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A Morphological and Molecular Approach to the Study Of A MORPHOLOGICAL AND MOLECULAR APPROACH TO THE STUDY OF PLANKTONIC PROTIST PHYLOGENETICS WITH A FOCUS ON TAXA FROM OLD WOMAN CREEK NATIONAL ESTUARINE RESEARCH RESERVE A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Hope Ball May, 2006 A MORPHOLOGICAL AND MOLECULAR APPROACH TO THE STUDY OF PLANKTONIC PROTIST PHYLOGENETICS WITH A FOCUS ON TAXA FROM OLD WOMAN CREEK NATIONAL ESTUARINE RESEARCH RESERVE Hope Ball Thesis Approved: Accepted: __________________________________ _________________________________ Advisor Dean of the College Dr. Robert Joel Duff Dr. Ronald F. Levant __________________________________ _________________________________ Committee Member Dean of the Graduate School Dr. Peter Lavrentyev Dr. George R. Newkome _________________________________ _________________________________ Committee Member Date Dr. Donald Ott _________________________________ Interim Department Chair Dr. Richard Londraville ii ABSTRACT Planktonic protists play a vital role in many aspects of the Microbial Food Web including nutrient cycling, grazing controls and primary and secondary production in a variety of different systems including both salt and freshwater. Despite the importance of these organisms, few morphological and molecular studies have been conducted. This study provides both morphological characterization and complete 18S rDNA sequences for a number of single cell isolates of important planktonic protist taxa. Single cell isolates of ten commonly encountered taxa were captured manually using micropipettes. DNA extraction, PCRs, cloning and sequence reactions were performed on these samples in order to obtain complete 18S rDNA sequences. The completed sequences were then manually aligned with existing 18S GenBank sequences of the same or related taxa. Phylogenetic analyses were performed on these alignments to assess the relationships of these sequences to other previously collected samples. Typically, the results of the analyses performed in this study supported the taxonomic relationships observed on GenBank. An analysis of community structure was performed at three of the primary collecting sites at Old Woman Creek National Estuarine Reserve in Huron, Ohio. At least 100 partial 18S sequences were acquired for each sampling location. These sequences were used to explore the community diversity and structure at each site as well iii as for the Old Woman Creek watershed itself. Sequences for common taxa identified from each site were aligned with other related sequences from GenBank. Each of the sampling sites was located at a unique position of the estuary. The diversity of 18S sequences observed from each site was generally reflective of those unique habitat types. The study involved the use of both molecular and morphological techniques to provide correct identification for the species examined. Currently, data available for many abundant planktonic taxa lack combined morphological and molecular information. This study aimed to draw on both detailed morphological and molecular data for species identification and community structure analysis. iv DEDICATION For my parents: Mary Jo and Lawrence Ball. Thank you for always being there. Your love, never ending support, confidence and advice mean the world to me. v ACKNOWLEDGMENTS I would like to express my gratitude to the members of my committee, Dr. Joel Duff, Dr. Peter Lavrentyev and Dr. Donald Ott. Thank you for giving me the opportunity to work on this project with you and for your continued guidance, advice, support and patience throughout the process. Also, I would also like to thank my lab co-workers Traci Branch, Laurie Kay, Chiara Benvenuto, Kristin Green and Ken Moats who offered support and advice and helped with the data acquisition. vi TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................ ix LIST OF FIGURES .............................................................................................................x CHAPTER I. INTRODUCTION...........................................................................................................1 Current Taxonomic Relationships Among Planktonic Eukaryotes ............................4 Importance of Proposed Research ..............................................................................7 II. MATERIALS AND METHODS.................................................................................10 Sampling Sites................................................................................................... ........10 Sampling Techniques.................................................................................................11 Sequence Analysis .....................................................................................................12 Single Cell Sample Treatment ...................................................................................15 Morphology................................................................................................................18 III. PROTIST COMMUNITY STRUCTURE RESULTS AND DISUCUSSION...........21 Individual Site Discussion of Community Structure ................................................22 Combined Site Discussion of Community Structure ...............................................26 Total Community Analysis of Sequences from Five Selected Taxa .......................27 IV. SINGLE CELL CHARACTERIZATION RESULTS AND DISCUSSION ............38 Morphological Analysis of Single Cell Isolates .......................................................42 vii Description and Phylogenies of Single Cell Isolates ................................................42 V. SUMMARY.................................................................................................................73 Community Studies....................................................................................................73 Single Cell analyses...................................................................................................75 REFERENCES ..................................................................................................................77 viii LIST OF TABLES Table Page 1 Primers Applied in Single Cell 18S Acquisition .....................................18 2 GenBank Accession and Collection Data, Where Available, for Samples Included in Strobilidium sp. Total Sequence Alignments...........28 3 GenBank Accession and Collection Data, Where Available, for Samples Included in Strombidium sp. Total Sequence Alignments..........30 4 GenBank Accession and Collection Data, Where Available, for Samples Included in Cryptomonas sp. Total Sequence Alignments.........32 5 GenBank Accession and Collection Data, Where Available, for Sample Included in Chlamydomonas sp. Total Sequence Alignments......34 6 GenBank Accession and Collection Data, Where Available, for Samples Included in Rotifer Total Sequence Alignments..........................36 7 OWC Sampling Information……………………………………………..39 ix LIST OF FIGURES Figure Page 1 Map of Old Woman Creek Sampling Locations.......................................11 2 Schematic for DNA Extraction Used for SSU Molecular Identification of Individual Protists……………………………...............17 3 Comparison of Community Composition from Site 1 Based on 18S Sequence BLAST Searches Grouped into Major Categories as Defined in the Text................................................................................23 4 Comparison of Community Composition from Site 3 Based on 18S Sequence BLAST Searches Grouped into Major Categories as Defined in the Text................................................................................24 5 Comparison of Community Composition from Site 4 Based on 18S Sequence BLAST Searches Grouped into Major Categories as Defined in the Text................................................................................25 6 Comparison of Community Composition from All Three Sites Based on 18S Sequence BLAST Searches Grouped into Major Categories as Defined in the Text................................................................................27 7 Phylogram Resulting from a Distance Analysis of Strobilidium sp. Sequences..................................................................................................29 8 Figure 8: Phylogram Resulting from a Distance Analysis of Strombidium sp. Sequences.......................................................................31 9 Phylogram Resulting from a Distance Analysis of Cryptomonas sp. Sequences..................................................................................................33 10 Figure 10: Phylogram Resulting from a Distance Analysis of Chlamydomonas sp. Sequences……………………………………........35 11 Phylogram Resulting from a Distance Analysis of Rotifer Sequences………………………………………………………………..37 x 12 Photograph of Lugol Preserved Dileptus sp. ….……….………………..44 13 Phylogeny for Dileptus sp. Based on a Maximum Parsimony Analysis. C.I. = 0.9160, Number of Shortest Trees = 1, Total Length = 250……………………………………………………….…….46 14 Photograph of Protargol stained Codonella sp…………….…………….47 15 Phylogeny for Codonella sp. Based on a Maximum Parsimony Analysis. C.I. = 0.8246, Number of Shortest Trees = 1, Total Length = 268……..………………………………………..…………….48 16 Photograph of Protargol stained Synura sp................................................50 17 Phylogeny for Synura
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