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Untersuchungen an Populationen Von Gibbaeum Nuciforme (Haw.) Glen & H.E.K

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Untersuchungen an Populationen Von Gibbaeum Nuciforme (Haw.) Glen & H.E.K Untersuchungen an Populationen von Gibbaeum nuciforme (Haw.) Glen & H.E.K. Hartmann (Aizoaceae) und zur Phylogenie der Gattung Gibbaeum N.B. Br. Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Department Biologie der Fakultät für Mathematik, Informatik und Naturwissenschaften an der Universität Hamburg vorgelegt von Gisela Bertram aus Hamburg Hamburg, Mai 2007 Drucklegung März 2008 Dank Mein Dank geht an meinen Doktorvater Prof. Dr. N. Jürgens für die Betreuung dieser Arbeit und die Möglichkeit Labor und molekulare Methoden zu etablieren. An U. Schmiedel geht mein Dank für die Ideen und vielen Hinweise zu meinem Thema sowie für die gemeinsame Tour durch die Kleine Karoo in Südafrika, ohne ihre Kenntnis von Orten, Pflanzen und Menschen wäre diese Arbeit nicht möglich gewesen. Verschiedenen Landbesitzern in der Kleinen Karoo (Südafrika) danke ich für ihre Erlaubnis auf ihrem Land Pflanzen zu untersuchen und zu sammeln. Dem Western Cape Nature Conservation Board sei für die Genehmigung zum Sammeln und zur Ausfuhr von Pflanzenmaterial gedankt. Für einen Reisekostenzuschuss danke ich der Deutschen Kakteen Gesellschaft e.V. Der gesamten Arbeitsgruppe Systematik und Vegetationsökologie am Biozentrum Klein Flottbek möchte ich für ihre großartige Unterstützung und Diskussionsbereitschaft danken. Danke für Pipettieren, Plattenputzen, Primerbestellen, Pakete annehmen, PCR-Rausnehmen, Fehler-Beratungen, Organisieren, Diskutieren, Reparieren, schnell Antworten, Korrekturlesen, noch mal Lesen, Teekochen und so weiter - ich hoffe Ihr wisst, dass Ihr alle gemeint seid. Zudem möchte ich mich bei A. Schierholt fürs Korrektur-Lesen bedanken. Ganz besonders möchte ich mich bei Manfred Haacks bedanken: Danke für Deine umfassende Unterstützung und die nie versiegende Teeversorgung! Mein abschließender Dank geht an meine Familie, Freunde und Kollegen, die hartnäckig genug waren, immer wieder nachzufragen, wann ich endlich meine Arbeit abgebe - hier ist sie. Hamburg im Mai 2007 Gisela Bertram Inhaltsverzeichnis INHALTSVERZEICHNIS 1 Einleitung ....................................................................................................... 1 1.1 Die Gattung Gibbaeum ..................................................................................................1 1.2 Die Art Gibbaeum nuciforme..........................................................................................4 1.3 Untersuchungsgebiet Kleine Karoo................................................................................6 1.4 Quarzflächen.................................................................................................................7 1.4.1 Vegetation und Ökologie von Quarzflächen im südlichen Afrika .................................7 1.4.2 Alter der Quarzflächen...............................................................................................8 1.5 Fragestellung.................................................................................................................8 1.5.1 Populationsuntersuchungen bei Gibbaeum nuciforme, landscape genetics................9 1.5.1.1 Fragestellungen für die Untersuchung an Populationen.....................................9 1.5.2 Phylogenie der Gattung Gibbaeum..........................................................................10 2 Material und Methoden................................................................................ 11 2.1 Blattmaterial ................................................................................................................11 2.1.1 Die Gattung Gibbaeum............................................................................................11 2.1.2 Blattmaterial aus Populationen von Gibbaeum nuciforme.........................................12 2.2 Bodenuntersuchungen.................................................................................................13 2.3 Vegetation...................................................................................................................14 2.4 Habitataufnahme.........................................................................................................14 2.5 Populationsgröße ........................................................................................................14 2.6 Messungen an den Blattpaaren ...................................................................................15 2.7 Molekularbiologische Methoden...................................................................................15 2.7.1 DNA-Isolierung........................................................................................................16 2.7.1.1 Vorversuch zur Lagerung von gesammeltem Blattmaterial auf Reisen.............16 2.7.2 DNA-Isolierung........................................................................................................16 2.7.3 „bulked“ DNA...........................................................................................................17 2.7.4 Chloroplasten PCR - Restriktionsanalyse ................................................................18 2.7.5 Random Amplified Polymorphic DNA Analysen, RAPD Analysen.............................20 2.7.6 Amplified Fragment Length Polymorphism, AFLP-Analysen.....................................21 2.7.7 Inter Simple Sequence Repeats, ISSR-Analysen.....................................................24 2.7.8 Consensus Chloroplast Simple Sequence Repeat-Analysen (ccSSR)......................25 2.7.9 ITS Sequenzen .......................................................................................................26 2.8 Auswertungsmethoden ................................................................................................28 2.8.1 Normalverteilung und Gruppenunterschiede ............................................................28 2.8.2 Gruppierungen ........................................................................................................28 2.8.2.1 Cluster-Analysen.............................................................................................28 2.8.2.2 Diskriminanzanalysen .....................................................................................29 2.8.3 Varianzanalyse........................................................................................................29 2.8.4 Genetische Diversität ..............................................................................................29 2.8.5 Distanzberechnungen..............................................................................................30 Inhaltsverzeichnis 2.8.6 AMOVA...................................................................................................................30 2.8.7 Mantel-Test .............................................................................................................30 2.8.8 Sequenzen..............................................................................................................31 2.8.9 Phylogenetische Auswertung...................................................................................31 3 Ergebnisse ................................................................................................... 31 3.1 Bodenanalysen in den Populationen von Gibbaeum nuciforme ....................................31 3.2 Vegetation in den Populationen von Gibbaeum nuciforme............................................32 3.3 Habitataufnahme für Populationen von Gibbaeum nuciforme .......................................33 3.4 Populationsgröße der Populationen von Gibbaeum nuciforme .....................................34 3.5 Messungen an Pflanzen und Blattpaaren von Gibbaeum nuciforme.............................37 3.5.1 Anzahl Früchte........................................................................................................37 3.5.2 Höhe der Blätter über dem Boden ...........................................................................39 3.5.3 Blattform .................................................................................................................40 3.5.4 Transparenter Bereich in Blattspitze ........................................................................41 3.5.5 Behaarung der Blätter .............................................................................................41 3.6 Überprüfung von Ost-West-Gruppierung anhand der Merkmale der Individuen ............44 3.6.1 Zusammenfassung für die Überprüfung der Ost- und der West-Gruppe mit Feld-Merkmalen 46 3.7 Blühende Pflanzen von Gibbaeum nuciforme im Gewächshaus...................................47 3.8 Molekulare Untersuchungen ........................................................................................48 3.8.1 Ergebnisse der Vorversuche zur Lagerung von Blattmaterial zur DNA- Gewinnung..48 3.8.2 Ergebnisse der molekularen Populationsuntersuchungen ........................................49 3.8.2.1 Ergebnisse der Chloroplasten PCR Restriktion,...............................................49 3.8.2.2 Ergebnisse der AFLP Populationsuntersuchungen ..........................................51 3.8.2.3 Testen der Zuordnung von Individuen an drei Populationen ............................51 3.8.2.4 Abgrenzung von Individuen und Populationen mit AFLPs................................54 3.8.2.5 Untersuchung der Populationen von Gibbaeum nuciforme mit AFLPs .............56 3.8.2.6 Populations-Untersuchungen mit AFLPs und „bulked“ DNA.............................64 3.8.2.7 Genetische Struktur ........................................................................................67
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