Restoration of plant populations and communities - insights from ecology and genetics for conservation

Biodiversity is defined as the variety of life on earth, comprising genetic diversity, species diversity and ecosystem diversity. It is crucial for human life since it is the foundation of functioning ecosystems, which provide necessary ecosystem services. In the past centuries, human activities have altered the world’s ecosystems and led to massive losses in biodiversity. To counteract the ongoing decrease in biological diversity therefore is a key issue in conservation. The reintroduction of native plant species is a common and useful practice in restoration and approaches range from reintroduction of plant populations to the creation of whole plant communities. Chapter One addresses the research question of this thesis and introduces the sci-entific context. The importance of biodiversity and its ongoing loss is described. Fur-ther, plant species reintroduction as an effective procedure to increase biodiversity on population and community level is presented with respect to the impact on genetic variation within restored populations and among natural and restored populations of herbaceous target species. Chapter Two focuses on the genetic diversity within and the genetic differentiation among populations of the rare and endemic plant species Cochlearia bavarica. Am-plified Fragment Length Polymorphisms (AFLP) were used to analyze the genetic variation of 32 remnant populations of the species. With respect to conservation management, recommendations are presented to increase the success of future population reintroduction and reinforcement. In Chapter Three the restoration of species-rich grassland communities by the transfer of green hay and threshed plant material was investigated. Species diversity and composition on restoration sites compared to their corresponding source sites was analyzed. Further genetic variation within and among populations on source and restoration sites of the common grassland species Knautia arvensis and Plantago lanceolata was examined using AFLPs. The study revealed no significant differences among source sites and their corresponding restoration sites, neither in species di-versity and composition nor in genetic variation within populations of the two plant species. Only marginal differences in genetic variation among populations on source sites and their corresponding restoration sites could be found. The transfer of local plant material is highly suited to preserve species composition of species-rich grass-lands and the natural genetic pattern of typical grassland plant species on a small geographical scale. Chapter Four and Five focus on the restoration of species-rich grassland communi-ties by sowing commercially produced regional seed mixtures. Even though this is a common approach in restoration ecology today, there are only few long-term studies investigating if local seed mixtures can actually be applied successfully to restore species-rich grassland communities. Further, it is often questioned whether commer-cially produced seed material is viable enough to establish vital populations and how the sowing may affect genetic variation of neighboring natural populations. Hence, the outcome of a large-scale grassland restoration project which started about 15 years ago in south-eastern Germany provides more information about the impact of this restoration measure on species diversity and genetic variation of plant species and populations on restored sites. The success of applying local seed mixtures to restore species-rich grasslands was analyzed within Chapter Four. Local seed mixtures can be used successfully to re-store species-rich grassland communities in practice: of all species that were present in the local seed mixtures 62 % were contained in the current and on average cov-ered up two thirds of the total vegetation cover. Moreover, restoration can be im-proved by using specific seed densities and species that are ecologically more suita-ble to restored sites. In Chapter Five deal with the impact of the restoration management on genetic vari-ation within and among restored and natural populations of typical grassland species. Genetic variation of three common plant species (K. arvensis, Silene vulgaris and P. lanceolata) was analyzed with AFLPs. The study revealed that using commercially produced seed mixtures in restoration caused no decease in genetic diversity within restored populations of common grassland species but did not match exactly the lo-cal genetic pattern of the study species. However, commercially produced seed ma-terial reflects the genetic potential of an entire seed transfer zone and provides seeds for the reestablishment of genetically viable populations. Finally, in Chapter Six the results of the four main chapters are reviewed in the con-text of nature conservation. Benefits and disadvantages of species (re)introductions are discussed. Recommendations are given to enhance the success of species reintroduction in restoration projects

Erweiterung der Grenzen der Hartstoffbeschichtung durch Nanostrukturierung

Hartstoffschichten ermöglichen die Verbesserung bewährter und die Entwicklung neuartiger Produkte. Bei geringstem Materialaufwand lassen sich mit solchen Schichten Wirkungen erzielen, die auf eine andere Weise nicht erreichbar wären. Dünne Hartstoffschichten bis 10 µm werden seit Jahrzehnten zum Verschleißschutz von Werkzeugen und Bauteilen eingesetzt. Der zu den PVD-Verfahren zählende Vakuumbogenprozess (Arc-PVD) wird in der Industrie in großem Umfang zur Abscheidung nitridischer Hartstoffschichten eingesetzt. Als besonders vorteilhaft sind dabei die hohe Ionenenergie im fast vollständig ionisierten Plasma, die damit verbundenen hervorragenden Schichteigenschaften (Mikrohärte, Haftung, Struktur) und die relativ unkomplizierte, robuste und flexible Anlagentechnik anzusehen. Im Rahmen dieser Arbeit wurde ein nanostrukturierte AlCr(Si)N/TiN Hartstoffsystem entwickelt, welches sich mit dem Arc-PVD-Prozess homogen in Schichtdicken größer 50 µm aufbringen lässt und damit neue Einsatzbereiche für die Hartstoffbeschichtung eröffnet. Durch das definierte Schichtdesign im Nanometermaßstab kann einerseits das Eigenspannungsniveau stark abgesenkt werden und zusätzlich wird das Wachstum von Defektstrukturen unterdrückt. Die gewonnen Erkenntnisse bei der Herstellung dicker Schichten ermöglichen auch Verbesserungen der Eigenschaften dünner Schichten bis 10 µm

Untersuchungen zur Klassierung von abnormal-repetitiven Verhaltensweisen bei Hunden

Abnormal-repetitive Verhalten (ARV) umfassen verschiedene Verhaltensstörungen, deren auffälligstes Merkmal die abnormal häufige Wiederholung von Verhaltensweisen ist. ARV treten auch bei Hunden auf und umfassen mitunter „Koprophagie“, „die eigene Rute jagen (Kreiseln)“, „Lichtreflexe jagen“, „Schatten anstarren“ und „psychogene Leckdermatitis“. In der Humanpsychologie werden ARV unter anderem in Stereotypien und Zwangsstörungen unterteilt. Bei Stereotypien wird ein bestimmtes Bewegungsmuster meist ziellos, jedoch gleichförmig wiederholt, während es bei Zwangsstörungen in gewissem Maße variabel, jedoch sehr zielgerichtet ausgeführt wird. Eine klare Zuordnung verschiedener ARV bei Hunden zu Stereotypien und Zwangsstörungen ist aufgrund ihrer phänomenologischen Eigenschaften jedoch meist nicht möglich. Stereotypien und Zwangsstörungen unterscheiden sich auch auf neurologischer Basis. In Studien an autistischen Kindern konnte gezeigt werden, dass sie mit unterschiedlichen Formen von Perseveration in spezifischen Verhaltenstests korrelieren. Während Stereotypien mit rekurrenter Perseveration, dem unangebrachten Wiederholen einer Verhaltensreaktion, verbunden waren, korrelierten Zwangsstörungen mit stuck-in-set Perseveration, dem unangebrachten Festhalten an bestimmten Verhaltensregeln. Ausgehend von diesen Erkenntnissen sowie von vergleichbaren Untersuchungen an Primaten und Labornagern wurden daher in der vorliegenden Arbeit Verhaltenstests zur Unterscheidung von rekurrenter und stuck-in-set Perseveration entwickelt und auf ihre Eignung zur Klassierung von ARV bei Hunden in Stereotypien und Zwangsstörungen getestet. Damit sollte eine zuverlässigere Diagnose dieser Verhaltensstörungen ermöglicht und eine gezieltere Therapie mit besseren Therapieerfolgen bei geringeren unerwünschten Nebenwirkungen angestrebt werden. Dazu wurden im Rahmen dieser Dissertation drei Studien durchgeführt. Weder bei den Hunden mit ARV insgesamt, noch bei Hunden mit bestimmten ARV bewirkte das mit Tryptophan angereicherte Futter eine signifikante Besserung in der Dauer oder Frequenz des auffälligen Verhaltens. Hunde mit Koprophagie zeigten jedoch unabhängig von der verabreichten Substanz eine signifikante Besserung während des Studienverlaufes. Zudem konnte bei diesen Hunden eine signifikante Korrelation zwischen der Häufigkeit der Kotaufnahme und der Häufigkeit der Konfrontation mit Kot festgestellt werden. Dieser Nebenbefund deutet darauf hin, dass eine Vermeidung des Kontakts mit Kot ein Ansatz zur Verminderung von Koprophagie darstellt. Dagegen konnten anhand der oralen Gabe von L-Tryptophan keine Rückschlüsse auf eine mögliche Diagnose von Zwangsstörungen gezogen werden. Dies könnte einerseits an der schwachen Ausprägung der ARV bei den untersuchten Hunden liegen, andererseits aber auch an einer zu kurzen Behandlungszeit. Entsprechend sind auch hierzu weitere Untersuchungen unter kontrollierten Bedingungen erforderlich.Abnormal-repetitive behaviours (ARBs) are a subset of behaviours that are frequently repeated, invariant in motor output, and independent of environmental interaction or goal. Furthermore they are apparently functionless, maladaptive, self-injurious, and inappropriate or odd. They are common in dogs and are shown as different behaviour patterns, such as coprophagia, lick granulomas, tail chasing, light chasing and shadow staring. Two of the most common types of ARBs are stereotypies and compulsive behaviours. In human medicine stereotypies and compulsive behaviour are distinguished based on measurable characteristics. For example, a stereotypy comprises the abnormal repetition of a motor pattern that has no obvious goal, whereas a compulsive behaviour is characterized by the abnormal repetition of a behavioural goal while the motor pattern may be variable. However, a clear differentiation of ARBs based on their phenomenological characteristics is often not possible. Stereotypies and compulsive disorders also differ with respect to the underlying neurobiology. Studies in humans have shown that both stereotypies and compulsive behaviours are associated with neural dysfunctions resulting in perseverative behaviour in test situations. However, while stereotypic behaviour is associated with recurrent perseveration - the inappropriate repetition of previous responses - compulsive behaviour is associated with stuck-in-set perseveration - the inappropriate repetition of attentional sets or goals. The aim of the present dissertation was to develop behavioural tests for use with dogs to assess these different types of perseveration. Furthermore, the tests should be used to classify ARBs in dogs into stereotypies and compulsive behaviour with the intent of improving strategies to prevent or treat ARBs in dogs. In order to meet these goals, three studies were conducted. There was no significant effect of treatment on the frequency or duration of ARBs. However, a significant improvement was shown in the dogs with coprophagia regardless of the substance applied. Furthermore, a significant correlation between the frequency of confrontation with feces and the frequency of coprophagia was found in these dogs. This incidental finding shows that the avoidance of feces may be an important first step in the behavioural therapy of coprophagia. The present results indicate that dietary supplementation with tryptophan is ineffective for detecting compulsive behaviour in dogs. However, several reasons may account for this finding. The dosage may have been too low and/or treatment too brief to demonstrate measurable improvements. Furthermore, the severity of the ARBs may again have been too mild to find differences to healthy dogs. Further research under controlled conditions is needed to clarify this

Identifizierung von Zielgenen des chimären Transkriptionsfaktors ETV6/RUNX1

Die häufigste Leukämieform im Kindesalter, mit einem Anteil von ca. 80% ist die akute lymphoblastische Leukämie (ALL). Bei ca.75% der ALL im Kindes- und Jugendalter werden chromosomale Aberrationen numerischer oder struktureller Art, wie z.B. Translokationen, Inversionen oder Deletionen nachgewiesen. Die am häufigsten vorkommende Translokation t(12;21)(p13;q22) mit einem Anteil von ca. 25% resultiert in der Bildung des chimären Transkriptionsfaktors ETV6/RUNX1. Die Transkriptionsfaktoren ETV6 und RUNX1 sind elementare Regulatoren der Hä-matopoese. Deren Fusionsprodukt ETV6/RUNX1 interferiert entscheidend mit dieser Regulation und bewirkt eine veränderte Funktion der hämatopoetischen Transkriptionsfaktoren ETV6 und RUNX1, die in der Struktur des Fusionsproteins begründet ist. Zum jetzigen Zeitpunkt geht man davon aus, dass ETV6/RUNX1 mit dem endogenen RUNX1 um dieselben Promotorbereiche ihrer Zielgene konkurriert und dass das N-terminal im Fusionsprotein gelegene ETV6 Co-Repressoren wie z.B. N-CoR, mSin3A und Histondeacetylasen (HDAC) rekrutiert. Die Rekrutierung dieser Co-Repressoren führt zu einer Kondensation der Chromatinstruktur und somit zu einer Repression der Zielgene. Dies bedeutet, dass die Expression von ETV6/RUNX1 v.a. eine Inaktivierung von Genen bewirkt, die normalerweise durch RUNX1 aktiviert werden. Verschiedene Studien im Mausmodell, mit eineiigen Zwillingen und retrospektive Studien mit Nabelschnurblut von Neugeborenen zeigten u.a. dass die Bildung des Fusionsgens ETV6/RUNX1 den initialen Schritt in der Pathogenese ETV6/RUNX1-positiver ALL dar (first hit) darstellt („Greaves-Hypothese“). Für die Ausbildung einer klinisch manifesten Leukämie sind jedoch weitere genetische Veränderungen, sogenannte second hits notwendig. Ein solcher second hit kann z.B. die Deletion des zweiten endogenen ETV6-Allels sein, die besonders häufig in ETV6/RUNX1 positiven ALL gefunden wird. Ziel dieser Promotionsarbeit war es, zur Aufklärung dieser molekularen Mechanis-men durch die genomweite Identifizierung von DNA-Bindungsstellen und damit von Zielgenen des chimären Transkriptionsfaktors ETV6/RUNX1 und deren Einordnung in die zellulären Prozesse beizutragen, die letztendlich zur Ausprägung der Leukämie führen. Um diese Fragestellung zu bearbeiten, wurden ChIP-Seq-Analysen von etablierten ETV6/RUNX1 positiven humanen Prä-B Leukämie-Zelllinien REH und UoC-B6 sowie von primären ALL-Blasten aus Knochenmarkproben von Kindern (ALL_#1 und ALL_#2) mit ETV6/RUNX1 positiven ALL durchgeführt um potenzielle ETV6/RUNX1 Zielgene zu identifizieren. Die Chromatin-Immunpräzipitation ist eine sensitive und spezifische Methode zur Untersuchung der Interaktionen zwischen Proteinen und DNA. Gegenüber herkömmlichen Genexpressionsanalysen hat die ChIP den entscheidenden Vorteil, dass durch die Anwendung spezifischer Antikörper und anschließender Sequenzierung regulatorische Elemente der DNA, wie z.B. Promotoren, direkt untersucht und Zielregionen bzw. potenzielle Zielgene von Transkriptionsfaktoren identifiziert werden können. Für das Gelingen der ChIP wurden die Versuchsbedingungen optimiert. Dazu gehörten die Bestimmung der optimalen Zellzahl pro Ansatz, der Art und Dauer der Fixierung sowie die Evaluation geeigneter ETV6 und RUNX1 Antikörper. Unter diesen für die ChIP-Seq optimierten Bedingungen wurden mit den ETV6/RUNX1 positiven humanen Prä-B Leukämie-Zelllinien REH und UoC-B6 sowie mit den ETV6/RUNX1 positiven ALL-Blasten aus dem KM von Kindern (ALL_#1 und ALL_#2) 935 potenzielle proteincodierende ETV6/RUNX1 Zielgene (Kerngenset), identifiziert. Durch die Zuordnung dieses Kerngensets zu biologischen Prozessen bzw. Signalwegen wurden 23 Signalwege identifiziert, die statistisch signifikant überrepräsentiert sind (p-value<0,05) und möglicherweise durch ETV6/RUNX1 fehlreguliert werden. Neben dem Signalweg Transendotheliale Migration der Leukozyten waren u.a der Signalweg, der das Aktin-Zytoskeletts reguliert und der B-Zell-Rezeptor Signalweg die am signifikantesten hervortretenden Signalwege (Tabelle 4.8. und Tabelle 5.3). Ein Vergleich des Kerngensets mit differenziell regulierten Genen, die von Fuka et al.184 durch Genexpressionsanlysen nach ETV6/RUNX1 knockdown in den Zelllinien REH und AT2 ermittelt wurden, ergab eine hohe Übereinstimmung an Genen (= 100 durch ETV6/RUNX1 in ihrer Expression beeinflusste Gene). Die Zuordnung dieser Gene zu Signalwegen zeigte ebenfalls den B-Zellrezeptor-Signalweg signifikant überrepräsentiert. Über die Identifizierung von DNA-Bindungsmotiven in den ETV6/RUNX1-Bindungsregionen (Peaks) des Kerngensets und der anschließenden Zuordnung dieser Motive zu Transkriptionsfaktoren wurden 110 DNA-Bindungsmotive, die 29 Transkriptionsfaktoren zugeordnet werden konnten, ermittelt. Von diesen wurden sieben Transkriptionsfaktoren (ERG, FEV, KLF5, MYOG, RUNX1, RUNX2 und SP1) sowohl in den ETV6/RUNX1 positiven Zelllinien REH und UoC-B6, als auch den den primären ALL-Blasten aus Knochenmarkproben von Kindern (ALL_#1 und ALL_#2) gefunden. Für ein zukünftig besseres Verständnis über die biologischen Folgen der chromosomalen Translokation (t12;21) sowie deren Auswirkungen auf die Entstehung von malignen Zellen sollten die von Fuka et al184 veröffentlichten und in der vorliegenden Arbeit gezeigten Ergebnisse durch einen effektiven ETV6/RUNX1 knockdown in REH- und UoC-B6 Zellen validiert werden. Über die direkten Auswirkungen eines stabilen ETV6/RUNX1 knockdowns in leukämischen Zellen ist wenig bekannt, daher wurde für den shRNA vermittelten ETV6/RUNX1 knockdown ein durch Tetrazyklin induzierbares System gewählt (Tet-on). Die Effizienz der Transduktion wurde mittels FACS und fluoreszenzmikroskopisch bestimmt: In den REH-Zellen war die Transduktion des TetR und der shRNAs zu 90%, in den UoC-B6-Zellen zu 60% bis 70% erfolgreich. Auf Proteinebene konnte jedoch kein knockdown von ETV6/RUNX1 festgestellt werden. Für die Fehleranalyse wurde die genomische DNA aus den transduzierten Zellen zunächst isoliert und anschließend sequenziert. In allen shRNAs zeigten sich Mutationen. Die Ursache für die Entstehung dieser Mutationen ist nicht bekannt. Nichtdestotrotz ist die Identifizierung von Zielgenen des chimären Transkriptionsfak-tors ETV6/RUNX1 und die Zuordnung dieser Gene zu Signalwegen ein wichtiger Schritt für das Verständnis der biologischen Prozesse die die Entwicklung einer kli-nisch manifesten Leukämie begünstigen können. Basierend auf den in der vorliegenden Arbeit gewonnenen Ergebnisse können weitere Forschungen durchgeführt werden, die es ermöglichen könnten direkt in betroffene Signalwege einzugreifen bzw. die Expression bestimmter Gene so zu manipulieren (z.B. durch CRISPR/CAS9), dass die Manifestation der Leukämie abgeschwächt oder sogar unterbunden werden kann.The most common form of leukemia in childhood, accounting for about 80% of all forms, is acute lymphoblastic leukemia (ALL). Chromosomal aberrations of a numeri-cal or structural type, e.g. translocations, inversions or deletions, are found in around 75% of cases of ALL in childhood and adolescence. The most common translocation t(12;21)(p13;q22), with a share of around 25%, results in the formation of the chimeric transcription factor ETV6/RUNX1. The transcription factors ETV6 and RUNX1 are elementary regulators of hemato-poiesis. Their fusion product ETV6/RUNX1 interferes decisively with this regulation and the structure of the fusion protein leads to a change in the function of the hematopoietic transcription factors ETV6 and RUNX1. It is currently assumed that ETV6/RUNX1 competes with the endogenous RUNX1 for the same promoter regions of their target genes and that the N-terminal recruits ETV6 co-repressors located in the fusion protein such as N-CoR, mSin3A, and histone deacetylases (HDAC). The recruitment of these co-repressors leads to condensation of the chromatin structure and thus to repression of the target genes. This means that ETV6/RUNX1 expression chiefly causes the inactivation of genes that are normally activated by RUNX. Different studies of a mouse model, with monozygotic twins, and retrospective stu-dies with umbilical cord blood of newborns show among other things that the formation of the fusion gene ETV6/RUNX1 is the initial step (first hit) in the pathogenesis of ETV6/RUNX1-positive ALL (“Greave’s hypothesis”). However, additional genetic changes, known as second hits, are necessary for the development of clinically manifest leukemia. One of these second hits can be the deletion of the second endogenous ETV6 allele, for example, which is found particularly frequently in ETV6/RUNX1-positive ALL. The objective of this doctoral thesis was to contribute to the explanation of these molecular mechanisms through the genome-wide identification of DNA binding sites and thus of target genes of the chimeric transcription factor ETV6/RUNX1 and its integration into cellular processes that ultimately lead to the manifestation of leukemia. To address this question, ChIP-Seq analyses of established ETV6/RUNX1-positive human pre-B leukemia cell lines REH and UoC-B6 and of primary ALL blasts from bone marrow samples from children (ALL_#1 and ALL_#2) with ETV6/RUNX1-positive ALL were conducted to identify potential ETV6/RUNX1 target genes. Chromatin immunoprecipitation is a sensitive and specific method for examining the interactions between proteins and DNA. Compared with conventional gene expression analyses, ChIP has the decisive advantage that by using specific antibodies and subsequent sequencing, regulatory elements of DNA such as promoters are examined directly and target regions and potential target genes of transcription factors can be identified. The test conditions were optimized to ensure the success of ChIP, including determination of the optimized cell count per batch, the type and duration of fixation, and the evaluation of suitable ETV6 and RUNX1 antibodies. Under these conditions optimized for ChIP-Seq, 935 potential protein-coding ETV6/RUNX1 target genes (core gene set) were identified with the ETV6/RUNX1 positive human pre-B leukemia cell lines REH and UoC-B6 and with the ETV6/RUNX1 positive ALL blasts from the bone marrow of children (ALL_#1 and ALL_#2). By allocating this core gene set to biological processes or signaling pathways, 23 signaling pathways were identified that are statistically significantly over-represented (p<0.05) and possibly dysregulated by ETV6/RUNX1. In addition to the leukocyte transendothelial migration signaling pathway, the signaling pathway that regulates the actin cytoskeleton and the B-cell receptor signaling pathway were the most significant signaling pathways occurring (Table 4.8 and Table 5.3). A comparison of the core gene set with differentially regulated genes detected by Fuka et al.184 in gene expression analyses using ETV6/RUNX1 knockdown in the REH and AT2 cell lines showed a high correspondence of genes (= 100 genes whose expression was influenced by ETV6/RUNX1). The allocation of these genes to signaling pathways also showed that the B-cell receptor signaling pathway was significantly over-represented. By identifying DNA-binding motifs in the ETV6/RUNX1 binding regions (peaks) of the core gene set and subsequently allocating these motifs to transcription factors, 110 DNA-binding motifs were identified that could be assigned to 29 transcription factors. Of these, seven transcription factors (ERG, FEV, KLF5, MYOG, RUNX1, RUNX2 and SP1) were found both in the ETV6/RUNX1 positive cell lines REH and UoC-B6 and in the primary ALL blasts from bone marrow samples from children (ALL_#1 and ALL_#2). In order to achieve a better understanding of the biological consequences of chromosomal translocation (t12;21) and the impact on the development of malignant cells in the future, the results published by Fuka et al.184 and presented in this study were to be validated by an effective ETV6/RUNX1 knockdown in REH and UoC-B6 cells. As little is known about the direct impact of a stable ETV6/RUNX1 knockdown in leukemia cells, a system that can be induced by tetracycline (Tet-on) was selected for the shRNA-mediated ETV6/RUNX1 knockdown. The effectiveness of transduction was determined using FACS and fluorescence microscopy: In the REH cells, transduction of TetR and shRNAs was 90% successful and 60% to 70% successful in the UoC-B6 cells. However, no knockdown of ETV6/RUNX1 was found at the protein level. For the error analysis, genomic DNA from transduced cells was first isolated and then sequenced. Mutations were found in all shRNAs. The cause of these mutations is not known. Nevertheless, the identification of target genes and the chimeric transcription factor ETV6/RUNX1 and the allocation of these genes to signaling pathways is an im-portant step toward understanding the biological processes that can lead to the development of clinically manifest leukemia. Based on the results of this study, additional research can be conducted that could allow the direct intervention in affected signaling pathways or manipulation of the expression of certain genes (e.g. by CRISPR/CAS9) so that the manifestation of leukemia can be attenuated or even prevented

Reintroduction of the endangered and endemic plant species Cochlearia bavarica -Implications from conservation genetics

Population reintroduction is a common practice in conservation, but often fails, also due to the effects of inbreeding or outbreeding depression. Cochlearia bavarica is a strongly endangered plant species endemic to Bavaria in Germany, constantly declining since the late 1980s. Therefore, population reintroduction is intended. In this study, we analyzed genetic diversity within and genetic differentiation between all 32 remnant populations of the species in Swabia and Upper Bavaria using amplified fragment length polymorphisms. Our aim was to increase reintroduction success by providing data to avoid negative effects of inbreeding and outbreeding and to preserve the natural genetic pattern of the species. Genetic diversity within populations was low but similar to other rare and endemic species and varied strongly between populations but did not depend on population size. Our analysis revealed a strong geographic pattern of genetic variation. Genetic differentiation was strongest between Swabia and Upper Bavaria and at the population level, whereas differentiation between subpopulations was comparatively low. Isolation by distance and genetic differentiation was stronger among populations from Upper Bavaria than from Swabia. From the results of our study, we derived recommendations for a successful reintroduction of the species. We suggest using rather genetically variable than large populations as reintroduction sources. Moreover, the exchange of plant material between Swabia and Upper Bavaria should be completely avoided. Within these regions, plant material from genetically similar populations should preferably be used for reintroduction, whereas the exchange among subpopulations seems to be possible without a negative impact on genetic variation due to natural gene flow

Restoration of species‐rich grasslands by transfer of local plant material and its impact on species diversity and genetic variation—Findings of a practical restoration project in southeastern Germany

Restoration of species-rich grasslands is a key issue of conservation. The transfer of seed-containing local plant material is a proven technique to restore species-rich grassland, since it potentially allows to establish genetically variable and locally adapted populations. In our study, we tested how the transfer of local plant material affected the species diversity and composition of restored grasslands and the genetic variation of the typical grassland plant species Knautia arvensis and Plantago lanceolata. For our study, we selected fifteen study sites in southeastern Germany. We analyzed species diversity and composition and used molecular markers to investigate genetic variation within and among populations of the study species from grasslands that served as source sites for restoration and grasslands, which were restored by transfer of green hay and threshed local plant material. The results revealed no significant differences in species diversity and composition between grasslands at source and restoration sites. Levels of genetic variation within populations of the study species Knautia arvensis and Plantago lanceolata were comparable at source and restoration sites and genetic variation among populations at source and their corresponding restoration sites were only marginal different. Our study suggests that the transfer of local plant material is a restoration approach highly suited to preserve the composition of species-rich grasslands and the natural genetic pattern of typical grassland plant species

Grassland restoration by local seed mixtures: New evidence from a practical 15‐year restoration study

Aim Local seed mixtures are frequently used to restore species-rich grasslands. However, it has hardly been tested whether local seed mixtures can actually be applied successfully in grassland restoration practice at larger scales and long-term. To close this gap, we report the results of a large-scale restoration study in which grasslands were restored about 15 years ago using different local seed mixtures. Location Bavaria, SE Germany. Methods To evaluate the efficacy of the local seed mixtures, we compared the species composition of seed mixtures and current vegetation. We then tested whether restoration success depends on site characteristics such as the size and shape (rectangle or stripe) of the grassland, restoration procedures such as topsoil removal, seed density and land use, or species habitat preferences for light, water and nutrients, and species life span (annual, perennial). Results n average, the current vegetation contained 62.4% of all species that were present in the local seed mixtures. Species from the local seed mixtures made up on average 69.1% of the total cover in the established vegetation, whereby the species composition of the local seed mixture and vegetation differed significantly from each other. The probability that a sown species would establish increased with seed density up to 300 seeds/m². Furthermore, habitat preferences significantly affected species establishment chances, with species requiring full illumination, dry and nutrient-poor soil being more successful during restoration, reflecting the high proportion of sites with topsoil removal prior to seeding in our study. Annual species had significantly lower establishment chances compared with their perennial counterparts. Conclusions Our study provides another piece of evidence that local seed mixtures can be applied successfully in large-scale grassland restoration projects. We provide several practical recommendations of how such practices can be further improved by using specific seed densities and creating new local seed mixtures using species that are ecologically more suitable to the restored sites

Neutron-proton mass difference in nuclear matter

Isospin-breaking effects in nuclear matter are studied in the framework of a medium-modified Skyrme model. The proposed effective Lagrangian incorporates both the medium influence of the surrounding nuclear environment on the single nucleon properties and an explicit isospin-breaking effect in the mesonic sector. The approach predicts that the neutron-proton mass difference decreases in isospin-symmetric nuclear matter but by a very small amount only.Comment: 8 pages, 4 figures, revised versio

Nucleon deformation in finite nuclei

The deformation of a nucleon embedded in various finite nuclei is considered by taking into account the distortion of the chiral profile functions under the action of an external field representing the nuclear density. The baryon charge distribution of the nucleon inside light, medium-heavy and heavy nuclei is discussed. The mass of the nucleon decreases as it is placed deeper inside the nucleus and reaches its minimum at the center of the nucleus. We discuss the quantization of non-spherical solitons and its consequences for the mass splitting of the delta states. We show that bound nucleons acquire an intrinsic quadrupole moment due to the deformation effects. These effects are maximal for densities of nuclei about \rho(R)\sim 0.3...0.35 \rho(0). We also point out that scale changes of the electromagnetic radii can not simply be described by an overall swelling factor.Comment: 29 pp, REVTeX, 8 figures, more detailed discussion on quantization and intrinsic quadrupole moments, references adde