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August 2010 Genetics of the snake star Astrobrachion constrictum ( Ophiuroidea: Asteroschematidae) in Fiordland, New Zealand Debbie Steel ~- '.> A thesis submitted for the degree of Master of Science i' in Marine Science at the University of Otago, Dunedin, ~ New Zealand "-.. >- April 1999 Abstract This thesis was initiated to address two of the questions posed by Stewart's 1995 study on the biology of Astrobrachion constrictum; Is gene flow between fiords restricted in this ophiuroid species and are the colour morphs as different genetically as they are visually? Specimens of A. constrictum were collected from four sites within Doubtful Sound and one site in each of Nancy Sound, Preservation Inlet and 7 Chalky Inlet. The genetics of these populations of the snake star were assessed using mitochondrial DNA and allozyme analysis. These results were analysed using G-test and AMOVA. Both G-test and AMOV A anlaysis showed that there was no significant genetic differentiation between populations within the same fiords (G[21J = 11.97; P > 0.9) (Fsc = -0.037, P > 0.05) or among fiords (G[21J = 23.32; P > 0.2) (FsT = 0.013, P > 0.05). This result was unexpected as the Fiordland environment appears to present several barriers to dispersal, and genetic differentiation of Fiordland populations has been demonstrated in other echinoderm species. Genetic differences between the five colour morphs of A. constrictum were also assessed using mitochondrial DNA and allozyme analysis. G-test and AMOVA analysis of these :~ results also showed no significant differences between colour morphs (G[lSJ = 13.88; P > 0.9) (FsT = -0.089, P > 0.05) indicating that they are conspecific. While studying the population genetics of the snake star two individuals were discovered which > appeared to be heteroplasmic. Through the use of PCR cloning, SSCP and sequencing, the presence of heteroplasmy within these two individuals was demonstrated. This is the first reported case of heteroplasmy in an ophiuroid species and probably arose - through paternal leakage. Cytochrome Oxidase I sequence obtained from the population analysis was also used in conjunction with sequences from Genbank to ;,- assess the phylogeny of echinoderms. Several studies have addressed echinoderm phylogeny over the past century but have failed to clarify the relationship between the echinozoa ( echinoids and holothuroids ), ophiuroids and asteroids. A relatively fast evolving gene was used in this study in an attempt to clarify this relationship. Of the nineteen trees generated with PAUP (Swofford, 1993), only one gave a topology 11 indicating monophyly for all five classes. This was trimmed and compared with three previous hypotheses using the Kishino-Hasegawa test. Results from this showed no significant difference between hypotheses. This is probably due the large amount of noise introduced into the study through the use of a rapidly evolving gene. 7 'l :, ,~ I>-..., }- ill Acknowledgements There are just a few people who I would like to thank for all their effort in helping me with this wee project of mine. First, my supervisors, Dr Brian Stewart, Dr Graham Wallis and Dr Steve Trewick, 7 thanks for all your patient guidance and words of wisdom over the years. ,, Thanks to those who helped with the collection of all my wonderful stars, and made trips into Fiordland an interesting and fun time, Paul Meredith, Les Raj and Brian. A special thanks to Pete Stratford who did most of the collecting while I sorted myself out underwater! Thanks also to Jo and Nicki for help in fetching my samples from the deep south. Big thanks to people who helped me in the lab, Karen I would have been lost without you, Marty you made the lab meetings almost fun to attend and PAUP easy .... .ish to understand!! Steve, without you pushing me all the way I doubt I ever would've ,. finished. Finally thanks to the Warrender St crew and Mikey who housed me during my write up, Susan that massage was fantastic, and a big thanks to Harne. ;,_ '--< Cheers!!! ;,- IV Table of Contents List of Tables Page No . .. .. • .• . .. .. .• .. .. .. •. .. .. .. .. .. .. .. .. • . .. · · · · · · ·· · Vil List of Figures . .. v111 List of Abbreviations . .. IX Chapter One: Introduction......................................................... 1 Methodology . 5 Chapter Two: Population genetics of the sea snake star Astrobrachion constrictum in Fiordland New Zealand. 7 Introduction . 7 Materials and Methods . 11 Sample collection and storage . 11 DNA Analysis . 13 Allozyme Electrophoresis . 16 Results.......................................................... 19 " DNA Analysis . 1 9 Allozyme Analysis . 2 4 ;> Discussion . .. 25 ~ c, Chapter Three: Evidence of Germ-line Heteroplasmy in the Mitochondrial DNA of the brittle star Astrobrachion ~>----< constrictum (Ophiuroidea: Asteroschematidae ) ......... 28 Introduction ............................ : . 28 Materials and Methods . 3 0 Results......................................................... 32 Discussion...................................................... 35 V Chapter Four: Echinoderm Phylogeny ... ... .. .. ... .. .. ... .... .. .. .. .. 37 Introduction . 37 Materials and Methods . 39 Results......................................................... 42 Discussion..................................................... 56 Chapter Five: General Discussion.............................................. 58 References . 61 AppendixA: Collection Information.............................................. 76 Appendix B: Buffer and Stain Recipes......................................... 77 Appendix C: Enzymes Assessed . .. 79 \> c, ... t, Vl List of Tables ,) Page No. Table 2.1: Summary of haplotype data. 19 Table 2.2: PGI allele frequency results for fiords. 24 ') Table 4.1: Species of echinoderm used in this analysis. 40 ,), Table 4.2: Summary of relationship between data and weighting schemes used and which analyses were conducted. 41 'I Table 4.3: Distance information for the reduced taxa list. 43 " Table 4.4: Results from Kishino-Hasegawa tests. 53 Table 4.5: Results from permutation tests and analysis of random trees. 54 ;, ~,,,. •·. ;·';,- ., vii List of Figures Page No. Figure 1.1: Striped colour morph of Astrobrachion constrictum. 4 Figure 2.1: Estuarine-like circulation pattern within Fiordland. 9 Figure 2.2: Map of the Fiordland area, South Island, New Zealand. 12 '! Figure 2.3: Region of ophiuroid mitochondrial genome showing ,_, location of amplified segments. 13 Figure 2.4: SSCP gel showing haplotypes A, B, D, E and F. 20 'f ., Figure 2.5: Sequence and amino acid data for the eight COI haplotypes. 21 ., Figure 2.6: Minimal spacing network constructed from sequence data. 22 Figure 3 .1: SSCP gel showing heteroplasmic individual and haplotypes. 33 '.• Figure 3.2: Sequence and amino acid data for the three haplotypes present in the two heteroplasmic individuals. 34 Figure 4.1: Echinoderm phylogenies proposed by previous authors. 38 Figure 4.2: Base by base transitions and transversions calculated from the tree in Fig. 4.4. 42 ',, Figure 4.3: Percentage of steps by codon from the tree shown in Fig 4.4. 44 Figure 4.4: MP tree from analysis of the full taxa list data set with non-synonymous weighting. 45 Figure 4.5: Bootstrap analysis of the parsimony tree shown in Fig 4.4. 46 Figure 4.6: An example of the most commonly produced topology ':- from parsimony analysis. 47 Figure 4.7: Tree trimmed from the tree shown in Fig. 4.4. 48 (' Figure 4.8: NJ tree produced from analysis of the full taxa list, unweighted. 49 '} Figure 4.9: Bootstrap of the tree shown in Fig. 4.8. 50 ..::-',"-<( Figure 4.10: NJ tree produced from analysis of the full taxa list j>....t weighted for non-synonymous changes. 51 Figure 4.11: ML analysis ofreduced taxa list weighted for -, non-synonymous changes. 52 Figure 4.12: DNA sequence variation for the region of COI assessed for different levels of taxonomy. 55 Vlll List of Abbreviations mtDNA mitochondrial DNA COI Cytochrome Oxidase subunit I con Cytochrome Oxidase subunit II -" SSCP Single Stranded Conformational Polymorphisms ·j' RFLP Restriction Fragment Length Polymorphism '/ TBE Tris Borate EDTA r -1 TEMED Tetramethy1-ethy lene-diamine AMOVA Analysis of Molecular Variance CAEA Citric acid-aminopropyl morpholine HK Hexokinase PGI Phosphoglucose isomerase PEP Peptidase SOD Superoxide dismutase cs Continental Shelf PE Perkin Elmer NJ Neighbour Joining MP Maximum Parsimony ·.i ML Maximum Likelihood BS Bootstrap \• CI Consistency Index •. ,. RI Retention Index ;,.,.., "~ Ti/Tv Transition/Transversion .. ·;:.·;r .J IX Chapter One Chapter One: Introduction The phylum Echinodermata is one of the best characterised and most distinctive phylum of the Animal Kingdom (Hyman, 1955). Species of this marine phylum are recognised by the _\ "! presence of pronounced radial symmetry, the notable absence