DISTRIBUTION, GENETIC DIFFERENTIATION, AND ASSORTATIVE MATING OF DISTINCT MORPHOTYPES OF DIAULULA SANDIEGENSIS, A NUDIBRANCH WITH HIGH DISPERSAL POTENTIAL By Julie Anne Kelly A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology Committee Membership Dr. Sean F. Craig, Committee Chair Dr. Edward C. Metz, Committee Member Dr. Erik S. Jules, Committee Member Dr. John O. Reiss, Committee Member Dr. Michael R. Mesler, Graduate Coordinator May 2013 ABSTRACT DISTRIBUTION, GENETIC DIFFERENTIATION, AND REPRODUCTIVE ISOLATION OF DISTINCT MORPHOTYPES OF DIAULULA SANDIEGENSIS, A NUDIBRANCH WITH HIGH DISPERSAL POTENTIAL Julie A. Kelly Diaulula sandiegensis (Cooper, 1862), a northern Pacific nudibranch, has considerable phenotypic variation in dorsal spotting pattern throughout its range. Experiments were conducted to test the possibility that D. sandiegensis is a complex of unrecognized species, even though it has a planktonic larva with a high dispersal potential. Field and laboratory common garden experiments were conducted to investigate whether length of the individual or diet influence spotting pattern. These investigations found individual D. sandiegensis maintained dorsal spotting morphology, indicating that their dorsal spotting pattern was genetically determined. Field investigations from California to British Columbia and a survey of D. sandiegensis images from the World Wide Web were conducted to describe the variation in spotting pattern of D. sandiegensis and to correlate this variation in dorsal spotting pattern with latitude, depth, and prey. Characteristics from 337 D. sandiegensis were categorized and found to separate individuals into two distinct morphotypic populations, the “many-spotted” (MS) and “few-spotted” (FS) morph, reliably distinguished by presence or absence of mantle-skirt spots, respectively. Dorsal spot number, spot type, ii and background color can also be used to distinguish between the morphotypes, but slight overlap of these characteristics exists between morphotypes. Morphotype frequencies of 433 D. sandiegensis showed a strong correlation to differences in latitude and depth. The FS morph was the only form found south of Fort Bragg (southern region) in intertidal, subtidal, and bay habitats. In addition, the FS morph was the primary morphotype found in the coastal subtidal habitats within the entire study area. The MS morph was the primary morphotype in the coastal intertidal habitat north of Bodega Bay (northern region), comprised of 88 percent MS and 12 percent FS morph. Field investigations also indicated a dietary difference. Diaulula sandiegensis (both morphotypes) from coastal intertidal habitats fed on Haliclona sp. A (Hartman, 1975). A fecal analysis indicated that D. sandiegensis collected from the subtidal habitat of Monterey Bay (FS morphotype) fed on Neopetrosia problematica (de Laubenfels 1930). A laboratory common garden experiment indicated a higher growth and survival rate for MS morph from the Crescent City intertidal habitat than FS morph from the Monterey Bay subtidal habitat, when fed Haliclona sp. A in the laboratory. A mating study and genetic analysis, using mtDNA sequences of the COI gene, were performed. The results showed that the MS and the FS morphs are reproductively incompatible. MS and FS morphs were reciprocally monophyletic at COI, with sequences differing by a p-distance percentage of between 5.9-7.9 percent. Examining behavior, habitat partitioning, and genetic variation clearly indicates that D. sandiegensis is recognizable as two species with distinct ranges, habitat, and prey. iii ACKNOWLEDGEMENTS This work was supported by the NSF grant DBI-0755466 given to Sean F. Craig. I especially thank my husband, Mike Kelly, and daughter, Jen Kelly, for their long hours spent in the tidepools. I also want to thank all of the following faculty, staff, and students from Humboldt State University, the Telonicher Marine Laboratory, and the Research Experience for Undergraduates Program for their guidance, assistance, hard work, and support: S. F. Craig, E. C. Metz, J. O. Reiss, E. S. Jules, G. Eberle, D. Hoskins, A. Baker, K. Korcheck, R. Koeppel, J. Koeppel, S. Monk, E. Martin, and A. Carter. I want to thank the faculty and students from San Jose State University for their assistance with the genetic analysis: J. Mackie and N. Taeidi. I want to thank Bret Grasse from the Monterey Bay Aquarium for his help with the collection of nudibranchs from Monterey Bay. I want to thank D. Behrens, author of Eastern Pacific Nudibranchs, and Dr. B. Penney from Saint Anselm College for the initial idea for this study. iv TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGEMENTS ............................................................................................... iv TABLE OF CONTENTS .................................................................................................... v LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ............................................................................................................ x INTRODUCTION .............................................................................................................. 1 METHODS ....................................................................................................................... 13 Study Organism - Diaulula sandiegensis ....................... 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Change in Spotting Pattern over Time .......................................................................... 13 Data acquisition - field investigations ....................................................................... 17 Data acquisition – laboratory common garden experiment ....................................... 17 Laboratory set up ....................................................................................................... 19 Data analysis .............................................................................................................. 20 Distinct Morphotypes .................................................................................................... 21 Data acquisition ......................................................................................................... 21 Data analysis .............................................................................................................. 21 Morphotypes of Diaulula sandiegensis Relative to Latitude and Depth ...................... 23 v Data acquisition ......................................................................................................... 23 Data analysis .............................................................................................................. 23 Sponge Use Variation.................................................................................................... 23 Data acquisition - field investigation/fecal analysis .................................................. 24 Data acquisition – laboratory common garden experiment ....................................... 25 Data analysis .............................................................................................................. 25 Reproductive Compatibility of Morphotypes Using Mating Studies............................ 26 Data acquisition – field study .................................................................................... 26 Data acquisition – laboratory mating study ............................................................... 26 Laboratory set up ....................................................................................................... 26 Data analysis .............................................................................................................. 27 Phylogenetic Analysis Using COI................................................................................. 28 Data acquisition ......................................................................................................... 28 DNA extraction, PCR, and sequencing ..................................................................... 28 Data sequence analysis .............................................................................................. 30 RESULTS ......................................................................................................................... 31 Change in Spotting Pattern over Time .......................................................................... 31 Laboratory common garden experiment .................................................................... 32 vi Field investigations .................................................................................................... 33 Distinct Morphotypes ..................................................... Error! Bookmark not defined. Spot type .................................................................................................................... 41 Background color ...................................................................................................... 43 Commensal scale worm ............................................................................................. 43 Radula .......................................................................................................................