Phylogeography and Cryptic Speciation in the Bivalved Sea

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

Biological Sciences

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 species (Krug et al., 2007; Lindsay and Valdés, 2016; Table 1). This is a common occurrence in various groups of heterobranch gastropods, particularly in taxa with large, disjunct ranges (Carmona et al., 2011; Lindsay and Valdés, 2016; Pola et al.,

2012; Valdés et al., 2018).

The genus Julia Gould, 1862 is a group of sacoglossans belonging to the family

Juliidae E. A. Smith, 1885, which includes morphologically unique gastropods possessing bivalve shells. When Juliidae shells were first discovered, they were mistakenly classified within Bivalvia based on morphological characteristics (Gould,

1862). It was not until the discovery of a live specimen that Juliidae was reclassified into

Gastropoda (Kawaguti and Baba, 1959; Kay, 1962). Analyses of morphological data were traditionally used to understand relationships between the extant genera (Berthelinia

Crosse, 1875 and Julia), and their species (Kawaguti and Yamasu, 1982). Recently, a

1 synergy of molecular and morphological data were used to create a species-level phylogeny for Juliidae (McCarthy, 2017). Based on this work there are six valid species of Julia (Julia exquisita Gould, 1862, Julia japonica Kuroda & Habe, 1951, Julia burni

Sarma, 1975, Julia zebra Kawaguchi, 1981, Julia mishimaensis Kawaguti & Yamasu,

1982, and Julia thecaphora Carpenter, 1857). There are also three additional undescribed candidate species recovered from molecular analyses (McCarthy, 2017; Table 1). All of these species were discovered and delimited using molecular data, but were based on a limited number of ethanol-preserved samples.

With newly modified protocols, we have been successful at procuring molecular data from dry museum samples that allow greater representation across the range of Julia species available (Figure 1). McCarthy (2017) used a very large collection of dry material to identify specimens to the species level based on morphological characteristics. This resulted in most species having unusually large geographic ranges, often overlapping and some stretching from the Hawaiian Islands to East Africa. This is problematic, because other studies on heterobranch sea slugs have revealed species complexes (Table 1) of cryptic and pseudocryptic species after analyzing molecular data and uncovering morphological differences in groups with large distributions (Carmona et al., 2011;

Lindsay and Valdés, 2016; Pola et al., 2012; Valdés et al., 2018). Therefore, investigating the genetic structure of species of Julia using two mitochondrial genes and one nuclear gene may reveal additional species in these widely distributed taxa.

Julia Candidate species • (McCarthy, 2017) 0 Julia burni • Julia mishimaensis 0 Julia zebra • Julia exquisita w

Midway Atoll, Hawaii 5

French Frigate Shoals, Hawaii

3 2 r, Oahu, Hawaii 4 3 6 4 Maui, Hawaii Philippines Saudi Arabia 3 2 2 12 2 5 3 J.~ Island of Hawaii, Hawaii Kauai, Hawaii Papua New Guinea

8 2 Aese, Mavea, Turtle Island, Vanuatu 9 9 8 4 Aoré, Tangoa, Malo, Palikulo, Urélapa, Tutuba, Segond, Vanuatu

4 Rapa, French Polynesia 6 Lifou, New Caledonia Noumea, New Caledonia

2 4 6 2 2 29 10 11 19 Koumac, New Caledonia 5 Madagascar 25 Mozambique

Table 1. Definitions of frequently used terms in this paper.

Species (Phylogenetic The smallest monophyletic group with a common ancestor Concept) (Queiroz and Donoghue, 1988)

Species complex A group of closely related species often difficult to differentiate

Clade A monophyletic group of organisms

Candidate Species A likely species based on sufficient evidence

Cryptic Species Species that look identical morphologically, but differ genetically

Pseudocryptic Species Species that are found to have morphological distinctions after applying genetic tools

MATERIALS AND METHODS

Source of Specimens

A total of 205 museum samples were successfully extracted and used for molecular analysis in this study (Table 2). Of those, 79 of these samples were preserved in 70-95% ethanol and the rest were dry and unpreserved. One Edenttellina specimen was used as an outgroup because it is the sister genus of Julia. The specimens providing wide geographical coverage were provided by a diversity expedition to Koumac, New

Caledonia and the following museums: National Museum of Natural History, in Paris

(MNHN), Natural History Museum of Los Angeles County (LACM), California

Academy of Sciences (CASIZ), and The Florida Museum of Natural History (FMHN).

Table 2. List of specimens used for this study including complex, species, isolate, locality and genbank accession number information.

Genbank Accession Numbers Complex Species Isolate Locality CO1 16S H3

SMM26 Libonana Beach, Madagascar pending pending pending

Sainte-Luce, SW Ilot Souillac, SMM21 pending pending pending Madagascar Candidate Species 1 Sainte-Luce, SW Ilot Souillac, SMM55 pending pending pending Madagascar

Sainte-Luce, SW Ilot Souillac, SMM181 pending pending pending Species Madagascar Complex 1 SMM39 Austral Islands, SW ile Rarapai Rapa pending pending pending

SMM49 Austral Islands, Pointe Mei, Rapa - pending - Candidate Species 2 SMM64 Austral Islands, Pointe Mei, Rapa - pending pending

SMM40 Austral Islands, Rapa - pending pending

SMM38 Rasch Passage, Papua New Guinea pending pending pending

Panglao Island, Pontod Lagoon, SMM57 - pending - Philippines

SMM197 Midway, Hawaii pending pending -

SMM190 Sand Island, Midway, Hawaii - pending pending

SMM199 Kapa‘a Beach Park, Hawaii pending pending pending

SMM191 Sand Island, Midway, Hawaii - pending -

Candidate SMM200 Hawaii pending pending pending Species 3

SMM193 Sand Island, Midway, Hawaii - pending -

SMM192 Sand Island, Midway, Hawaii - pending -

SMM201 Puako Bay, Hawaii pending pending pending

SMM198 Puako Bay, Hawaii - pending -

SMM194 New Caledonia - pending -

SMM36 N Sek I., Papua New Guinea - pending pending

SMM22 Segond Channel, Vanuatu pending pending pending

SMM349 Koumac, New Caledonia pending - -

Panglao Island, Pontod Lagoon, SMM58 - pending - Philippines

Panglao Island, Sungcolan Bay, SMM70 - pending pending Candidate Philippines Species 4 SMM186 W. Tangoa I., Vanuatu pending pending pending

SMM23 W. Ella I., Vanuatu pending pending pending

SMM24 W. Tangoa I., Vanuatu pending pending pending

SMM30 Cape Barschtch, Papua New Guinea pending pending pending

SMM353 Koumac, New Caledonia pending - -

SMM35 W Malo I., Vanuatu - pending pending

Palikulo Bay, old Japanese Fisheries, SMM48 pending pending pending Vanuatu

SMM62 W. Ella I., Vanuatu pending pending pending

SMM180 N Sek I., Papua New Guinea - pending pending

SMM31 E. Aoré I., Mambeto Point, Vanuatu - pending pending

SMM53 NW Urélapa I., Vanuatu - pending pending

SMM56 N Sek I., Papua New Guinea pending pending pending

Palikulo Bay, old Japanese Fisheries, SMM60 pending pending pending Vanuatu

SMM63 W. Tangoa I., Vanuatu pending pending pending

SMM347 Koumac, New Caledonia pending - -

SMM351 Koumac, New Caledonia pending - -

SMM344 Koumac, New Caledonia pending - -

SMM346 Koumac, New Caledonia pending - -

SMM343 Koumac, New Caledonia pending - -

SMM350 Koumac, New Caledonia pending - -

SMM342 Koumac, New Caledonia pending - -

SMM9 Quest Cap Antsirabe, Madagascar pending pending pending

Candidate SMM208 Libanona Beach, Madagascar pending pending pending Species 5 Red Sea: Ablo Island reef, Saudi SMM178 pending pending pending Arabia

SMM156 N Sek I., Papua New Guinea pending pending pending Julia burni Species SMM205 N Sek I., Papua New Guinea pending pending pending Complex

SMM252 Pamilican I., Philipines pending pending pending Candidate Species 6 SMM362 Koumac, New Caledonia pending - -

SMM363 Koumac, New Caledonia pending - -

SMM177 Kauai, Hoai Bay, Hawaii pending pending pending

SMM130 Baie des Lokaro, Madagascar pending pending -

SMM169 Ile d'Inhaca, Mozambique pending pending -

SMM133 Ile d'Inhaca, Mozambique pending pending -

SMM19 Libanona Beach, Madagascar pending pending -

SMM132 Plage de Lavanono, Madagascar pending pending -

Candidate SMM239 Pinte Flacourt, Madagascar pending pending pending Species 7

SMM115 Libanona Beach, Madagascar pending pending -

SMM134 Ile d'Inhaca, Mozambique pending pending -

SMM109 Ile d'Inhaca, Mozambique - pending -

SMM131 Ile d'Inhaca, Mozambique pending pending -

SMM107 Baie des Galions, Madagascar pending pending -

Julia SMM76 Aoré Island, Aimbué Bay, Vanuatu pending pending - mishimaensis Species SMM322 Koumac, New Caledonia pending - - Complex

SMM126 S Aoré Island, Vanuatu pending pending -

SMM304 Koumac, New Caledonia pending - -

SMM85 Paliko Bay, Vanuatu - pending -

SMM98 Cape Barschtch, Papua New Guinea pending pending pending

Candidate SMM99 E Aoré I., Vanuatu - pending - Species 8

SMM124 NE Tutuba Island, Vanuatu pending - pending

SMM276 SE Matewulu, Vanuatu - pending -

SMM326 Koumac, New Caledonia pending - -

SMM309 Koumac, New Caledonia pending - -

SMM324 Koumac, New Caledonia pending - -

SMM337 Koumac, New Caledonia pending - -

SMM329 Koumac, New Caledonia pending - -

SMM327 Koumac, New Caledonia pending - -

SMM333 Koumac, New Caledonia pending - -

SMM365 Koumac, New Caledonia pending - -

SMM367 Koumac, New Caledonia pending - -

SMM369 Koumac, New Caledonia pending - -

SMM321 Koumac, New Caledonia pending - -

SMM332 Koumac, New Caledonia pending - -

SMM335 Koumac, New Caledonia pending - -

SMM341 Koumac, New Caledonia pending - -

SMM323 Koumac, New Caledonia pending - -

SMM368 Koumac, New Caledonia pending - -

SMM364 Koumac, New Caledonia pending - -

SMM330 Koumac, New Caledonia pending - -

SMM370 Koumac, New Caledonia pending - -

SMM325 Koumac, New Caledonia pending - -

SMM331 Koumac, New Caledonia pending - -

SMM340 Koumac, New Caledonia pending - -

SMM348 Koumac, New Caledonia pending - -

SMM312 Koumac, New Caledonia pending - -

SMM336 Koumac, New Caledonia pending - -

SMM95 NW Urélap a Island, Vanatu pending pending -

SMM103 S Sek I., Papua New Guinea - pending -

SMM83 SW Urélapa Island, Vanuatu pending pending -

SMM97 W Mavéa Island, Vanuatu pending pending -

SMM93 SE Aésé Island, Vanuatu pending pending -

SMM94 Panglao Island, Napaling, Philippines pending pending pending

SMM116 Palikulo Bay, Vanuatu pending pending pending

SMM81 Pamilacan I., Philippines pending pending pending

SMM144 Pamilacan I., Philippines pending pending pending

Candidate SMM141 Panglao Island, Napaling, Philippines pending - - Species 9

SMM140 Panglao Island, Napaling, Philippines pending - -

SMM173 Pamilacan I., Philippines pending pending -

SMM102 W. Malo Island, Vanuatu pending pending pending

SMM92 N Sek I., Papua New Guinea - pending -

SMM82 Kranket I., Papua New Guinea pending pending pending

SMM96 S Sek I., Papua New Guinea pending pending pending

SMM101 Tangoa Island, Vanuatu - pending -

SMM151 N Sek I., Papua New Guinea pending pending pending

SMM152 N Sek I., Papua New Guinea pending pending pending

Candidate SMM74 S. Turtle Island, Vanuatu pending pending - Species 10

SMM84 Elia Island, Vanuatu pending pending pending

SMM91 N Sek I., Papua New Guinea pending pending pending

SMM104 E Malo Island, Vanuatu pending pending -

SMM150 S Sek I., Papua New Guinea pending pending pending

SMM153 Cape Barschtch, Papua New Guinea pending pending pending

SMM154 S Sek I., Papua New Guinea pending pending pending

SMM298 S Sek I., Papua New Guinea pending pending pending

SMM260 Iles Loyaute, Lifou - - pending

SMM263 Iles Loyaute, Lifou - - pending

SMM267 Iles Loyaute, Lifou - - pending

SMM270 Koumac, New Caledonia - - pending

SMM296 Baie du Santal, Lifou - - pending

SMM297 Baie du Santal, Lifou - - pending

SMM262 Koumac, New Caledonia - - pending

SMM268 Iles Loyaute, Lifou - - pending

SMM78 Tangoa Is., Vanuatu pending pending -

SMM264 Tutuba I., Vanuatu pending pending -

SMM284 SE Malo I., Vanuatu - pending -

SMM275 E. Kranket I., Papua New Guinea pending pending pending

SMM283 E Aoré Island, Vanuatu pending pending pending

SMM290 N Sek. I., Papua New Guinea pending pending pending

SMM291 Ile d'Inhaca, Mozambique pending pending pending

SMM293 Mavéa Island, Vanuatu pending pending pending

Julia zebra Candidate Species SMM310 Koumac, New Caledonia pending - - Species 11 Complex SMM300 Koumac, New Caledonia pending - -

SMM316 Koumac, New Caledonia pending - -

SMM359 Koumac, New Caledonia pending - -

SMM311 Koumac, New Caledonia pending - -

SMM306 Koumac, New Caledonia pending - -

SMM303 Koumac, New Caledonia pending - -

SMM320 Koumac, New Caledonia pending - -

SMM317 Koumac, New Caledonia pending - -

SMM315 Koumac, New Caledonia pending - -

SMM314 Koumac, New Caledonia pending - -

SMM319 Koumac, New Caledonia pending - -

SMM299 Koumac, New Caledonia pending - -

SMM301 Koumac, New Caledonia pending - -

SMM307 Koumac, New Caledonia pending - -

SMM308 Koumac, New Caledonia pending - -

SMM313 Koumac, New Caledonia pending - -

SMM318 Koumac, New Caledonia pending - -

SMM305 Koumac, New Caledonia pending - -

SMM265 Bruat Channel, Vanuatu - - pending

SMM294 Lagon de Noumea, New Caledonia - - pending

SMM286 Hawaiian Islands, Hawaii - pending pending

Candidate SMM287 Hawaiian Islands, Hawaii - pending pending Species 12

SMM289 Oahu, Hawaii pending pending pending

SMM209 Palikulo Bay, Vanuatu pending pending pending

SMM358 Koumac, New Caledonia pending - -

SMM214 W. Tangoa I., Vanuatu pending pending pending

SMM18 E. Aoré Island, Vanuatu pending pending pending Julia exquisita Candidate Species Species 13 Complex SMM12 W Tangoa I., Vanuatu pending pending pending

SMM354 Koumac, New Caledonia pending - -

SMM360 Koumac, New Caledonia pending - -

SMM355 Koumac, New Caledonia pending - -

SMM366 Koumac, New Caledonia pending - -

SMM15 E. Luganville, Vanuatu pending pending -

SMM206 E. Aore Island, Vanuatu pending pending pending

SMM228 NE Tutuba I., Vanuatu pending - pending

SMM334 Koumac, New Caledonia pending - -

SMM356 Koumac, New Caledonia pending - -

SMM219 French Frigate Shoals, Hawaii pending pending pending Candidate Species 14 SMM225 Maui, Hawaii pending pending pending

SMM1 Pamilacan I., Philippines pending pending pending

SMM14 Pamilacan I., Philippines pending pending pending

SMM215 Pamilacan I., Philippines pending pending pending

SMM222 Maui, Hawaii pending pending pending

SMM230 Puako Bay, Hawaii pending pending pending

SMM223 Maui, Hawaii pending pending pending Candidate Species 15 SMM224 Madang Province, Papua New Guinea pending pending pending

SMM235 Pamilacan I., Philippines pending pending -

SMM229 Kapa'a Beach Park, Hawaii pending pending pending

SMM226 Maui, Hawaii pending pending pending

SMM231 Midway, Hawaii pending pending pending

SMM220 French Frigate Shoals, Hawaii pending - pending

SMM240 Ile d'Inhaca, Mozambique pending pending pending

Candidate SMM238 Baie de Lokaro, Madagascar pending pending pending Species 16

SMM241 Baie de Galions, Madagascar pending pending pending

Candidate SMM207 E. Luganville, Vanuatu pending pending - Species 17

Candidate SMM361 Koumac, New Caledonia pending - - Species 18

SMM338 Koumac, New Caledonia pending - -

Candidate SMM302 Koumac, New Caledonia pending - - Species 19

SMM339 Koumac, New Caledonia pending - -

SMM236 N BilBil I., Papua New Guinea pending pending pending Candidate Species 20 SMM237 Cape Barschtch, Papua New Guinea pending pending pending

DNA Extractions

DNA from 205 Julia samples was extracted from a majority of dry museum specimens and a handful of ethanol preserved specimens using an E.Z.N.A mollusk kit

(Omega bio-tek, Norcross, GA). Tissue from dry specimens was collected by prying the bivalve shell open under a dissecting scope (Leica EZ4D) and using the whole body for extraction due to the minute sizes of Julia and the high possibility of degraded DNA. For preserved samples, approximately 0.5 mg of tissue was removed from the foot before being minced using a sterile blade. Removed tissue was placed in a 1.7 microcentrifuge tube with ML1 Buffer and Proteinase K Solution then vortexed. Both sample types were left overnight in a hot water bath set at 60ºC to ensure the tissues completely dissolve for better downstream results. After completing the first centrifuge step, if whole bodies were used, the pellet was saved and resuspended in DI H2O to later locate the radulae under the dissecting scope for future research. E.Z.N.A. protocols after this step remain unchanged and are followed through using the manufacturer’s instructions.

DNA Amplification and Purification

Polymerase Chain Reaction (PCR) was used to amplify 2 mitochondrial genes

(CO1 and 16S) and the nuclear gene Histone H3. Universal primers for Histone H3 were used (Colgan et al., 1998) and genus-specific internal primers for CO1 and 16S were designed to increase the amplification success after having low success rates using universal primers (Table 3). The PCR master mix for each sample consisted of 50 µL of the following reagents: 37.25 µl of H2O, 5.00 µl of DreamTaq PCR Buffer, 1.00 µl of 40 mM dNTPs, 2.5 µl of 10mg/ml bovine serum albumin, 1.00 µl of each forward and reverse primer, 0.25 µl of 5mg/ml DreamTaq, and 2 µl of the extracted DNA. To amplify the 410 base pair (bp) internal region of CO1, the following conditions were applied:

95ºC initial denaturation phase for 3 minutes followed by 35 cycles of a 94ºC denaturation phase for 45 seconds, a 39ºC annealing phase for 45 seconds, and a 72ºC elongation phase for 2 minutes, concluded by a final elongation phase at 72ºC for 10 minutes. To amplify the 364 bp region for 16S and the 328 bp region for H3, the following conditions were applied: 94ºC initial denaturation phase for 2 minutes followed by 30 cycles of a 94ºC denaturation phase for 30 seconds, a 59.2ºC annealing phase for

30 seconds, a 68ºC elongation phase for 1 minute, concluded by a final elongation phase at 68ºC for 7 minutes. Successful PCR yields were confirmed through gel electrophoresis with 1% agarose and 2 µl of ethidium bromide. PCR products were run for 30 minutes at

101 volts then checked under a transilluminator for bands. Samples showing successful gene amplification were subsequently purified using a Genejet Purification kit (Thermo-

Scientific, Waltham, MA) following the manufacturers protocols with the exceptions of using 500 µl of wash buffer and 35 µl of elution solution. DNA yields were quantified

using a Nanodrop 1000 spectrophotometer before being sent to Source Bioscience (Santa

Fe Springs, California) for sanger sequencing.

Table 3. Universal primers for Histone H3 and genus-designed primers for CO1 and 16S

Gene Primers Sequence

5’– ATG GCT CGT ACC AAG CAG ACG GC– HexAF 3’ Histone H3 (Colgan et al., 1998) HexAR 5’– ATA TCC TTG GGC ATG ATG GTG AC–3’

5’–ATA ATT TTT GGA ATA TGA TGT GGT Jul1intCO1 CO1 C–3’ (cytochrome c oxidase subunit I) 5’– GAC CAC ATC ATA TTC CAA AAA TTA Jul2intCO1-r T –3’

5’– GTA AAT GGC CGC AGT ACC TTG ACT Jul1int16S G –3’ 16S (ribosomal RNA) 16Sbr-H 5’– CCG GTC TGA ACT CAG ATC ACG T–3’

Species Delimitation Analysis

To perform an Automatic Barcode Gap Discovery (ABGD) analysis, CO1 sequences were placed in the program MEGAX v. 10.1.8 (Kumar et al., 2018) using the

Kimara-2 parameter model to determine genetic distances among those samples. The distance matrix generated by MEGA was input into the ABGD webpage (Puillandre et al., 2012) using the simple distance option to be further analyzed for hypothetical species.

Phylogenetic Analyses

Returned sequences were subsequently cleaned and aligned through Geneious v.

11.1.5 (Kearse et al., 2012) using the MUSCLE v. 3.8.425 (Edgar, 2004) alignment option. Signs of contaminations were checked using the NCBI Basic Local Alignment

Search Tool (BLAST).

The concatenated dataset was analyzed using a Bayesian analysis with the program Mr. Bayes v. 3.2 (Ronquist et al., 2012). The GTR model of evolution with no rate variation across sites (nst=6 rates=equal) was used for the alignments CO1, 16S, and

H3. The analysis ran for 4 chains, 5 million generations with sampling every 100 generations, and a burn-in of 25% using a Markov Chain Monte Carlo method. A

Maximum Likelihood (ML) analysis was also conducted on the concatenated dataset using RAxMLGUI v.1.0 (Silvestro and Michalak, 2012) with 10,000 replications and the

GTR model of evolution. Significant clades were determined based on a posterior probability of 0.90 or greater and bootstrap values of 70 or greater (Hillis and Bull, 1993;

Huelsenbeck and Rannala, 2004; Table 1).

Biogeography

Haplotype networks were created in the program Population Analysis with

Reticulate Trees v. 1.7 (PopART; Leigh and Bryant, 2015), with the TCS package

(Clement et al., 2002) to visualize geographic distributions using the mitochondrial gene

CO1.

Morphological Comparisons

Shell morphological characteristics such as shape, size, color, and pattern were compared to determine if there were consistent differences that could potentially be species-specific. Multiple lateral photos of shells were taken using a Leica EZ4D dissecting microscope and merged together using Photoshop v. 20.0.6 to obtain a detailed image. Both left and right valves (if available) were used to make sufficient comparisons.

RESULTS

Species delimitation

Using the available CO1 sequences, the ABGD analysis recovered 20 candidate species. The candidate species that were recovered coincide with the concatenated

Bayesian and Maximum Likelihood tree of mtDNA and nDNA (Figure 2).

Phylogenetic Analyses

Both the Bayesian and Maximum Likelihood analyses recovered 20 clades consistent with the groups recovered in the ABGD analysis (Figure 2). All resulting clades representing candidate species were well supported, with the exception of one unsupported clade in the Maximum Likelihood phylogeny (PP= 0.98, BS= 56). Within

McCarthy’s (2017) candidate species 2, the phylogenetic analyses conducted here recovered four clades, which together are designated as species complex 1 in this thesis.

Species complex 1 is sister to all other Julia species and was found to include four candidate species: candidate species 1 (Indian Ocean, PP = 1, BS = 100), candidate species 2 (Western Pacific, PP= 1, BS = 98), candidate species 3 (Central Pacific, PP= 1,

BS = 80), and candidate species 4 (Western Pacific, PP = 1, BS = 100). The J. burni species complex considered by McCarthy’s (2017) as a single species, is composed of 2 candidate species: candidate species 5 (Indian Ocean and Red Sea, PP= 1, BS= 91), and candidate species 6 (Western and Central Pacific, PP= 1, BS= 98). Julia mishimaensis, also regarded by McCarthy’s (2017) as a single species, is a species complex with four candidate species: candidate species 7 (Indian Ocean, PP= 1, BS = 100), candidate

species 8 (Western Pacific, PP= 1, BS = 100), candidate species 9 (Western Pacific,

PP=1, BS= 88), and candidate species 10 (Western Pacific, PP= 1, BS= 87). Julia zebra is also a species complex with two candidate species: candidate species 11 (Widespread in the Indo-Pacific, PP= 0.99, BS= 83) and candidate species 12 (Central Pacific, PP= 1,

BS= 94). Finally, J. exquisita was also found to be a complex sister to the J. burni, J mishimaensis, and J. zebra complexes, and is composed of eight candidate species: candidate species 13 (Western Pacific, PP=1, BS= 94), candidate species 14 (Central

Pacific, PP= 1, BS= 100), candidate species 15 (Western and Central Pacific, PP= 0.98,

BS= 56), candidate species 16 (Indian Ocean, PP= 1, BS= 74), candidate species 17 from a single sample (Western Pacific), candidate species 18 from a single sample (Western

Pacific) candidate species 19 (Western Pacific, PP= 1, BS= 100), and candidate species

20 (Western Pacific, PP= 1, BS= 100).

There are many species with overlapping distributions (Figure 3). Julia candidate species 1, 5, 7, and 16 all have representatives in the Indian Ocean, all overlapping in

Madagascar. Julia candidate sp. 7, 11 and 16 overlap in Mozambique, and sp. 5 includes the only representative found in the Red Sea in Saudi Arabia. In the Western Pacific, extensive overlap occurs in the Philippines with candidate sp. 2, 4, 6, 8, 9, and 15.

Overlap also occurs in Papua New Guinea with candidate sp. 2, 4, 6, 8, 10, 11, 15, and

20. Overlap in the Western Pacific also extends to Vanuatu in candidate sp. 4, 8, 10, 11,

13, and 17. Overlap in New Caledonia occurs in sp. 4, 6, 8, 10, 11, 13, 18, and 19. There is one representative located in Rapa, French Polynesia in candidate sp. 2, and one representative located in Lifou in candidate sp. 10. In the Central Pacific, representatives of sp. 3, 6, 12, 14, and 15 inhabit the Hawaiian Islands.

.Juti.can.:naa .. Af) . t

Jul/a species - complex 1 -- ~

J . burni species complex

Jf J . mlshlmaensis species complex •

J . zebra species complex

J. exquisita species complex

Candidate species 1 •Candidate species 2 •0 Candidate species 3 0 Candidate species 4 0 Candidate species 5 0 Candidate species 6 Candidate species 7 •Candidate species 8 •0 Candidate species 9 Candidate species 10 •0 Candidate species 11 Candidate species 12 •Candidate species 13 •® Candidate species 14 @ Candidate species 15 0 Candidate species 16 Candidate species 17 •e Candidate species 18 Midway Atoll, Hawaii 5 Candidate species 19 Candidate species 20

22 •e French Frigate Shoals, Hawaii

Oahu, Hawaii 4 5 2 3 Maui, Hawaii Philippines Saudi Arabia 3 2 Island of Hawaii, Hawaii 2 10 2 2 4 2 Kauai, Hawaii Papua New Guinea

5 3 2 Aese, Mavea, Turtle Island, Vanuatu 8 9 7 4 Aoré, Tangoa, Malo, Palikulo, Urélapa, Tutuba, Segond, Vanuatu

6 Lifou, New Caledonia 4 Rapa, French Polynesia Noumea, New Caledonia

4 6 2 2 2 27 2 10 7 19 3 Koumac, New Caledonia 5 Madagascar 25 Mozambique

Figure 3. Distributions of newly recovered Julia species based on the phylogenetic and ABGD analyses. Colors represent candidate species and numbers indicate the number of individuals of that candidate species if more than 1.

Haplotype Networks

The haplotype network analyses of the COI sequence data recovered haplogroups that correspond to the ABGD analysis and the consensus phylogenetic tree (Figure 4).

The data are highly polymorphic, with many distinct haplotypes within the same haplogroup and few to one representative species for each haplotype.

The haplotype network for Julia species complex 1 contains four distinct haplogroups. Clear geographic structure is found within haplotypes from the Indian

Ocean (Madagascar) and the Central Pacific (Hawaiian Islands). There is no clear structure within the specimens of the Western Pacific.

In the J. burni species complex, there is also differentiation between the haplogroup represented by samples in the Indian Ocean and Red Sea, and the haplogroup represented by samples in the Western and Central Pacific.

In the J. mishimaensis species complex, geographic structure between the Indian

Ocean haplogroup and the three Western Pacific haplogroups is evident. Candidate species 9 only includes samples from the Philippines, however candidate species 8 and 10 include a mixture of localities from the Western Pacific.

The J. zebra species complex includes a haplogroup represented by samples covering the Indo-Pacific and a single diverging sample representing the Central Pacific.

Lastly, within the species complex of J. exquisita, there are a total of eight haplogroups. There is no clear geographic differentiation between each of these haplogroups within the Western and Central Pacific. The only exception is within the candidate species 16 haplogroup, which contains samples from the Indian Ocean.

_,,exqulslta species complex i T --_.. Julia zebra species complex -- ·•--~--•---- •-~•___,__~ -_,. I-lpada11 -- _,. ~I~- --~ _,. - -IIIKiNl2 - -_, ---· Julia mlshlmsensisspecies complex .. .

_, Julia species complex 1 ---· -~10 - ···... .. ·• . _, .. - ---· ······......

Julia bum/ speciescomplex

.!. ! Sep;aration linu . Candldatespecies !. . Loulitlu .. e vi.nuatu-· . . e Pepua,NewGulnea .. ___..._._ ...... • PhillPPfl8S .. ---· .. Rapa . . ·• Hawlllllln-- Islands . . • Mozambique . . e Madaga,

Morphological Analyses

The only morphological traits available for comparisons between candidate species were the shape and color of the external shell, and only one representative per clade was photographed. Differences between species complexes are clear and consistent while differences among the candidate species within the species complexes are not as apparent. Shell sizes were not provided until more data become available. Descriptions based on individuals are illustrated (Figure 2) and described below.

Species Complex 1 Shell Description:

Shell profile ovoid with highest point slightly anterior to center. Rounded anterior, posterior, and dorsal margins; ventral margin rounded and slightly more flattened.

Posterior margins contain shallow notch in middle area separating ventral and dorsal divisions. Dorsal posterior ends of left valves contain protoconch on protruding ends slanted downward. Dorsal ventral ends of left valves rounded and extended past protoconch. Dorsal and ventral posterior ends of right valves rounded with larger ventral ends slightly extending past dorsal end. Shell color bright green to olive green with some having faint lines perpendicular to growth lines.

Julia burni Species Complex Shell Description:

Shell profile ovoid with highest point in center. Dorsal and anterior margins rounded, ventral margin flattened; posterior margin centers contain deep notch separating posterior ventral and dorsal areas. Posterior dorsal margins curve downward and left valves contain protoconch on ends. Posterior ventral end curves slightly upward and distal end comes to a point extending past posterior dorsal end. Shell color bright green to olive green with small white spots spread throughout shell; few faint lines perpendicular to growth lines sometimes present near center.

Julia mishimaensis Complex Shell Description:

Shell profile ovoid with highest point anterior to center. Dorsal and anterior margins rounded, ventral margin slightly to greatly flattened; posterior margin centers contain deep notch separating posterior ventral and dorsal areas. Posterior dorsal margins curve

downward and left valves contain protoconch on dorsal end. Posterior ventral margins curve slightly upward and distal end comes to a point extending past posterior dorsal end.

Shell color light green to olive green with faded lines perpendicular to growth lines; lines sometimes thick, thin, or both present and can be alternating. Faded circular spots present either in between or overlapping the faded lines, ranging from small to large in size; brownish-red spots sometimes present at posterior dorsal end.

Julia zebra Complex Shell Description:

Shell profile ovoid with highest point anterior to center. Dorsal, anterior, and ventral margins rounded; posterior margin centers contain shallow notch separating posterior ventral and dorsal areas with protoconch on end of left valve. Posterior dorsal end short, slanted down. Posterior ventral end slants upward, extending past dorsal end, comes to a rounded point. Shell color light green on posterior end and brownish-red on anterior end, sometimes with brownish-red spot near notch and on posterior dorsal extension. Faint lines clustered on anterior dorsal side perpendicular to growth lines; few lines wider and more spaced out in center; faint circular spots with greyish centers dispersed on ventral area.

Julia exquisita Complex Shell Description:

Shell profile ovoid with highest point in center. Margins highly variable; anterior and dorsal margins rounded, ventral margins greatly rounded to flattened. Notches variable from deep to shallow separating posterior dorsal and ventral areas with protoconch on posterior dorsal end of left valve; posterior dorsal end curved downwards, short to

medium length. Posterior ventral ends variable; slightly to greatly slanted upwards, comes to a point; end can be angular to rounded, extends past dorsal posterior end. Shell color highly variable from off-white to light green to rusty brown; sometimes a brownish- red spot is present near posterior dorsal end. Small spots sometimes present in colors of dark green to dark brownish-red; spots dispersed in a scattered pattern or an organized linear pattern; some with many dots, some with few to none; some spots have short, pale white lines attached and traveling dorsally. Few pale lines perpendicular to growth lines in center of shell can be visible or not present.

DISCUSSION

A basic understanding of the biology and systematics of Julia has eluded scientists since this group was first discovered and placed in the wrong class, Bivalvia.

This problem has been exacerbated by somewhat limited sampling and poor soft tissue preservation. With the increased availability of museum samples and modified methods of DNA extraction from dried remnants of tissue attached to shells, this study represents the first robust attempt at understanding the evolution and biodiversity of this genus by examining its population structure using morphological and molecular data. This study utilizes a large collection of unpreserved samples to reveal that four currently recognized species and one putative species from McCarthy (2017) are all species complexes comprising a total of 20 candidate species. The phylogenetic analyses showed 20 monophyletic clades which agreed with the results of the species delimitation analysis.

Unfortunately, specimens of J. japonica, J. thecaphora, and 2 other recovered candidate species from McCarthy (2017) were unavailable for examination. The results are not entirely unexpected as many of the recognized species of Julia were previously identified solely on morphology, however the sheer amount of species recovered is unusual. Our genetic analyses reveal a vast underestimation of currently recognized diversity within

Julia.

Morphology

The molecular data used in this study have revealed that genetic structure is clearly present in each of the candidate species recovered by McCarthy (2017). However, among these species complexes, morphological differentiation was not evident as most of

the clades within each complex (candidate species) share similar characteristics, with the

J. exquisita complex being the exception. Within the J. complex 1, photographed samples are similar in shape and coloration, with some possessing faint bands perpendicular to growth lines. Representative samples within the J. burni complex have similar shell profiles, with copious light spots spreading throughout the shell, and faint banding perpendicular to growth lines. Members of the J. mishimaensis complex also possess similar shell profiles, coloration, faint banding perpendicular to growth lines that is more detectable in this complex than others, and light-colored spots with variable sizes forming a radiating pattern. Within the J. zebra complex, specimens of both candidate species share similar shapes, have faint colored anterior-dorsal banding perpendicular to growth lines on a brownish-red area, and faint colored spotting located on the center of the shell.

Members of the J. exquisita complex, however, show no consistent morphological characteristics observed among the candidate species. Although many of these candidate species have noticeable morphological differences which could suggest the occurrence of pseudocryptic speciation, we still need to examine more shells to determine if these characteristics are reliable. Since there is only one representative sample examined per candidate species, more representatives of each clade will help in determining whether or not increased morphological information across will reveal consistent morphological patterns and potential identification of synapomorphies for each candidate species.

Biogeography

Many of the species of Julia observed in this study are found sympatrically, with many candidate species collected from multiple locations (Figure 3). As described in our

results, candidate species have overlapping ranges in the Indian Ocean, Western Pacific,

Central Pacific, and some include individuals widespread through the Indo-Pacific. No records of Recent Julia have been reported in the Atlantic Ocean.

To make things slightly more complicated, little ecological information is available on this genus making it difficult to make inferences about their biogeographic distributions. Julia species have been found on different types of green algae (Kawaguti and Yamasu, 1962; Kay, 1962; McCarthy, 2017), and although they have not been observed consuming them, McCarthy (2017) hypothesized that species of Julia are opportunistic grazers. This assumption could potentially explain the overlap in species distributions by minimizing resource competition, allowing them to share resources and prevent competitive exclusion, the concept that species cannot coexist within the same habitat when utilizing the same resource (Hardin, 1960; McCarthy, 2017). However, with the discovery of many new species unbeknownst to previous researchers, it is difficult to determine whether the records of individuals found on different algal hosts were of the same species or one of the newly identified candidate species.

Variability in radular morphology in gastropods can likely be associated with the specialization of an algal host (Ekimova et al., 2019; Jensen, 1997). Preliminary examination of few representative radulae suggest that possible morphological differences may be present, however further study is needed. Therefore, acquiring a sufficient representation of radulae from this genus and identifying any differing characteristics could help researchers understand if species prefer different algal types and could therefore facilitate speciation through niche partitioning.

Larval development is also unexplored in this group. A larval mode of lecithotrophy would mean an organism relies on a supply of yolk before it establishes itself in a habitat. A reliance on a limited supply of nutrition constrains dispersal capabilities (Bouchet and Warén, 1994). In planktotrophic larval modes where organisms feed on plankton and typically have a longer larval stage, some evidence suggests a greater dispersal potential (Bradbury et al., 2008). Investigation into the larval modes of these species could help in understanding how these organisms are able to disperse. A planktotrophic larval mode seems most likely as it could explain the genetically similar but highly widespread populations through gene flow. Multiple candidate species with separated populations are showing few substitutions displayed in the haplotype networks.

Fortunately, Juliidae has a fossil record that has been helpful in attempts to understand biogeographic histories. Members of the subfamily Juliinae, considered ancestors of Julia, were first documented from Oligocene deposits in northern France and diversified during the Neogene period based on fossil dating, distributions, and morphology (Schneider et al., 2008). A previous study that used a molecular clock analysis showed a correlation of divergence times of extant Julia species and the formation of the Hawaiian Islands (McCarthy, 2017). In that same study, it is speculated that historical geological changes caused the distribution of species we see today, however this was with a limited sample size of Julia for a broader scaled question of relationships within the family Juliiidae. Further molecular clock analyses using a larger sample size could support this hypothesis as well bring insight into divergences with land masses outside of volcanic island formations.

Other studies have indicated that the closure of the Tethys seaway, an area connecting the Atlantic and Pacific Oceans, during the Miocene resulted in the cooling regional climates and oceanic environments (Hamon et al., 2013; Harzhauser et al., 2007;

Shevenell et al., 2004). It is also speculated that the changing of oceanic conditions forced organisms to retreat south-easterly to occupy areas that are tropical to subtropical today which may have resembled earlier climates in the Paratethys and Tethys Sea

(Carpenter et al., 2011; Schneider et al., 2008). It is possible that Julia species were able to diversify after populating new habitats in the Indo-Pacific areas where high species diversity is recorded in a variety of marine taxa (Frey and Vermeij, 2008; Gaither et al.,

2011; Malaquias and Reid, 2009).

Numerous Julia species occur within the Coral Triangle biodiversity hotspot, co- existing with one another, as well as in areas outside of the boundary (New Caledonia,

Vanuatu, and Hawaii). Studies of distribution patterns in this hotspot suggest that changes in sea levels during the Pleistocene exposed continental shelves, which may have separated populations leading to vicariant events resulting in diversification of marine taxa (Barber et al., 2016; Carpenter et al., 2011; Gaither et al., 2011). This hypothesis could also be used to explain the high diversity of Julia in this hotspot. Further work using divergence time estimation is required, however, to evaluate this hypothesis.

Examining the reproductive anatomy of these species could also inform us as to why their sympatric relationships persist. Prezygotic mechanical barriers of differing reproductive organs has been noted to prevent admixture within other groups of sea slugs, such as the presence or absence of the bursa copulatrix in Glaucus species (Churchill et al., 2013), and could potentially maintain the coexistence of distinct species in the same

habitats (Coyne and Orr, 2004). Perhaps a future study on Julia that includes dissections could reveal internal variations. Unfortunately, examining internal anatomical characteristics using unpreserved tissue from old samples would prove to be difficult and therefore was not a possibility for this study.

Conclusions

The application of molecular data to groups such as Julia has shown that many cryptic and pseudocryptic species remain undiscovered (Carmona et al., 2011; Krug et al., 2013; Lindsay and Valdés, 2016; Pola et al., 2012; Valdés et al., 2018). As new species are discovered, questions about mechanisms of speciation and biogeographic distributions become more relevant. This study has used a robust sample size from a large collection of mostly unpreserved samples to build a phylogeny recognizing much higher levels of diversity than previously known. This study can be used as a foundation to answer questions pertaining to Julia and possibly to other organisms that share similar geographic distributions. Future studies of this genus include sequencing H3 and 16S data from the New Caledonia specimens as well as exploring morphological variability of shells and radulae to further distinguish whether species are cryptic or pseudocryptic.

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