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A Barrel of Sponges: a Xestospongia Testudinaria Species Complex in the Verde Island Passage, Philippines”

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A Barrel of Sponges: a Xestospongia Testudinaria Species Complex in the Verde Island Passage, Philippines” “A BARREL OF SPONGES: A XESTOSPONGIA TESTUDINARIA SPECIES COMPLEX IN THE VERDE ISLAND PASSAGE, PHILIPPINES” A Thesis submitted to the faculty of A* San Francisco State University In partial fulfillment of the requirements for ‘fylOlr the Degree Master of Science In Biology: Ecology, Evolution, and Conservation Biology by Joseph Elliz B. Comendador San Francisco, California January 2020 Copyright by Joseph Elliz B. Comendador 2020 CERTIFICATION OF APPROVAL I certify that I have read A BARREL OF SPONGES: A XESTOSPONGIA TESTUDINARIA SPECIES COMPLEX IN THE VERDE ISLAND PASSAGE, PHILIPPINES by Joseph Elliz B. Comendador, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Biology: Ecology Evolution and Conservation at San Francisco State University. Terrence M. Gosliner, Ph.D. Research Professor Rich Mooi, Ph.D. Research Professor Sarah Cohen, Ph.D. Professor A BARREL OF SPONGES: A XESTOSPONGIA TESTUDINARIA SPECIES COMPLEX IN THE VERDE ISLAND PASSAGE, PHILIPPINES Joseph Elliz B. Comendador San Francisco, California 2020 The giant barrel sponge Xestospongia testudinaria is a dominant member of the coral reef community in the Verde Island Passage (VIP) in the Philippines. However, little is known about its morphology and gene flow. Barrel sponge morphotypes, osculum diameter, body circumference, body length, spicule length, and spicule width were all recorded, analyzed and combined with mitochondrial COI gene results to establish a possible pattern useful to understand or refute the morphological concept of barrel sponge classification. ABGD analysis revealed four haplotypes for the VIP. Spicule variation showed no significant differences and no correlation among sites and haplotypes. On the other hand, colony dimensions showed no significant difference and no correlation among sites and haplotypes. Colony form showed significant variation within and among sites and significant differences linked with haplotypes. My research findings agree with global studies on barrel sponges, showing the vast majority of Xestospongia (including X. muta from the Caribbean) are of the same haplotype. The lack of variation between the common haplotype of X. testudinaria and X. muta suggests that X muta from the Caribbean is the same species as X testudinaria. Interestingly, the Coral Triangle region of the Indo-Pacific showed greater variation in haplotypes, especially within Indonesia and the Philippines than between these areas of the Coral Triangle and the rest of the world. I certify that the Abstract is a correct representation of the content of this thesis. October 28,2019 Date iv ACKNOWLEDGEMENTS I would like to thank first and foremost the Almighty God, for giving me this opportunity and for keeping me safe throughout my two years of stay in San Francisco, California. I extend my thanks to my thesis committee, Dr. Rich Mooi, Dr. Sarah Cohen, Dr. Dirk Erpenbeck (LMU-Germany) and especially to Dr. Terrence Gosliner, for all the time, patience and guidance that they have provided throughout this project. I wish also to thank my dive guide, PJ Aristorenas, and his wife Jaichelle, who helped during fieldwork. Also, I would like to thank the Center for Comparative Genomics, especially Dr. Athena Lam and Boni Cruz for assistance in analyzing the molecular data, troubleshooting during sequencing and for patiently guiding me. I would like to extend my deepest gratitude to the CAS wet laboratory team, headed by Christina Piotrowski, with Elizabeth Kools and Johanna Loacker, for providing me lab space, chemicals, and compound microscope. Special mention goes to Dr. Gary Williams for help in the SEM laboratory. I thank my Gosliner/Mooi lab mates: Lynn Bonomo, Alexis Coles, Lisa Paggeot, Kelly Larkin, Kelly Markello, Francis Armstrong, Emily Otstott and especially to Dmitri Smirnoff and Sam Donohoo for their ideas, expertise, and guidance. The management of the National Museum of the Philippines allowed me to pursue my degree abroad. The endless support of my family, especially of my dear wife Jam, is forever appreciated. Lastly, this research would never have been possible without the support of the Lakeside Foundation, which funded my entire graduate school program at San Francisco State University and California Academy of Sciences. Thank you very much. v TABLE OF CONTENTS List of Tables.......................................................................................................................... vii List of Figures ..............................................................................................................viii List of Appendices.....................................................................................................................x Introduction................................................................................................................................ 1 Impact Statement...........................................................................................................5 Hypothesis..................................................................................................................... 6 Methods..................................................................................................................................... 7 Rationale........................................................................................................................7 Collection and Examination of Barrel Sponges in the Field.................................... 7 Identification of Sponge Morphological Types........................................................11 Slide Preparation, Spicule Measurement and Scanning Electron Microscopy 12 Molecular Work: DNA Extraction, PCR, Gel Electrophoresis, PCR Purification, ExoSAP-IT, Cycle Sequencing and Ethanol Precipitation......................................15 Preparing Sequence Reactions for the ABI Prism 3130..........................................19 Mitochondrial Data Analysis and Interpretation...................................................... 19 Results...................................................................................................................................... 20 A. Morphological Types .........................................................................................20 B. Spicule Measurements.........................................................................................25 C. Mitochondrial DNA Analysis..............................................................................32 Discussions...............................................................................................................................40 References................................................................................................................................47 Appendices...............................................................................................................................52 LIST OF TABLES Table Page 1. Recipe sample for a 25 p.L PCR reaction....................................................................18 2. One Way ANOVA analysis of mean body height for each dive site.............22 3. One Way ANOVA analysis of mean sponge osculum diameter per dive site . 22 4. One Way ANOVA analysis of mean sponge body circumference per dive site . 22 5. One Way ANOVA analysis of mean spicule length and width per dive site . 25 6. Tukey’ s post hoc test (spicule length).........................................................................26 7. Tukey’s post hoc test (spicule width)......................................................................... 26 8. One Way ANOVA ofX. testudinaria spicule length and width from El Pinoy 28 9. Tukey test of X. testudinaria spicule length and width from El Pinoy....................39 10. One Way ANOVA ofXestospongia testudinaria spicule length and width from Dead Palm...............................................................................................................................30 11. Tukey test of X. testudinaria spicule length and width from Dead Palm................ 31 12. Haplotypes of X. testudinaria from the Verde Island Passage Philippines 32 13. Nucleotide difference for mitochondrial marker COI...............................................43 14. VIP Haplotypes in relation to morphotypes, spicule length, and spicule width 44 LIST OF FIGURES Figures Page 1. An example of a bleached and normal barrel sponge...............................................5 2. Field collection sites in Mabini-Tingloy and Isla Verde in Batangas Province 9 3. Sponge fragments being taken using an apple corer and ecological parameters being recorded...............................................................................................................9 4. Sponge body circumference (left) and osculum diameter ( ght) being measured and recorded...............................................................................................................10 5. Sponge sample taken with an apple corer................................................... 10 6. Morphological types of Xestospongia testudinaria in the Verde Island Passage....................................................................................................................... 11 7. Spicule length measured with a calibrated eyepiece graticule............................... 13 8. An SEM image of oxea spicules.............................................................................
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