf morphology in generations of radioactively-exposed plants was undertaken with semi-autonomous image analysis software (LAMINA- Leaf shApe deterMINAtion). The same leaves were analysed for antioxidant changes via an assay for glutathione. No transgenerational trends were identified in any of the endpoints examined. Significant changes were detected in development and morphology in some treatments in some generations and are in line with stochastic effects expected to occur over generations in the conditions. No significant differences were found in glutathione concentrations. No significant differences in root length were found between treatments or between generations. Lastly, seeds from two species of crop plants Eruca sativa and Solanum lycopersicum (rocket and tomato) that had flown aboard the International Space Station, and that had received a cosmic radiation dose in low-Earth orbit of similar magnitude to the Arabidopsis, were grown in Cs-137 contaminated soil. Previous exposure to cosmic radiation as seeds did not change uptake characteristics of either species or total growth or root length. In conclusion, while some effects have been observed, nothing was found to suggest that doses of ionising radiation at ‘low’ doses has a significant, negative impact on generations and populations of plants throughout these experiments. This contrasts with many reports from the field at similar dose rates and provides evidence that: a) current radioprotection limits are satisfactory at protecting plant populations, b) many effects on plants of low-dose radiation reported from the field are due to either past exposures to populations or other environmental factors and c) what constitutes a low dose of ionising radiation to plants could be redefined

Cell Nuclear Morphology Analysis Using 3D Shape Modeling, Machine Learning and Visual Analytics

Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with cell differentiation, development, proliferation, and disease. Changes in the nuclear form are associated with reorganization of chromatin architecture related to altered functional properties such as gene regulation and expression. Understanding these processes through quantitative analysis of morphological changes is important not only for investigating nuclear organization, but also has clinical implications, for example, in detection and treatment of pathological conditions such as cancer. While efforts have been made to characterize nuclear shapes in two or pseudo-three dimensions, several studies have demonstrated that three dimensional (3D) representations provide better nuclear shape description, in part due to the high variability of nuclear morphologies. 3D shape descriptors that permit robust morphological analysis and facilitate human interpretation are still under active investigation. A few methods have been proposed to classify nuclear morphologies in 3D, however, there is a lack of publicly available 3D data for the evaluation and comparison of such algorithms. There is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analyses. In this work, we address a number of these existing limitations. First, we present a largest publicly available, to-date, 3D microscopy imaging dataset for cell nuclear morphology analysis and classification. We provide a detailed description of the image analysis protocol, from segmentation to baseline evaluation of a number of popular classification algorithms using 2D and 3D voxel-based morphometric measures. We proposed a specific cross-validation scheme that accounts for possible batch effects in data. Second, we propose a new technique that combines mathematical modeling, machine learning, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. Employing robust and smooth surface reconstruction methods to accurately approximate 3D object boundary enables the establishment of homologies between different biological shapes. Then, we compute geometric morphological measures characterizing the form of cell nuclei and nucleoli. We combine these methods into a highly parallel computational pipeline workflow for automated morphological analysis of thousands of nuclei and nucleoli in 3D. We also describe the use of visual analytics and deep learning techniques for the analysis of nuclear morphology data. Third, we evaluate proposed methods for 3D surface morphometric analysis of our data. We improved the performance of morphological classification between epithelial vs mesenchymal human prostate cancer cells compared to the previously reported results due to the more accurate shape representation and the use of combined nuclear and nucleolar morphometry. We confirmed previously reported relevant morphological characteristics, and also reported new features that can provide insight in the underlying biological mechanisms of pathology of prostate cancer. We also assessed nuclear morphology changes associated with chromatin remodeling in drug-induced cellular reprogramming. We computed temporal trajectories reflecting morphological differences in astroglial cell sub-populations administered with 2 different treatments vs controls. We described specific changes in nuclear morphology that are characteristic of chromatin re-organization under each treatment, which previously has been only tentatively hypothesized in literature. Our approach demonstrated high classification performance on each of 3 different cell lines and reported the most salient morphometric characteristics. We conclude with the discussion of the potential impact of method development in nuclear morphology analysis on clinical decision-making and fundamental investigation of 3D nuclear architecture. We consider some open problems and future trends in this field.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147598/1/akalinin_1.pd

Virtual Morphology and Evolutionary Morphometrics in the new millenium.

EDITED VOLUME; abstract N/

A novel image analysis approach to characterise the effects of dietary components on intestinal morphology and immune system in Atlantic salmon

The intestinal tract of salmonids provides a dynamic interface that not only mediates nutrient uptake but also functions as the first line of defence against ingested pathogens. Exposure of the immune system to beneficial microorganisms and different dietary immunostimulants via the intestine has been shown to prime the immune system and help in the development of immune competence. Furthermore, the morphology and function of teleostean intestines are known to respond to feed components and to ingested and resident bacterial communities. Histological appraisal is still generally considered to be the gold standard for sensitive assessment of the effects of such dietary modulation. The aim of the present study was to improve understanding of salmonid intestinal function, structure and dynamics and to use the knowledge gained to develop a model for analysis, which would allow intestinal health to be assessed with respect to different intestinal communities and feed components. Virtual histology, the process of assessing digital images of histological slides, is gaining momentum as an approach to supplement traditional histological evaluation methodologies and at the same time, image analysis of digitised histological sections provides a practical means for quantifiable assessment of structural and functional changes in tissues, being both objective and reproducible. This project focused on the development of a rapid, practical analytical methodology based on advanced image analysis, that was able to measure and characterise a range of features of the intestinal histology of Atlantic salmon in a quantitative manner. In the first research chapter, the development of a novel histological assessment system based upon advanced image analysis was described, this being developed with the help of a soybean feed model known to induce enteropathy in Atlantic salmon. This tool targeted the evaluation of the extent of morphological changes occurring in the distal intestine of Atlantic salmon following dietary modulation. The final analytical methodology arrived at, could be conducted with minimal user-interaction, allowing rapid and objective assessment of 12 continuous variables per histological frame analysed. The processing time required for each histological frame was roughly 20-25 min, which greatly improved the efficiency of conducting such a quantitative assessment with respect to the time taken for a subjective semi-quantitative alternative approach. Significant agreement between the fully automated and the manual morphometric image segmentation was achieved, however, the strength of this quantitative approach was enhanced by the employment of interactive procedures, which enabled the operator / observer to rectify preceding automated segmentation steps, and account for the specimen’s variations. Results indicated that image analysis provided a viable alternative to a pathologist’s manual scoring, being more practical and time-efficient. In the second research chapter, feeding Atlantic salmon a high inclusion level of unrefined SBM (25 %) produced an inflammatory response in the distal intestine as previously described by other authors. The model feed trial successfully generated differentiable states, although these were not, for the most part, systemically differentiable through the majority of standard immunological procedures used, being only detectable morphologically. Quantitation of morphometric parameters associated with histological sections using the newly developed image analysis tool successfully allowed identification of major morphological changes. Image analysis was thus shown to provide a powerful tool for describing the histomorphological structure of Atlantic salmon distal intestine. In turn, the semi-automated image analysis methods were able to distinguish normal intestinal mucosa from those affected by enteritis. While individual parameters were less discriminatory, use of multivariate techniques allowed better discrimination of states and is likely to prove the most productive approach in further studies. Work described in the third research chapter sought to validate the semi-automated image analysis system to establish that it was measuring the parameters it was purported to be measuring, and to provide reassurance that it could reliably measure pre-determined features. This study, using the same sections for semi-quantitative and quantitative analyses, demonstrated that the quantitative indices performed well when compared to analogous semi-quantitative descriptive parameters of assessment for enteritis prognosis. The excellent reproducibility and accuracy performance levels indicated that the image analysis system was a useful and reliable morphometric method for the quantification of SB-induced enteritis in salmon. Other characteristics such as rapidity, simplicity and adaptability favour this method for image analysis, and are particularly useful where less experienced interpreters are performing the analysis. The work described in the fourth research chapter characterised changes in the morphology of the intestinal epithelial cells occurring as a result of dietary modulation and aspects of inflammatory infiltration, using a selected panel of enzyme and IHC markers. To accomplish this, image analysis techniques were used to evaluate and systematically optimise a quantitative immunolabelling assessment protocol. Digital computer-assisted quantification of labelling for cell proliferation and regeneration; programmed cell death or apoptosis; EGCs and t-cell like infiltrates; mobilisation of stress-related protein regenerative processes and facilitation of nutrient uptake and ion transport provided encouraging results. Through the description of the intestinal cellular responses at a molecular level, such IHC expression profiling further characterised the inflammatory reaction generated by the enteropathic diet. In addition, a number of potential diagnostic parameters were described for fish intestinal health e.g. the relative levels of antigenicity and the spatial distribution of antigens in tissues. Work described in the final research chapter focused on detailed characterisation of intestinal MCs / EGCs in order to try to elucidate their functional role in the intestinal immune responses. Through an understanding of their distribution, composition and ultrastructure, the intention was to better characterise these cells and their functional properties. The general morphology, histochemical characteristics and tissue distribution of these cells were explored in detail using histochemical, IHC and immunogold staining / labelling, visualised using light, confocal and TEM microscopy. Despite these extensive investigations, their physiological function and the content of their granules still remain somewhat obscure, although a role as immunodulatory cells reacting to various exogeneous signals through a finely regulated process and comparable to that causing the degranulation of mammalian MCs is suggested. The histochemical staining properties demonstrated for salmonid MCs / EGCs seem to resemble those of mammalian mucosal mast cells, with both acidophilic and basophilic components in their granules, and a granule content containing neuromodulator / neurotransmitter-peptides such as serotonin, met-enkephalin and substance-p. Consequently, distinguishable bio-chromogenic markers have been identified that are of utility in generating a discriminatory profile for image analysis of such cells

An Integrative Assessment Of The Ex Situ Conservation And Reintroduction Of An Endangered Species Of Pupfish (Cyprinodon bovinus): Investigation Of Genetic, Morphological and Behavioral Variation.

The overarching objective of this thesis was to: (I) synthesize and review the conservation history of an endangered species of pupfish (Cyprinodon bovinus), (II) test for genetic and phenotypic divergence between a captive and wild population, (III) examine the wild population for signs of introgression with its congener, C. variegatus, (IV) evaluate evidence for the exhibition of maladaptive behaviors following the release of captive animals into the wild, and (V) examine how the presence of a putative egg predator (Gambusia nobilis) may affect density-dependent behavior and the future persistence of C. bovinus in the wild. The first chapter illustrated that while there has been a contemporary increase in the number of territorial C. bovinus, there may be unintended consequences of habitat restoration projects on reproductive success. In the second chapter, landmark-based geometric morphometrics revealed considerable morphological divergence in body shape and examination of both neutral and adaptive variation revealed significant levels of genomic divergence as well as evidence for local adaptation, possibly relating to differences in salinity between environments. While the captive population showed higher levels of genetic diversity, the wild population has maintained substantial genetic variation despite its small estimated effective population size. The third chapter revealed that the wild population failed to show substantial evidence of introgression or contemporary hybridization with the congener, C. variegatus, which was demonstrated by a lack of morphological overlap with C. variegatus, distinct genotypic clustering, and high levels of genetic divergence with the conger. The fourth chapter illustrated that following the release of captive C. bovinus into their ancestral habitat, the behavior of the reintroduced population was both quantitatively and qualitatively similar to that of the wild population; the reintroduced captive and wild populations exhibited comparable levels of reproduction, foraging and agnostic behavior. The fifth chapter demonstrated that G. nobilis failed to exert a negative density-dependent effect on C. bovinus reproductive behavior or fecundity

Recipes for calibration and validation of agent-based models in cancer biomedicine

Computational models and simulations are not just appealing because of their intrinsic characteristics across spatiotemporal scales, scalability, and predictive power, but also because the set of problems in cancer biomedicine that can be addressed computationally exceeds the set of those amenable to analytical solutions. Agent-based models and simulations are especially interesting candidates among computational modelling strategies in cancer research due to their capabilities to replicate realistic local and global interaction dynamics at a convenient and relevant scale. Yet, the absence of methods to validate the consistency of the results across scales can hinder adoption by turning fine-tuned models into black boxes. This review compiles relevant literature to explore strategies to leverage high-fidelity simulations of multi-scale, or multi-level, cancer models with a focus on validation approached as simulation calibration. We argue that simulation calibration goes beyond parameter optimization by embedding informative priors to generate plausible parameter configurations across multiple dimensions

Untying Gordian knots: The evolution and biogeography of the large European apomictic polyploid Ranunculus auricomus plant complex

Polyploidie, das Vorhandensein von zwei oder mehr vollständigen Chromosomensätzen, tritt wiederholt über den gesamten Baum des Lebens auf. Bei Pflanzen ist die wirtschaftliche, aber vor allem auch die evolutionäre Bedeutung überwältigend. Polyploidisierungen, wahrscheinlich verbunden mit Schlüsselinnovationen (z.B. die Entwicklung der Gefäßelemente oder des Fruchtblattes), traten in der Evolution der Blütenpflanzen häufig auf. Blütenpflanzen sind die artenreichste Gruppe im Pflanzenreich mit ca. 370,000 Arten und umfassen 30–70% Neopolyploide. Es wird angenommen, dass Polyploidie und Hybridisierung (Allopolyploidie) besonders zur Entstehung von Biotypen mit neuartiger genomischer Zusammensetzung beitragen und damit Schlüsselfaktoren für nachfolgende Artbildungen und Makroevolution sind. Bei Pflanzen sind beide Prozesse häufig mit Apomixis, der Reproduktion über asexuell gebildete Samen, verbunden. Das rätselhafte Phänomen der von Polyploidie und Apomixis begleiteten Artbildung ist jedoch trotz enormer Fortschritte auf dem Gebiet der Genomik noch immer kaum verstanden. Die Frage „Was ist eine Art?“ hat für Evolutionsbiologen höchste Priorität: Arten sind die Grundlage der Biodiversitätsforschung, und die evolutionäre und ökologische Forschung stützt sich auf gut definierte Einheiten. Evolutionär junge Artkomplexe bieten eine einzigartige Möglichkeit die Artbildung bei Pflanzen und deren begleitende Prozesse zu erforschen und zu verstehen. Sie umfassen meist wenige sexuelle Stammarten und zahlreiche polyploide, teilweise apomiktische, hybridogene Derivate. Das Fehlen von Rekombination und Kreuzbestäubung in apomiktischen Linien kann zu einer Vielzahl klonaler Hybridlinien mit fixierten morphologischen und ökologischen Merkmalen führen (Agamospezies). Selbst das Erkennen und Abgrenzen der sexuellen Stammarten ist aufgrund geringer genetischer Divergenz, eventuellen hybridogenen Ursprüngen, stetigem Genfluss und/oder unvollständiger genetischer Auftrennung der Abstammungslinien (ILS) methodisch herausfordernd. Integrative Ansätze, die sowohl genomische als auch morphometrische Daten verwenden, um die jungen Stammarten aufzutrennen, fehlen bisher. Die Biogeographie und Evolution der Artkomplexe ist weitaus komplexer. Apomikten besetzen im Vergleich zu ihren sexuellen Verwandten häufig größere Areale oder sind in nördlicheren Regionen verbreitet, ein Phänomen, das als Geographische Parthenogenese (GP) bezeichnet wird. GP-Muster haben meist einen pleistozänen Kontext. Klimatische Schwankungen in den gemäßigten und borealen Zonen boten häufig Möglichkeiten zur interspezifischen Hybridisierung, was wahrscheinlich auch zur Entstehung von Apomixis auf der Nordhalbkugel geführt hat. Faktoren, die diese Muster erzeugen, werden immer noch kontrovers diskutiert. GP-Muster wurden bisher oft den Vorteilen apomiktischer Populationen aufgrund von (Allo)polyploidie und uniparentaler Fortpflanzung zugeschrieben: Fixierte, hohe Heterozygotie führt zu einer erhöhten Stresstoleranz, und Selbstfertilität bedingt eine bessere Kolonisierungsfähigkeit. Einerseits sind die komplexen Wechselwirkungen von genomweiter Heterozygotie, Ploidie, Reproduktionssmodi (sexuell versus asexuell) und klimatischer Umweltfaktoren auf GP-Muster nicht ausreichend untersucht worden, andererseits wurden potentielle Nachteile sexueller Stammarten aufgrund ihres Fortpflanzungssystems auf Fitness und genetische Vielfalt bisher kaum betrachtet. Schließlich sind neben der Biogeographie die retikulate Evolution und die genomische Zusammensetzung und Evolution junger, großer polyploider Pflanzenartenkomplexe noch nicht detailliert entschlüsselt worden. Neben Herausforderungen, die auf eine hohe Anzahl an Polyploidisierungs- und Hybridisierungsereignissen zurückzuführen sind, werden bioinformatische Analysen oft durch fehlende Informationen zu sexuellen Stammarten, Ploidiegraden und Reproduktionsmodi erschwert. Der europäische, polyploid-apomiktische Ranunculus auricomus (Gold-Hahnenfuß) Pflanzenkomplex ist gut geeignet, um alle aufgeworfenen Fragestellungen zu untersuchen. Der Komplex entstand wahrscheinlich durch unzählige Hybridisierungen weniger sexueller Stammarten. Bisher wurden mehr als 800 morphologisch sehr diverse Agamospezies (Derivate) beschriebenen. Die sexuellen Stammarten werden weniger als 1.0 Millionen Jahren alt geschätzt, und die Agamospezies sind wahrscheinlich noch viel jünger. In meiner Dissertation habe ich unter Verwendung des R. auricomus Komplexes als Modellsystem die bisher wenig verstandenen phylogenetischen, genomischen und biogeographischen Beziehungen junger, polyploider Pflanzengruppen untersucht. Ich habe einen umfassenden theoretischen und bioinformatischen Workflow entwicklelt, beginnend mit der Untersuchung der Evolution der sexuellen Stammarten, über die Entschlüsselung der Reproduktionsmodi und Biogeographie polyploid-apomiktischer Derivate bis hin zur Aufdeckung der retikulaten Ursprünge und Genomzusammensetzung und -evolution des Polyploidkomplexes. Diese Arbeit umfasst 251 Populationen und 87 R. auricomus Taxa europaweit. Die Analysen basieren auf 97,312 genomischen Loci (RADseq), 663 Kerngenen (target enrichment) und 71 Plastidenregionen, und 1,474 Blattploidie-, 4,669 Reproduktions- Samen-, 284 Kreuzungs- (Samenansatz), und 1,593 Morphometrie-Messungen. Phylogenomische Daten basierend auf RADseq, Kerngenen und geometrischer Morphometrie unterstützten die Zusammenlegung der zwölf sexuellen Morphospezies in fünf neu klassifizierte Stammarten. Diese Arten stellen klar unterscheidbare genetische Hauptlinien oder Cluster dar, die sowohl geographisch gut isoliert als auch morphologisch klar differenziert sind: R. cassubicifolius s.l., R. envalirensis s.l., R. flabellifolius, R. marsicus und R. notabilis s.l. Enorme retikulate Beziehungen innerhalb der Kladen, die nicht-vorhandene geographische Isolation und das Fehlen markanter morphologischer Merkmale haben zu diesem taxonomischen Konzept geführt. Allopatrische Artbildungsereignisse fanden interessanterweise vor ca. 0.83–0.58 Millionen Jahren während enormer klimatischer Schwankungen statt und wurden wahrscheinlich durch Vikarianzprozesse aus einer weit verbreiteten europäischen Stammart ausgelöst. Darüber hinaus wurde die neue Umschreibung der sexuellen Stammarten durch Populationskreuzungsexperimente unterstützt. Kreuzungen zeigten neben Inzuchtdepression, Auszuchtvorteilen und plötzlicher Selbstkompatibilität auch völlig fehlende Reproduktionsbarrieren zwischen einigen Morphospezies. Darüber hinaus wurden durchflusszytometrische Ploidy- und Reproduktions-, genomweite RADseq- und klimatische Umweltdaten in einer genetisch-informierten Pfadanalyse basierend auf Generalisierten Linearen Gemischten Modellen (GLMMs) kombiniert. Die Analyse hat ein komplexes europäisches GP-Szenario aufgedeckt, in der Diploide im Vergleich zu Polyploiden eine signifikant höhere Sexualität (Prozent sexueller Samen), mehr Blütenblätter (petaloide Nektarblätter) und bis zu dreimal weniger genomweite Heterozygotie zeigten. Die Sexualität war überaschenderweise positiv mit Sonneneinstrahlung und Isothermalität verbunden, und die Heterozygotie zeigte einen positiven Zusammenhang mit der Temperatursaisonalität. Die Ergebnisse stimmen mit der südlichen Verbreitung diploid-sexueller Populationen überein und deuten auf eine höhere Resistenz polyploid-apomiktischer Populationen gegenüber extremeren klimatischen Bedingungen hin. Ein neu entwickelter, multidisziplinärer Workflow, der alle bisherigen Daten einbezieht, deckte zum ersten Mal den weitestgehend allopolyploiden Ursprung und die Genomzusammensetzung und -evolution des R. auricomus Komplexes auf. Die Taxa waren in nur drei bis fünf unterstützten, nord-süd verbreiteten Kladen oder Clustern organisiert, die jeweils meistens diploid-sexuelle Stammarten enthielten. Allopolyploidisierungsereignisse bezogen jeweils zwei bis drei verschiedene, diploid-sexuelle Subgenome ein. Es wurde nur ein autotetraploides Ereignis nachgewiesen. Allotetraploide Genome sind gekennzeichnet durch Subgenomdominanz und einer enormen Evolution nach ihrer Entstehung (z.B. Mendelsche Segregation der Hybridgenerationen, Rückkreuzungen zu Elternarten und Genfluss aufgrund fakultativer Sexualität der Apomikten). Die über 800 Taxa des europäischen R. auricomus-Komplexes sind vermutlich aus vier diploiden Stammarten und eine bisher unbekannte, aktuell wahrscheinlich ausgestorbene Stammart, entstanden. Analysen zeigten auch, dass die Mehrzahl der beschriebenen polyploiden Agamospezies nicht monophyletisch ist und ähnliche Morphotypen wahrscheinlich mehrfach entstanden sind. Eine umfassende taxonomische Überarbeitung des gesamten Komplexes ist daher angebracht. In der Allgemeinen Diskussion kombiniere ich die Ergebnisse meiner Dissertation mit bereits existierenden Pflanzenstudien zur diploid-sexuellen und polyploid-apomiktischen Phylogenetik, Biogeographie und Genomzusammensetzung und -evolution junger Artkomplexe. Ich gebe zudem taxonomische Schlussfolgerungen und erkläre wie Artkomplexe mikro- und makroevolutionäre Prozesse miteinander verbinden. Abschließend gebe ich ein Fazit über die Ergebnisse meiner Dissertation und einen Ausblick für das laufende Forschungsprojekt und der Forschungsdisziplin der polyploiden Phylogenetik.Polyploidy, the presence of two or more full genomic complements, repeatedly occurs across the tree of life. In plants, not only the economic but particularly the evolutionary importance is overwhelming. Polyploidization events, probably connected to key innovations (e.g., vessel elements or the carpel), occurred frequently in the evolutionary history of flowering plants, which are the most species-rich group in the plant kingdom (ca. 370,000 species) and contain 30–70% neopolyploids. Polyploidy and hybridization (i.e., allopolyploidy) are particularly considered to create biotypes with novel genomic compositions and to be key factors for subsequent speciation and macroevolution. In plants, both processes are frequently connected to apomixis, i.e., the reproduction via asexually-formed seeds. However, the enigmatic phenomenon of plant speciation accompanied by polyploidy and apomixis is still poorly understood despite tremendous progress in the field of genomics. The question of “What is a species?” is of highest priority for evolutionary biologists: Species are the fundamental units for biodiversity, and further evolutionary and ecological research relies on well-defined entities. Evolutionarily young plant species complexes offer a unique opportunity to study plant speciation and accompanying processes. They usually comprise a few sexual progenitor species, and numerous polyploid, partly apomictic, hybrid derivatives. In apomictic lineages, the lack of recombination and cross-fertilization can result in numerous clonal lineages with fixed morphological and ecological traits (agamospecies). Nevertheless, even recognizing and delimiting the sexual progenitors of species complexes is methodically challenging due to low genetic divergence, possible hybrid origins, ongoing gene flow, and/or incomplete lineage sorting (ILS). Integrative approaches using both genomic and morphometric data for disentangling the young progenitors are still lacking so far. The biogeography and evolution of those plant complexes is even more challenging. Apomicts frequently occupy larger areas or more northern regions compared to their sexual relatives, a phenomenon called geographical parthenogenesis (GP). GP patterns usually have a Pleistocene context because climatic range shifts in temperate to boreal zones offered frequent opportunities for interspecific hybridization, probably giving rise to apomixis in the Northern Hemisphere. Factors shaping GP patterns are still controversially discussed. GP has been widely attributed to advantages of apomicts caused by polyploidy and uniparental reproduction, i.e., fixed levels of high heterozygosity leading to increased stress tolerance, and self-fertility leading to better colonizing capabilities. On the one hand, complex interactions of genome-wide heterozygosity, ploidy, reproduction mode (sexual versus asexual), and climatic environmental factors shaping GP have not been studied enough. On the other hand, potential disadvantages of sexual progenitors due to their breeding system on fitness and genetic diversity have received even less attention. Finally, alongside biogeography, the reticulate relationships and genome composition and evolution of young, large polyploid plant species complexes have not yet been deciphered comprehensively. Besides challenges attributed to numerous numbers of polyploidization and hybridization events, bioinformatic analyses are also often hampered by missing information on progenitors, ploidy levels, and reproduction modes. The European apomictic polyploid Ranunculus auricomus (goldilock buttercup) plant complex is well-suited to study all the aforementioned issues. The majority of goldilock buttercups probably arose from hybridization of a few sexual progenitors, leading to more than 800 described, morphologically highly diverse agamospecies. Sexuals are estimated to have speciated less than 1.0 million years ago, and agamospecies are probably much younger. In this thesis, using R. auricomus as a model system, I examined the recalcitrant and hitherto poorly understood phylogenetic, genomic, and biogeographical relationships of young polyploid apomictic plant complexes. I developed a comprehensive theoretical and bioinformatic workflow, starting with analyzing the evolution of the sexual progenitor species, continuing with unraveling reproduction modes and biogeography of apomictic polyploids, and ending up with revealing the reticulate origins and genome composition and evolution of the polyploid complex. Spanning up to 251 populations and 87 R. auricomus taxa Europe-wide, this work gathered data of 97,312 genomic loci (RADseq), 663 nuclear genes (target enrichment), and 71 plastid regions, and 1,474 leaf ploidy, 4,669 reproductive seed, 284 reproductive crossing (seed sets), as well as 1,593 geometric morphometric measurements. First of all, phylogenomics based on RADseq, nuclear gene, and geometric morphometric data supported the lumping of the twelve described sexual morphospecies into five newly circumscribed progenitor species. These species represent clearly distinguishable genetic main lineages or clusters, which are both well geographically isolated and morphologically differentiated: R. cassubicifolius s.l., R. envalirensis s.l., R. flabellifolius, R. marsicus, and R. notabilis s.l. Mainly within-clade reticulate relationships, missing geographical isolation, and a lack of distinctive morphological characters led to this taxonomic treatment. Interestingly, allopatric speciation events took place ca. 0.83–0.58 million years ago during a period of severe climatic oscillations, and were probably triggered by vicariance processes of a widespread European forest-understory ancestor. Sexual species re-circumscriptions were additionally supported by population crossing experiments. Besides inbreeding depression, outbreeding benefits, and sudden self-compatibility, crossings also revealed a lack of reproductive barriers among some of the formerly described morphospecies. Moreover, flow cytometric ploidy and reproductive, RADseq, and environmental data were combined into a genetically informed path analysis based on Generalized Linear Mixed Models (GLMMs). The analysis unveiled a complex European GP scenario, whereby diploids compared to polyploids showed significantly higher sexuality (percent of sexual seeds), more petals (petaloid nectary leaves), and up to three times less genome-wide heterozygosity. Surprisingly, sexuality was positively associated with solar radiation and isothermality, and heterozygosity was positively related to temperature seasonality. Results fit the southern distribution of diploid sexuals and suggest a higher resistance of polyploid apomicts to more extreme climatic conditions. Finally, a self-developed, multidisciplinary workflow incorporating all previously gathered data demonstrated, for the first time, the predominantly allopolyploid origin, genome composition, and post-origin genome evolution of the R. auricomus complex. Taxa were organized in only three to five supported, north-south distributed clades or cluster, each usually containing diploid sexual progenitor species. Allopolyploidizations involved two to three different diploid sexual subgenomes per event. Only one autotetraploid event was detected. Allotetraploids were characterized by subgenome dominance and enormous post-origin evolution, i.e., Mendelian segregation of hybrid generations, back-crossing to parents, and/or gene flow due to facultative sexuality of apomicts. Four diploid sexual progenitors and a previously unknown, nowadays extinct progenitor, probably gave rise to the more than 800 taxa of the European R. auricomus complex. Analyses also showed that the majority of analyzed polyploid agamospecies are non-monophyletic and similar morphotypes probably originated multiple times. The lack of monophyly suggests a comprehensive taxonomic revision of the entire complex. In the General Discussion, I combine my thesis results with existing plant studies on diploid sexual and polyploid apomictic phylogenetics, biogeography, and composition and genome evolution of young species complexes. I explain the taxonomic conclusions and how species complexes link micro- and macroevolutionary processes. Finally, I give conclusions of my thesis and an outlook of the project and the field of polyploid phylogenetics.2021-10-2

Adaptations in allopatric populations of Triakis megalopterus isolated by the Benguela Current: steps towards understanding evolutionary processes affecting regional biodiversity

This study was initiated to gain a better understanding of evolution and adaptation of elasmobranchs by investigating how a putative biogeographic barrier, the Benguela Current, had influenced populations of a demersal shark species, Triakis megalopterus. It was hypothesized that the Benguela Current formed a biogeographic barrier in the distribution of T. megalopterus and was responsible for the divergence between South African (SA) and Angolan (AN) populations. Since elasmobranchs are generally characterized by a slow rate of evolutionary change and conservative morphology and life history traits, it was hypothesized that there would be limited genetic, morphological and life history divergence between the populations. Both mtDNA Control Region (mtCR) and microsatellites (nDNA) were used to assess population connectivity and structure of T. megalopterus. The mtCR predominantly showed a northern (Angola, AN, and Namibia, NA) versus southern (Western Cape, WC, and Eastern Cape, EC) Benguela subsystem arrangement. This suggested that the formation of the Benguela Current had an influence on the genetic structure of T. megalopterus during the early Pleistocene. The nDNA, however, showed a distinct transoceanic, Atlantic (AN, NA, WC) versus Indian Ocean (EC) arrangement, and this was attributed to the more recent exposure of the Agulhas Bank and reduced rocky shore habitat during the glaciations of the late Pleistocene. Traditional morphological analyses on full body and tooth morphology were used to assess phenotypic plasticity and/or adaptability of T. megalopterus. A novel method of geometric morphology, with potential for non-lethal application, was developed and tested to examine interpopulation divergence in shape. Traditional morphometrics showed significant divergence between populations and this variation was congruous with the mtCR haplotypes. However, the divergence in the truss variables was not concomitant to the haplotypes and suggested that differences in shape may be attributed to phenotypic plasticity. There was limited divergence in the tooth morphology between populations. The divergence in several morphological characters associated with swimming speed and manoeuvrability may be attributed to both habitat structure and dominant prey in the different biogeographic zones. The diet of T. megalopterus consisted primarily of crustaceans, teleosts and molluscs. The significant variation in the diet between populations suggested a generalist tooth configuration and broad trophic adaptability. There was significant divergence in the interpopulation life history parameters. The AN population had the fastest growth, smallest size at maturity, and shortest longevity. Individuals in the EC population had the youngest age at maturity, while the WC population had the earliest parturition. This divergence may be attributed to the contrasting thermal regimes in the three biogeographic regions and the dissimilar exploitation rates of the three populations. The results of this thesis demonstrated that a combination of the formation of the Benguela Current and sea level change most likely contributed to vicariance of three populations of T. megalopterus. The significant interpopulation morphological and life history divergence appeared to be both phenotypic and genetic, and suggested that contrasting environmental drivers can result in relatively rapid change in elasmobranchs