Open Research Online The Open University’s repository of research publications and other research outputs Molecular phylogeny, phylogeography and population genetics of the red seaweed genus Asparagopsis Thesis How to cite: Andreakis, Nikolaos (2006). Molecular phylogeny, phylogeography and population genetics of the red seaweed genus Asparagopsis. PhD thesis The Open University. For guidance on citations see FAQs. c 2006 The Author https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000d56a 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 Molecular Phylogeny, Phylogeography and Population Genetics of the red seaweed genus Asparagopsis A thesis submitted to the OPEN University of UKfor the degree of Doctor of Philosophy by Nikolaos Andreakis Laurea in Biological Sciences - University "Federico II" of Naples - Italy Sponsoring Establishment: Stazione Zoologica "Anton Dohrn" - Naples, Italy May 2006 Av-nto e, \-J 0 W \ ';-S? "5'""0 3 1)'"'-1£ D~ s~~'n\~<;\otJ Ob MeJL- .2.00S wo -2- DA-TE 0 ~ ftvJM.. \) I \ S'&1E"ttE:-< 200b Che if Mediterraneo sia, que/la nave che va da so/a, Ira ilfuturo la poesia, nella scia di quei marinai, e quell 'onda che non smette mai, che il Mediterraneo sia... Eugenio Bennato CONTENTS THESIS ABSTRACT-- .----------- 6- CHAPTER I-GENERAL INTRODUCTION ·---------------------7- INTRODUCTION ------------- - 7 - THE GENUS ASPARAGOPSIS ----------------------------------------------------- - 9 - INV ASIJ-E SPECIES -------------------------------------------------------------------- 15 - THE MEDITERRANEAN BASIN ----------------------------- - 17 - THE HISTORY OF ASPARAGOPSIS IN THE MEDITERIUNEAN----------------------- 19- MOLECULAR SYSTEMATICS AND PHYLOGEOGIUPHY ------------------------- 19- PHYLOGENETIC ANALYSIS MARKERS ---------------------------------------- 21 - POPUU T/oN GENETICS MARKERS ------------------------------------------------------- 24 - USEOFTHEMARKERS------------------------------------------------------------------ 26 - RESEARCH STIUTEGY---------------------------------------------------- 27 - CHAPTER II -ASPARAGOPSIS TAXIFORMIS AND ASPARAGOPSISARMATA (BONNEMAISONIALES, RHODOPHYTA): GENETIC AND MORPHOLOGICAL IDENTIFICATION OF MEDITERRANEAN POPULATIONS - 29- ABSTRACT ------------------------------------------------------------------------------------ 29- INTRODUCTION ---------------------------------- - 30- MATERIALS AND METHODS------------------------------------------------------------------------ 33 - Sample collection and preservation ------------------------------------------------------ 33 - DNA extraction and purification -------------------------------------- ------- 36 - PCR amplification and sequencing of DNA marker regions------------------------ 37- Data analysis---------------------,---------------------------------------------------- 39 - REsULTS ------------------------------------------------------------------ 40 - Morphological data ------------------------------------------------------------------------------- 40 - LSU rDNA ---------------------------------------------------------------------- 40 - RuBisCo spacer----------------------------------------------------------- 43 - cox2 -3 spacer ------------------------------------------------------------------------------------------- 44 - DISCUSSION ------------------------------------------------- 46- Genetic identification of tetrasporophytes---------------------------------------------------------- 46 - Cryptic diversity ------------------------------------------------------------------------- 47 - Coexistence of gametophytes and tetrasporophytes of the same species ---------------- 48 - Distribution of the species in the Mediterranean Sea-------------------------------- 49- Comparison among molecular markers ---------------------------------------------------- 52- CHAPTER III - PHYLOGEOGRAPHY OF THE INVASIVE SEA'VEED ASPARAGOPSIS (BONNEMAISONIALES, RHODOPHYTA) REVEALS CRYPTIC DIVERSITY - 54 - ABSTIUCT ------------------------------- - 54- INTRODUCTION -------------------------------------------------------------------------- SS - MATERIALS AND METHODS ------------------------- - 58 - RESULTS ---------------------------------------------- - 64 - Asparagopsis armata---------------------------------------------------- 64 - Asparagopsis taxiformis ---------------------------------------- 66 - Rate constancy among lineages------------------------------------- 69 - Relative rates among the three markers ---------------------------________________ 7J - Molecular clock reconstructions----------------------------------- 72 - DISCUSSION ----------------------------------------------__________ - 73 • Congruence among three markers -------------------------------- ----- 73- - 3 - Phylogeography of A. armata------------------------------------------------------------- 73 - Phylogeography of A. taxiformis. ------------------------------------------------------------------------ 74 - Hybrid origin and incomplete lineage sorting of lineages J and 2----------------------------- 75- Molecular clock calculations in A. taxiformis ------------------------------------------------------ 76- Timing of the divergence between A. taxiformis lineages ----------------------------------------------- 77 - Cryptic invasion of A. taxiformis in the Mediterranean ----------------------------- 78 - CONCLUSIONS-------------------------------------------------------------------------------- 81 - CHAPTER IV - MICROSATELLITE MARKERS IN AN INVASIVE STRAIN OF ASPARAGOPSISTAXIFORMIS (BONNEMAISONIALES, RHODOPHYTA): INSIGHTS IN PLOIDY LEVEL AND SEXUAL REPRODUCTION - 82 - ABSTRACT ------------------------------------------------------------------------------------------- 82 • INTRODUCTION ---------------------------------- •• -.---------------------.--------------------. 83 - MATERIALS AND METHODS --------------- ••--------.------------.-----.------------ 85 - Collection of material and unialgal culture--------------·---------------------------------------------- 85 • DNA extraction -.--------.------------------------------------------------------------------ 86 • Microsatellite development-----------------------------------------------------------------. 86 - PCR optimization and microsatellite amplification and detection ------.---------------------- 87 - Analysis of banding patterns -----------.---------------------------------------------- 89 • RESUL TS --.-------.--.---------.-.---------------- ••---.---.--.-------.---.------.----------- 89 • DISCUSSION -----.------------------------.- •• --------------------------.-.---- •• -.--------------- 92 • CHAPTER V - MICROSATELLITE DIVERSITY IN THE MEDITERRANEAN INVASIVE SEAWEED ASPARAGOPSIS TAXIFORMIS (BONNEMAISONIALES RHODOPHYTA). - 97 - ABSTRACT ----.-.------ ••• ----.---------------------------------------------.----------------- 97 • INTRODUCTION --------------.----- ••---------------------------------------------------------- 98 - MATERIALS AND METHODS---·-----··--····-------------------------------------------------------------- - 101 - Sample collection and storage ---------------------------------------------------------- - J 01 - DNA extraction ------------------------------------------------------------------------------- - 103 - Scoring of polymorphisms --------------------------------------------------------------------------- - 104 - Analysis of genotypic diversity---------------------------------------------------------- - J 05 - Analysis of genetic structure ------------------------------------------------------------------- - J 08 - Molecular variance----------------------------------------------------------------------------- - J 08 - Bayesian analysis of molecular variance------------------------------------------ - J 08 - Bayesian clustering---------------------------------------------------------------------------------- - J 09 - Principal coordinate analysis. spatial autocorrelation. Mantel tests. assignment tests ------- - J 10- REsULTS ---------- -------------------------- - 110 - Analysis of genetic structure ------------------ - 1/6 - Molecular variance---------------------------------------------------------------- - 1/6 - Bayes ian clustering------ --------------------------- - 1/9 - Spatial analysis ------- ---------------------------------- - J20- DISCUSSION ------------ ---------------- - 122 - Genetic structure --------- ---------------------- - 125 - SUMMAR Y----.------------------- ---------------------------------- - 129 - CONCLUSIVE REMARKS AND FUTURE PROSPECTS----------------------------- - 133- REFERENCES----------------,------------------------135- APPENDIX 1 · 165_ PROGRAMS AND EQUATIONS USED FOR PHYLOGENETIC AND POPULATION GENETICS ANALYSIS---- - 165 - Software ----------------------------------- - 165 - -4- Equations--------------------------------------------------------------------------------------- - J 66 - APPENDIX 2 --- -----------------168 - NEI'S GENETIC DISTANCE AMONG POPULA TIONS CALCULA. TED IN CHAPTER V--------------- -168 - APPENDIX 3 ----------- --------------------------169- TECHNICAL REMARKS ------------------------------------------------------------------------- -169- Sampling strategy-------------------------------------------------------------------------------------