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Leptocylindrus Open Research Online The Open University’s repository of research publications and other research outputs Genetic, physiological and ecological diversity of the diatom genus Leptocylindrus Thesis How to cite: Nanjappa, Deepak (2012). Genetic, physiological and ecological diversity of the diatom genus Leptocylindrus. PhD thesis The Open University. For guidance on citations see FAQs. c 2012 The Author Version: Version of Record Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk >- :~ The Open University, London c ~ ~ Stazione Zoologica Anton Dohrn, o cu .r:. I- Naples Genetic, physiological and ecological diversity of the diatom genus Leptocylindrus OEEPAK NANJAPPA Thesis submitted for the degree of DoctorOJl Philosophy (PhD) in Life and Biomolecular Sciences September 2012 [ ~ ":- -"-: .:.. ~ , ~ :... .. 7. :_.:. ':.":.":" -_ ...... -""'''''' ":..':.~ .... ~ , ......... ' ~, .. - . .. \.... ' .. : ",.' ~ , '-... - .....---., ~. .- ...~"" '::'-- -.', - o· • , • , : , , : .' " ". '. I I •••• I • • • .. • , " '. ":"', ". "-''-'.'. ".'. :-...... ,. .~..." . '.. "-- • -. .. "I '. • . ' • •• . • • • .. " " , ..:>- :. ;:':-'. .... ::'.\',,: '; ;.'::'::.. ':'. :-:.. ::.:' .......' .. , ' .. , ..... :.. '. .. ' .. ' .. " ... ... ' .. ' ' '.' .. ' '"'' '. '. '. ". •• •• '.p' ..';0 ...b" . .... .'. .....' .. ":. ."; .;. '.:::'., ....:.. .' , .... .. ...... ~'.'.',. ' ... ~ . "'.:. .......... ~. .' .' ." " '.... ". " ' ' ' '. .... : ..... .'. " , . '... ...'... ..... .. '''''....••• , ~" • ~. I .~~ "0 • I',· I' .".. 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Let tM prt'vin, ft1,1Asc.tes, nerves, ever1j ?t'vrt elf 1j"1Ar P"~1j' pe jvttt "I t~t'vt i~et'v, t'vn~j1Ast let'vve ever1j "tMr i~et'v t'vl"ne. I~is is tM wt'v1j tel S1Ac-c-ess. - Swt'vft1,i ViveK.t'vnt'vn~t'v Thesis supervision committee Director of Studies Dr Adriana Zingone Senior Scientist Stazione Zoologica Anton Dohrn Villa Comunale, Naples, Italy Internal Supervisor Dr Wiebe H.C.F. Kooistra Senior Scientist Stazione Zoologica Anton Dohrn Villa Comunale, Naples, Italy External Supervisor Professor David G. Mann Senior Principal Research Scientist Royal Botanic Garden Edinburgh Edinburgh EH3 SLR Scotland, UK Table of contents Abstract Acknowledgements ii List of tables iv List of figures vi List of abbrevations ix Chapter' I General Introduction 1.1 Phytoplankton I 1.2 Diatoms 3 1.3 General biology 5 104 Diatom classification 6 104.1 Centric diatoms 7 104.2 Pennate diatoms 7 1.5 Life cycle features 8 1.6 The study model Leptocylindrus danicus 10 I .6.1 The life cycle of Leptocylindrus 14 1.6.2 Population dynamics 16 1.6.3 Physiology 17 1.7 The Gulf of Naples 18 1.8 Motivation for the study 18 1.9 Goals and thesis framework 20 Chapter' II Morphological and molecular diversity of Leptocylindrus species 22 from the Gulf of Naples 2.1 Introduction 22 2.2 Methods 25 2.2.1 Strain isolation 25 2.2.2 Microscopic observations 28 2.2.3 DNA extraction, peR amplification, sequencing, and phylogenetic analyses 29 2.204 Spore induction 33 2.3 Results 33 2.3.1 Leptocylindrus aporus 34 2.3.2 Leptocylindrus convexus 40 2.3.3 Leptocylindrus danicus 43 2.304 Leptocylindrus hargravesii 47 2.3.5 Tenuicylindrus be/gicus 50 2.3.6 Seasonal distribution 53 2.3.7 Molecular phylogenies 54 204 Discussion 64 204.1 Molecular phylogenies 65 204.2 Morphological and life-cycle features of Tenuicylindrus and Leptocylindrus 67 204.3 Variation of plastid morphology and number 73 204 A Temporal and spatial distribution of the species. 73 2.5 Conclusion 74 Chapter- III Molecular detection and distribution of Leptocylindrus species using metagenomic databases 77 3.1 Introduction 77 3.2 Methods 84 3.2.1 Sampling and databases 84 3.2.2 Sequence Retrieval 86 3.2.3 Alignment of obtained sequences 89 3.2.4 Phylogenetic analysis of obtained sequences 90 3.3 Results 90 3.3.1 Pairwise dissimilarity 90 3.3.2 The BioMarKs data 91 i Sequence abundance 91 ii Alignment and phylogenetic inferences 91 iii Phylogeography and additional metadata 98 3.3.3 Tara Oceans data 100 Sequence abundance 100 ii Alignment and phylogenetic inferences 101 3.3.4 Biogeography 105 3.4 Discussion 106 3.4.1 Support for the different recognized species in Chapter II 109 3.4.2 Possible evidence for additional Leptocylindrus species III 3.4.3 Biogeography 113 3.5 Conclusion 114 Chapter- IV Diversity of oxylipins and their biosynthetic pathways in the centric genera, Leptocylindrus and Tenuicylindrus 128 4.1 Introduction 128 4.2 Methods 133 4.2.1 Cultures 133 4.2.2 Oxylipins analysis 134 4.3 Results 135 4.3.1 Growth 135 4.3.2 Oxylipin profiles 136 4.3.3 Oxylipin characterisation 139 4.4 Discussion 146 4.4.1 Support for the species recognized in Chapter II. 146 4.4.2 Diatom taxonomic classification 147 4.4.3 Chemotaxonomy in Diatoms 147 4.4.4 Oxylipins as biomarkers 149 4.4.5 Intraspecific variations in oxylipins 150 4.5 Conclusion 151 Chapter- V Physiological responses and biochemical phenotypes in the diatom species Leptocylindrus danicus and Leptocylindrus aporus exposed to three different temperatures 153 5.1 Introduction 153 5.2 Methods 155 5.2.1 Cultures 159 5.2.2 Acclimation, batch cultures and experimental conditions 159 i Acclimation 159 ii Growth experiment with mUltiple strains 161 iii Growth and metabolomic experiment with two strains 161 5.2.3 Calculation of maximum growth rate, biovolume and biomass 162 5.2.4 Oxylipin analysis 162 5.3 Results 162 5.3.1 Acclimation 162 5.3.2 Effect of temperature on growth and physiological characteristics 163 i Maximum growth rate 166 ii Cell density 170 iii Days to complete the exponential growth phase 171 iv Cell size and morphology 171 v Biomass 172 5.3.3 Oxylipin and fatty acid profiles 177 5.4 Discussion 178 5.4.1 Maximum growth rates as a function of temperature 179 5.4.2 Cell volume and morphology as a function of temperature 181 5.4.3 Cell number and biomass as a function of temperature 182 5.4.4 Lipids, Polyunsaturated fatty acids as a function of temperature 184 5.4.5 Oxylipins as a function of temperature 186 5.4.6 Intraspecific variations in physiological and biochemical responses 187 5.4.7 Temporal isolation of the species explained by the temperature 187 5.4.8 Genetic and physiological distances between species 188 5.4.9 Trade-off in biomass bUild-up, time and metabolite accumulation 188 5.5 Conclusion 189 Chapter VI Conclusions and perspectives 191 Bibliography 198 1 Student details Your full name: Deepak Nanjappa I Personal identifier (PI): B2978976 Affiliated Research Centre (ARC) (if applicable): Stazione Zoologica A. Dohrn Department: Ecology and Evolution of Planktons ~:~~~::~~e~r~:netic , physiological and ecological diversity in the diatom genus 2 Authorisation I confirm that I am willing for m th . t b statement Open University Library, and t~at i~~~/beep~~~e a~adilable ~o readers by The discretion of the Librarian cople , subJect to the Signed: .... /V.~~n.~~~.. ~ .. ............ .... ..... ..,...... .................... Print name: Deepak Nanjappa Date: 21/01/2013 DD/MM/yY ! The Open University has agreed that a copy of your thesis can be made British Library available on loan to the British Library Thesis Service on a voluntary basis. The Authorisation British Library may make the thesis available online. Please indicate your (PhD and EdD candidates only) preference below: I:8J I am willing for The Open University to loan the British Library a copy of my thesis OR o I do not wish The Open University to loan the British Library a copy of my thesis I' I; Ii Abstract Diatoms are among the most productive photoautotrophic organisms
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