PHYLOGEOGRAPHY AND CRYPTIC SPECIATION IN THE BIVALVED SEA SLUG GENUS JULIA GOULD, 1862 A Project Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Sandra Muro 2020 SIGNATURE PAGE PROJECT: PHYLOGEOGRAPHY AND CRYPTIC SPECIATION IN THE BIVALVED SEA SLUG GENUS JULIA GOULD, 1862 AUTHOR: Sandra Muro DATE SUBMITTED: Summer 2020 Department of Biological Sciences Dr. Ángel Valdés _______________________________________ Project Committee Chair Department of Biological Sciences Dr. Carla Stout _______________________________________ Department of Biological Sciences Dr. Elizabeth Scordato _______________________________________ Department of Biological Sciences ii ACKNOWLEDGEMENTS First and foremost, thank you to my committee chair, Dr. Ángel Valdés, who has been incredibly helpful throughout the past few years with his constant guidance, encouragement, and patience. I am extremely grateful to have been a part of this lab, as I have learned so much about this field and also myself. I cannot thank Dr. Valdés enough for his leadership and support! Thank you to Dr. Carla Stout for all the help she has given me and inspiring me with her passion for science. Her advice, particularly during these last few months, have been invaluable. I would also like to thank Dr. Elizabeth Scordato for her unwavering encouragement in me pursuing a higher education. I am so appreciative of her help and feedback during the process of my research. To Kendall, Karina, Kim, Ka’ala, and Eric, I want to express my deepest gratitude. You have all been unbelievably supportive and I could not have asked for a better group of people to work with. I would like to acknowledge Sydney and Kairi for taking part in my research while working on their own education. Further, I want to extend a special thanks to my friend Jennifer Alexander for being the person who influenced me to dive into this master’s program. This research was funded by the following: MENTORES (Mentoring, Educating, Networking, and Thematic Opportunities for Research in Engineering and Science), The Society of Systematic Biologists (COA), CSU Council on Ocean Affairs, Science & Technology (COAST), and the Conchologists of America (COA). And finally, thank you to the museums that provided the specimens used for this study as well as the researchers that collected samples from Koumac, New Caledonia. iii ABSTRACT Juliidae E.A. Smith, 1885 is a family of Sacoglossan bivalve gastropods mistakenly placed in Bivalvia when initially discovered. These are the only known gastropods with bivalve shells, making them a morphologically unique group. A recent morphological and molecular study for the entire family Juliidae incorporated 21 samples of Julia (J. exquisita, J. zebra, and J. sp.) from six general localities worldwide and found support for unrecognized species diversity within the genus (three candidate species). Julia species were also previously reported to have interesting disjunct geographic distributions across the Indo-Pacific, often with overlapping ranges. These preliminary molecular results, in addition to their widespread distributions, warranted further investigation into Julia, especially since other recent studies on heterobranch sea slugs have revealed cryptic and pseudocryptic species in groups with large distributions. The objectives of this study were to use molecular sequence data to identify and delineate species of Julia using a more comprehensive representation of individuals across their ranges and supplement these data with morphological analyses of the bivalve shells to aid in potential species descriptions. The majority of the molecular data for this study were obtained from historical collections from several natural history museums, using DNA extraction methods that allowed the use of these dried, unpreserved specimens that were previously thought to yield insufficient or no DNA. One nuclear (H3) and two mitochondrial genes (CO1 and 16S) were used to establish a Bayesian and Maximum Likelihood phylogenetic hypothesis for members of Julia. Haplotype networks using the CO1 gene were created to visualize geographic differentiation. An Automatic Barcode Gap Discovery (ABGD) analysis recovered a total of 20 candidate species that coincide iv with monophyletic clades on the phylogenetic trees. Candidate species of Julia were found to be sympatric and haplotype networks showed little geographic differentiation among disjunct individuals within the same species. Morphological characteristics were found to be different among species complexes, however more data are needed to make conclusions about defining characteristics for candidate species recovered with genetic data. This study not only highlights the importance of museum collections in documenting species diversity, but also provides a framework for studying the evolution and biogeographical patterns of a group whose taxonomy, ecology, and overall biology have proven to be obscure since its initial discovery. This study provides an updated taxonomy of a morphologically interesting group that can help in future research of biogeographic and speciation patterns in other groups of organisms that share similar diversity and distributions. v TABLE OF CONTENTS SIGNATURE PAGE .......................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii ABSTRACT ....................................................................................................................... iv LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii INTRODUCTION .............................................................................................................. 1 MATERIALS AND METHODS ........................................................................................ 5 RESULTS ......................................................................................................................... 19 DISCUSSION ................................................................................................................... 28 LITERATURE CITED ..................................................................................................... 34 vi LIST OF TABLES Table 1. Definitions of frequently used terms in this paper. .............................................. 4 Table 2. List of specimens used for this study including complex, species, isolate, locality and genbank accession number information. ......................................................... 5 Table 3. Universal primers for Histone H3 and genus-designed primers for CO1 and 16S ........................................................................................................................................... 16 vii LIST OF FIGURES Figure 1. Distributions of available Julia species based on morphological identification. Colors represent species and numbers in circles indicate the number of individuals of that species if more than 1. ........................................................................................................ 3 Figure 2. Bayesian tree produced from the analysis of the concatenated genes CO1, 16S, and H3. Clades are outlined to show candidate species found based on the ABGD analysis results. Each supported clade is represented by a photo of one individual. Support values are shown on each branch with posterior probabilities on top of the branch and bootstrap values from the maximum likelihood analysis below. The ABGD histogram below the key shows the interspecific and intraspecific distances .................. 21 Figure 3. Distributions of newly recovered Julia species based on the phylogenetic ABGD analyses. Colors represent candidate species and numbers indicate the number of individuals of that candidate species if more than 1. ........................................................ 22 Figure 4. Haplotype networks produced from all available CO1 sequence data. Colors represent different localities, sizes of circles indicate the number of samples represented in that haplotype, thick dashed lines separate species complexes found from the phylogenetic analyses, and the thinner dashed lines separate the candidate species found from both the phylogenetic and ABGD analyses. ............................................................ 24 viii INTRODUCTION Researchers have expanded knowledge of marine biodiversity and biogeography using a variety of different techniques (Goodheart et al., 2015). However, the diversity of many groups remains greatly underestimated. One example is the order Sacoglossa, which typically includes small and cryptically colored heterobranch sea slug species (Wong and Sigwart, 2019). Many of the recognized species of sacoglossans have previously been identified solely based on morphological characteristics (Jensen, 2006). These characteristics have been also used to build several phylogenetic hypotheses available to date, however many morphology-based classification schemes have underestimated the true number of species (Krug et al., 2008). Reevaluation of morphology-based taxonomies using molecular markers has revealed cryptic and pseudocryptic