Preliminary Characteristics of Two Pseudocryptic Hermissenda Sea Slug Species

PRELIMINARY CHARACTERISTICS OF TWO PSEUDOCRYPTIC

HERMISSENDA SEA SLUG SPECIES

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Biological Sciences

Austin L. Kaʿala Estores-Pacheco

2020

SIGNATURE PAGE

THESIS: PRELIMINARY CHARACTERISTICS OF TWO PSEUDOCRYPTIC HERMISSENDA SEA SLUG SPECIES

AUTHOR: Austin L. Kaʿala Estores-Pacheco

DATE SUBMITTED: Spring 2020

Department of Biological Sciences

Dr. Ángel A. Valdés Thesis Committee Chair Biological Sciences

Dr. Jayson R. Smith Biological Sciences

Dr. Paul M. Beardsley Biological Sciences

ACKNOWLEDGEMENTS

Mahalo nui loa to my advisor Dr. Ángel Valdés for taking me under his wing and giving me the opportunity to conduct research at Cal Poly Pomona. I have learned so much from him in the last few years. He has been very kind to me from the beginning. I had a rough start to the Master’s program due to an injury that occurred in my first few months in the lab. He guided me every step of the way, and that support never stopped after I healed. I also appreciated the fun and light-hearted environment that he encouraged in the lab. I feel so fortunate to have had the best advisor for me.

I also want to thank Dr. Russell Wyeth for hosting me at the Bamfield Marine

Sciences Centre and for helping me to design and analyze the behavioral experiments that were included in this project. Mahalo to the MBRS-RISE program for my funding, training, and support! Through this program, I was able to travel to Bamfield to conduct the behavioral experiments, and later I had the opportunity to share my research in my hometown of Honolulu. Thank you to Dr. Carla Stout for graciously helping me when I hit a speed bump with my project and for her overwhelming support before, during, and after the writing process. Big thank you to my committee members Dr. Jayson Smith and

Dr. Paul Beardsley for reviewing my thesis and providing helpful feedback. Additional thanks to Jay for letting me conduct behavioral pilots in the aquarium room before I left for Bamfield.

I’d also like to thank my lab mates! This cohort of students (plus honorary member) made my experience in the lab so wonderful and full of laughter. Are we still getting sea slug tattoos? Last but not least, thank you to my family in California, Hawaiʻi,

North Carolina, Colorado, and worldwide for encouraging me to pursue my dreams.

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ABSTRACT

A recent study characterized the common and charismatic sea slug species

Hermissenda crassicornis (Eschscholtz, 1831) as a species complex of three distinct species. Hermissenda crassicornis and H. opalescens (Cooper, 1831) are two pseudocryptic sister species that occur in the Eastern Pacific with overlapping ranges in

Northern California and beyond. These species are considered to be pseudocryptic because their morphological differences were discovered after molecular data was obtained. DNA sequencing revealed genetic divergence between the taxa, and morphological studies showed differences in the cerata coloration and arrangement between species. In this study, we examined molecular and ecological differences between H. crassicornis and H. opalescens to explore the extent of the range overlap, characterize the niche of each species, and investigate behavioral differences that may serve to keep these sympatric taxa reproductively isolated. A haplotype network of the mitochondrial gene COI confirmed that the northern boundary of the range of H. opalescens expanded to British Columbia during the most recent El Niño event in 2015-

2016, and persisted after the strong El Niño event ended. Feeding experiments showed that H. crassicornis and H. opalescens exhibit a preference when consuming prey despite generalist feeding behavior. Mating experiments showed that both H. crassicornis and H. opalescens display assortative mating within species, which suggests the existence of a pre-zygotic reproductive barrier. Species of Hermissenda are important model organisms in neuroscience and other fields. Therefore, understanding the recent evolution of this group has broader impacts in other areas of science.

TABLE OF CONTENTS

SIGNATURE PAGE ...... ii

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... vi

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 6

Animal Collection and Husbandry Parameters ...... 6

Mating Experiments ...... 7

Feeding Experiments ...... 8

Video Review and Behavior Analyses ...... 9

DNA Extraction, Amplification, Sequencing ...... 10

Haplotype Network Reconstruction ...... 12

RESULTS ...... 13

Mating Experiments ...... 13

Feeding Experiments ...... 13

Haplotype Network Reconstruction ...... 14

DISCUSSION ...... 15

REFERENCES ...... 22

TABLES ...... 33

FIGURES ...... 40

LIST OF TABLES

Table 1. Criteria of slug health used in feeding and mating experiments...... 33

Table 2. Criteria of slug behavior used during mating experiment video review...... 34

Table 3. Summary of statistical significance separated by model for mating behavior in

conspecific and heterospecific Hermissenda pairings. Models used individuals as the

null and tested for effect of pairing and individual and effect of pairing by experiment

and individual. There was a significant difference in jousting, aggression, and

copulation behavior based on pairing type. There was also a significant difference in

aggressive behavior based on experiment. Abbreviations: HOHO: conspecific H.

opalescens pairs, HCHC: conspecific H. crassicornis pairs, HCHO: heterospecific

pairs...... 35

Table 4. Results of two-tailed t-tests for two paired invertebrate diets during a series of

two-choice feeding preference trials for Hermissenda crassicornis and H. opalescens.

Hermissenda crassicornis spent significantly more time feeding on Mytilus edulis

over Bugula spp., M. edulis over Metridium senile, and Plumularia setacea over M.

senile. No significant difference was found between the remaining pairs. There was

no significant difference in feeding time for H. opalescens. Abbreviations: BUME:

Bugula spp. vs Mytilus edulis; MEBS: M. edulis vs. Botryllus schlosseri; MEMS: M.

edulis vs. Metridium senile; MSPS: M. senile vs. Plumularia setacea; MEPS: M.

edulis vs. P. setacea, BSPS: B. schlosseri vs. P. setacea; MSBS: M. senile vs. B.

schlosseri; - denotes data not recorded...... 36

Table 5. List of specimens sequenced for this study, including locality, voucher number,

isolate code, and GenBank accession numbers. Abbreviations: CPIC: California State

Polytechnic University Invertebrate Collection; LACM: Los Angeles County

Museum of Natural History; UP16: Bamfield Marine Sciences Centre Undergraduate

Programs; *denotes sequences downloaded from GenBank...... 37

vii

LIST OF FIGURES

Fig. 1. Map of known range of Hermissenda crassicornis, H. opalescens, and H. emurai.

Purple triangles represent range of H. crassicornis. Orange circles represent range of

H. opalescens. Maroon circles represent range of H. emurai...... 40

Fig. 2. Mating Experiment 1 and 2 comparison of Hermissenda pairing treatments with

the proportion of time spent jousting of the oral tentacles. Jousting behavior

determined by criteria in Table 2. Conspecific H. crassicornis pairs and

heterospecific pairs exhibited significantly more jousting than conspecific H.

opalescens pairs...... 41

Fig. 3. Mating Experiment 1 and 2 comparison of Hermissenda pairing treatments with

the proportion of time spent copulating. Copulation behavior was based on mating

criteria in Table 2. Conspecific H. crassicornis and H. opalescens pairs spent a

significant time copulating. Heterospecific pairs rarely copulated. Outlier in H.

crassicornis and H. opalescens heterospecific trials exhibited copulation based on

criteria described...... 42

Fig. 4. Mating Experiment 1 and 2 comparison of Hermissenda pairing treatments with

the proportion of time exhibiting aggression. Aggressive behavior determined by

criteria in Table 2. Conspecific H. crassicornis pairs and heterospecific pairs

exhibited significantly more aggression than conspecific H. opalescens pairs...... 43

viii

Fig. 5. Proportion of time spent feeding (+/- 1SE) on individually offered invertebrate

foods during single diet feeding trials for Hermissenda crassicornis and H.

opalescens. Hermissenda crassicornis spent significantly more time feeding on

Plumularia and Mytilus over the remaining possible diets (Kruskal-Wallis,

Wilcoxon). Hermissenda opalescens spent significantly more time feeding on

Botryllus over Bugula and Mytilus as well as Mytilus over Metridium (Kruskal-

Wallis, Wilcoxon)...... 44

Fig. 6. Proportion of time spent feeding (+/- 1SE) on two paired invertebrate diets during

a series of two-choice feeding preference trials for Hermissenda crassicornis.

Hermissenda crassicornis spent significantly more time feeding on Mytilus edulis

over Bugula spp. M. edulis over Metridium senile, and Plumularia setacea over M.

senile (T-test). Significant pairs are noted with an asterisk (*). No significant

difference was found between the remaining pairs...... 45

Fig. 7. Proportion of time spent feeding (+/- 1SE) on two paired invertebrate diets during

a series of two-choice feeding preference trials for Hermissenda opalescens. There

was no significant difference in feeding pairs (T-test)...... 46

Fig. 8. Haplotype network for Hermissenda crassicornis and H. opalescens using the

mitochondrial gene COI. Circle size is proportional to the frequency of haplotypes

detected. Dashes represent mutations between haplotypes. Black circles indicate

potential unsampled haplotypes...... 47

INTRODUCTION

Cryptic species, or distinct species that were previously classified under the same name, are the result of speciation without changes in morphology. If cryptic species are not identified or separated correctly, this can have adverse effects on the ability to, for example, accurately measure biodiversity, develop conservation plans, treat diseases, and farm crops, among many other things (Bickford et al., 2007). In turn, inaccurate classification of taxa can have economic repercussions, result in loss of biodiversity, or result in failure of management actions. For example, mosquitoes originally believed to be Anopheles gambiae were found to consist of seven cryptic species within the complex, with only some of those species being vectors of malaria and detrimental to human health

(Besansky, 1999). Therefore, species of Anopheles that were not vectors of malaria were being targeted for population control to contain malaria, which may have detrimentally affected their populations.

Sometimes morphological changes that occur with speciation are subtle and difficult to describe until more data is found. Pseudocryptic species are species that are previously thought to be cryptic, but morphological characteristics are found a posteriori that distinguish them from other species (Sáez et al., 2003). For example, the nudibranch

Hermissenda crassicornis (Gastropoda: Heterobranchia) (Eschscholtz, 1831) has been used as a model organism in many fields of research, including studies in classical conditioning (Lederhendler et al., 1986; Etcheberrigaray et al., 1992; Blackwell, 2006), associative learning (Crow and Alkon, 1978; Crow and Alkon, 1980; Richards and

Farley, 1987; Werness et al., 1992; Epstein et al., 2000), and other areas of neuroscience

(Hodge and Adelman, 1980; Takeda, 1982; Crow et al., 2013). Hermissenda crassicornis was recently characterized as a species complex of three distinct species based on molecular and morphological characteristics (Lindsay and Valdés, 2016). The three species were distinguished based on two mitochondrial genes (cytochrome oxidase subunit I or COI, and 16S ribosomal RNA) and one nuclear gene (histone H3).

Morphologically the species differ based on cerata coloration and arrangement.

Hermissenda crassicornis has a white longitudinal stripe along the cerata that is not present in H. opalescens (Cooper, 1831). Hermissenda emurai Baba, 1937 has more distinctly grouped cerata and orange coloration than the other two species. The recent classification of H. crassicornis as a species complex means that all of the studies that use it as a model system now have to be reevaluated.

In addition to molecular and morphological differences, all three species of

Hermissenda differ geographically. Hermissenda crassicornis and H. opalescens are found on floating docks and on rocky habitats from the intertidal zone to 30 meters in depth (Morris et al., 1980) in the Eastern Pacific while H. emurai occurs in the same habitats in the Western Pacific. Lindsay and Valdés (2016) suggested H. crassicornis, ranging from Alaska to northern California, and H. opalescens, ranging from the Sea of

Cortez in Mexico to northern California, exhibited a range overlap over a small geographic area from approximately Point Reyes to Bodega Bay. However, a recent study indicated a range expansion of H. opalescens north of this range into British

Columbia, Canada (Merlo et al., 2018), thus increasing the size of the geographic overlap

(Fig. 1).

Prior to the division of Hermissenda into a species complex, numerous studies had examined the ecology and life history of the taxon (mostly as H. crassicornis).

Hermissenda is known to be a hermaphrodite with long-living planktotrophic larvae

(Harrigan and Alkon, 1978). Zack (1975) first described the interactions between H. crassicornis pairs and found that antagonistic behavior, although not as common as other interactions, may be related to hunger drive or sexual state of the animals. Longley and

Longley (1982) further described the mating behavior of H. crassicornis, including estimated time for contact, foot alignment, and copulation. The copulation time, from alignment of the gonopores to sperm emission and foot separation, lasts about one to two seconds (Longley and Longley, 1982), which is much shorter than the hours or days required for other opisthobranchs (Costello, 1938). Rutowski (1983) claimed that the short copulation time could be an adaptation to minimize contact between animals and avoid cannibalistic behavior. Hermissenda is a generalist feeder (Harrigan and Alkon,

1978), consuming hydroids, tunicates, mussels, other nudibranchs, as well as exhibiting cannibalism (McDonald and Nybakken, 1980; Tyndale, 1994). Despite this broad diet, gut analyses and behavioral studies have indicated that H. crassicornis exhibits some prey selectivity, preferring, for example, Aurelia jellyfish and Disaplia or Aplidium tunicates (Megina et al., 2007; Hoover et al., 2012). However, these previous studies do not distinguish between newly established species of Hermissenda and need to be reevaluated in order to determine if these generalities remain true.

Furthermore, given the range overlap of H. crassicornis and H. opalescens, studies need to be conducted to elucidate the interactions between these species, such as potential reproductive capability, potential for hybridization, and whether they are

utilizing similar resources (niche overlap) or different resources (niche partitioning). The range overlap area may be a hybrid zone, or an area where the ranges of two closely related taxa overlap and hybrid offspring are produced (Barton, 1979). Investigating the reproductive capability and potential hybridization of Hermissenda species may be important for understanding how barriers between closely related species are formed and maintained (Pascarella, 2007; Good et al., 2008; Cristescu et al., 2010; Mitsui et al.,

2011). Since the niches of H. crassicornis and H. opalescens are currently unclear, they may be competing for similar resources or have specialized behavioral characteristics within the range overlap that reduces competition between species (May and MacArthur,

1972; Wilson and Yoshimura, 1994). For example, Doridella steinbergae and Corambe pacifica are two species of dorid nudibranchs that co-occur in Southern California and compete for their bryozoan prey Membranipora membranacea (Yoshioka, 1986). Despite both species having differences in developmental time and feeding rates, the abundance of M. membranacea is variable, which affects the recruitment of D. steinbergae and C. pacifica in the kelp beds and drives competition between species (Yoshioka, 1986). On the other hand, Bloom (1981) investigated six species of dorid nudibranchs in the Pacific

Northwest that exhibited resource partitioning due to differing prey distribution and adaptations to consume sponge prey with various skeletal complexities. Interactions like those seen in other nudibranchs may provide more information about how H. crassicornis and H. opalescens are able to coexist in the range overlap area.

To initiate a better understanding of potential interactions of these two sea slugs, I focused on two specific components. First, I investigated the degree of reproductive isolation between H. crassicornis and H. opalescens through mating behavior

experiments, comparing intra- and inter-specific mating and aggression behaviors. If speciation is complete, then it was expected that the two species would not mate with one another (Coyne and Orr, 2004) and exhibit higher levels of interspecies aggression. If speciation is incomplete, then the species would exhibit increased patterns of interspecific mating, or hybridization (Via, 2001; Coyne and Orr, 2004). Second, I attempted to better understand invertebrate diet utilization of H. crassicornis and H. opalescens through both single food and two-choice preference feeding trials. These prey trials could help to determine whether H. crassicornis and H. opalescens are potential competitors for similar food sources or whether differential consumption, or resource partitioning, may be a driving mechanism for co-existence. In addition to looking at potential interactions between Hermissenda species, I also increased the sample size of the animals from the range overlap area for the gene fragment COI in order to analyze the genetic diversity and the extent of the range overlap. Since Hermissenda crassicornis was recently characterized as a species complex, further delineating these species by investigating intra- and interspecific interactions and genetic diversity may help us to better understand the mechanism of speciation that caused these species to diverge.

MATERIALS AND METHODS

Animal Collection and Husbandry Parameters

Animal collection and care for feeding and mating experiments was approved by the Bamfield Marine Sciences Centre (BMSC) in Bamfield, British Columbia. 56

Hermissenda crassicornis were collected from floating docks, intertidal zones, and subtidal habitats in Barkley Sound, British Columbia between June 17 and 21, 2017.

Although H. opalescens was reported as being abundant in British Columbia in 2016

(Merlo et al., 2018), only one individual was found during multiple collecting efforts.

Therefore, H. opalescens specimens (44 individuals) were obtained by MAC Bio-Marine in Monterey, CA, shipped to Bamfield, and then placed in quarantine in self-contained aquaria. Shipped animals were slowly introduced to quarantine tanks in order to acclimate to new conditions. Hermissenda crassicornis specimens were placed in a circulating, flow-through sea water system from the ocean that received a natural light to dark cycle. For the quarantine aquaria with H. opalescens, sea water was pumped from the ocean and drained onto gravel outside of the facility to prevent contamination of the circulating sea water system. During care and upkeep, animals were individually separated in mesh containers (inner diameter, depth = 4.3, 5.5 cm, respectively). Sea water was maintained at 12°C, 33 ppt, and had air bubbling throughout the tanks. Every other day, animals were fed fresh mussel carcasses (Mytilus edulis) collected from docks at BMSC. Health of the slugs was monitored during and between experiments based on criteria listed in Table 1.

Mating Experiments

To examine behavioral interactions between and within Hermissenda species, a series of mating experiments were conducted in sea tables with the same water conditions and setup as described previously for the quarantine aquaria. A pair of conspecific or heterospecific animals were placed in individual plastic containers with mesh windows

(inner length, width, height = 13, 13, 5 cm, respectively). Lamps were placed above the sea tables to ensure even illumination across the containers. Two GoPro Hero 4 cameras were fixed above the sea tables with time lapse videos recorded at two frames per second for the four-hour duration of the experiments. Six containers were placed below each camera for a total of twelve containers per recording session. Size-matched animals were introduced to randomly-assigned opposite corners of the tub before the trials began.

In the first set of mating trials, eight conspecific H. crassicornis, eight conspecific

H. opalescens, and eight heterospecific pairings were tested for a total of 24 trials. These trials lasted for four hours each and were conducted over the course of two days. In order to prevent potential effects of interactions in subsequent trials, animals were only used once per day and never underwent the same treatment more than once. For example, animals used in conspecific trials on the first day were used in heterospecific trials on the second day and vice versa. Due to the limited number of animals available, a second set of mating trials was conducted with six conspecific H. crassicornis pairs, six conspecific

H. opalescens pairs, and 12 heterospecific pairs for a total of 24 trials. These trials were also conducted over the course of two days to ensure that animals never underwent the same treatment more than once.

Feeding Experiments

In order to compare diets between H. crassicornis and H. opalescens, single- choice and two-choice feeding experiments were conducted in the sea tables described above. Two GoPro Hero 4 cameras were fixed above the sea tables and recorded time lapse photos every 30 seconds for the two-hour duration of the experiments. Each camera recorded six plastic containers at a time. Slugs were starved for 24 hours and placed into individual plastic containers before each trial. In order to prevent effects of previous feeding trials, sea slugs were only used once per day and starved for 24 hours in between usage.

In single-food experiments, approximately six trials of each prey type for both

Hermissenda species were conducted (total n=51). Prey items consisting of anemones

(Metridium senile), tunicates (Botryllus schlosseri), mussels (Mytilus edulis), or bryozoans (Bugula spp.) were placed in the center of each plastic container; an additional prey item, the hydroids (Plumularia setacea), was also used in feeding trials with H. crassicornis only.

For two-choice feeding experiments, combinations of two different prey items were placed in randomly assigned, opposite corners of the plastic containers.

Approximately twelve replicates of combinations of food items were conducted, although there were several occasions with fewer replicates due to slug mortality (total n= 113).

Prey pairings for both Hermissenda species included Bugula spp. vs. M. edulis, M. senile vs. B. schlosseri, M. edulis vs. B. schlosseri, B. schlosseri vs. P. setacea, and M. edulis vs. P. setacea. Due to slug mortality, Mytilus edulis vs. Metridium senile and M. senile vs. P. setacea were only tested for H. crassicornis.

Video Review and Behavior Analyses

Each four-hour mating experiment video was reviewed in VirtualDub and FIJI

(Schindelin et al., 2012), either frame-by-frame or at higher speed. The videos were reviewed to quantify the frequency and durations of typical aeolid reproductive behavior

(Longley and Longley, 1982; Rutowski, 1983), including: a.) jousting, where individuals touch and move their oral tentacles as a type of species recognition strategy (Zack, 1975); b.) copulation, whereby jousting pairs align their feet with the anterior end of one slug lining up with the posterior end of the other slug, after which pairs create a wheel-like formation and raise their cerata as a result of penis ejection, and c.) aggression, defined as any antagonistic behavior between slug pairs that lasts for longer than four seconds, including, but not limited to, tail biting, biting of body, or body slamming, etc. This aggressive behavior may or may not result in copulation.

The duration and frequency data from the mating experiments were analyzed in R v3.4.3 (R Core Team, 2018). Since the same animals were used twice in consecutive experiments, once in conspecific treatments and once in heterospecific treatments, the animals were not considered independent of one another and did not meet the assumptions of standard statistical tests. A linear mixed effects model was used to ensure that there was a random effect for Hermissenda. The model checked if conspecific and heterospecific pairings showed significant differences in behavior and also accounted for anomalous individual behavior. For example, if one individual specimen was particularly aggressive, the model accounted for that behavior. Since two mating experiments were conducted, effect of experiment was also checked to make sure that it was not a significant factor in their behavior.

Feeding videos were analyzed in VirtualDub and FIJI (Schindelin et al., 2012), either frame-by-frame or at higher speed. The amount of time that the sea slug spent on the prey items was used as a proxy for feeding. Any amount of time longer than one frame (two seconds) was recorded and quantified as a feeding interaction. Due to low sample sizes and high variation in the single-choice feeding experiment, a Kruskal-Wallis

Test was performed to see the time each species of Hermissenda spent feeding among the diet options, followed by a comparison of feeding time using the Wilcoxon Method. For the two-choice feeding experiment, paired t-tests were conducted for each prey pairing.

DNA Extraction, Amplification, Sequencing

Samples were collected from several localities within the species ranges:

Hermissenda crassicornis samples were obtained between Point Reyes, California to

Alaska, and H. opalescens were obtained from the Sea of Cortez to British Columbia. All samples were obtained through SCUBA diving, snorkeling, or on floating docks by the authors or were donated by colleagues. Specimens were photographed alive and preserved in 95% ethanol.

DNA was extracted using Chelex©, DNEasy Blood and Tissue kits (Qiagen), and

EZNA Mollusc kits (Omega Bio-tek). For the Chelex extraction protocol, a small piece

of the foot of each animal was cut into fine pieces using a sterilized razor blade. The

tissue was placed into a 1.7 mL microcentrifuge tube filled with 1 mL 1X TE buffer. The

samples were placed on a rotating block for at least 20 minutes to rehydrate the tissue and

to assist in the dissociation process. Samples were vortexed for at least five seconds and

centrifuged at 12578 × for 3 minutes. Then 975 μL TE buffer was removed from the

𝑔𝑔 10

microcentrifuge tube without disturbing the supernatant, and 125 μL of Chelex solution was added to each tube. A water bath set at 56°C was used for at least 20 minutes. After leaving samples in the water bath, the samples were vortexed and placed on a 100°C heating block for eight minutes. For DNEasy and EZNA extractions, the manufacturers’ protocols were used.

The resulting supernatant from the extractions were used for polymerase chain reaction (PCR) amplification. PCR was performed using the universal primers for the mitochondrial gene COI (LCO490 5’-GGTCAACAAATCATAAAGATATTGG-3’,

HCO2198 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al., 1994). The

universal primers did not yield bands for all samples, so additional primers were designed

for Hermissenda (KEP1F 5’-TGGGAATATGATGTGGGTTG-3’, KEP1R 5’-

AGAATAGGATCTCCACCTCC-3’). The following PCR conditions were used to

amplify a 695 base pair region of COI: 95 °C initial denaturation for three minutes, 35

cycles of 1.) 94 °C denaturation for 45 seconds, 2.) 45 °C annealing for 45 seconds, and

3.) 72 °C elongation for two minutes, followed by a final elongation at 72 °C for ten minutes. PCR protocol for designed primers was altered to have an annealing temperature of 55.5 °C. The PCR master mix was prepared with 38.50 μL of deionized water, 5.00 μL of DreamTaq PCR buffer (Fisher Scientific, Hampton, NH), 1.25 μL of 10 mg/μL bovine serum albumin (BSA), 1.00 μL of 10 mM deoxynucleotide triphosphates (dNTPs), 1.00

μL of 10 μM forward primer, 1.00 μL of 10 μM reverse primer, 0.25 μL of 5 mg/μL

Dream Taq (Fisher Scientific, Hampton, NH), and 2.00 μL of extracted DNA template.

Amplification of DNA was confirmed using 1% agarose gel electrophoresis with

ethidium bromide. PCR products were purified using the Thermo Scientific GeneJet

purification kit (Waltham, MA) using the manufacturer’s protocols. DNA concentrations from purified PCR products were measured using a NanoDrop 1000 spectrophotometer

(Thermo Scientific, Waltham, MA). Primers were diluted to 4.00 μM and purified

samples were diluted to 5 –20 ng/μL for sequencing. Diluted primers and purified

samples were sent to Source Bioscience for Sanger sequencing (Santa Fe Springs, CA,

USA).

Haplotype Network Reconstruction

Raw sequences were assembled and edited by eye in Geneious 11.1.5 (Kearse et

al., 2012) and aligned using the MAFFT option for multiple alignments (Katoh et al.,

2002). Haplotype network was generated using COI sequences in PopART 1.7 using the

TCS option (Clement et al., 2010; Leigh & Bryant, 2015).

RESULTS

Mating Experiments

Mating behavior experiments of conspecific and heterospecific Hermissenda pairs displayed some significant differences in the proportion of time spent jousting, exhibiting aggression, and copulating (Linear Mixed Model in R; Table 3). Conspecific H. crassicornis and heterospecific pairs exhibited significantly more jousting behavior than conspecific H. opalescens pairs (Linear Mixed Model in R; Fig. 2). Conspecific H. opalescens pairs exhibit significantly less aggression than conspecific H. crassicornis pairs and heterospecific pairs (Fig. 4). Conspecific H. crassicornis and H. opalescens pairs spent significantly more time copulating while copulation between heterospecific pairs was extremely rare and significantly lower than conspecific copulation (Fig. 3).

Heterospecific pairs would begin the mating process, starting with jousting of the oral tentacles and alignment of the foot, but interactions typically ended in aggression without copulation.

Feeding Experiments

In single-food feeding experiments, the proportion of time H. crassicornis spent on different prey items varied significantly (Wilcoxon Test, p<0.001, df = 4, Chi-squared

= 18.8) with significantly more time spent on Plumularia hydroids and Mytilus mussels than the remaining taxa (Nonparametric Wilcoxon pairwise tests; Fig. 5). Hermissenda opalescens also exhibited significant differences in the proportion of time spent on different food items (Wilcoxon Test, p = 0.007, df = 3, Chi-squared = 12.2). Generally, more time was spent consuming Mytilus and Botryllus tunicates (Nonparametric

Wilcoxon pairwise tests; Fig. 5). Comparing between Hermissenda species suggests that both species share selectivity for Mytilus and avoidance of Metridium. Botryllus is only preferred by H. opalescens. Hermissenda crassicornis preferentially consumes

Plumularia; unfortunately, feeding experiments could not be conducted with this prey for

H. opalescens.

In two-choice feeding preference trials, Hermissenda crassicornis spent significantly more time feeding on mussels over both bryozoans and anemones, and on hydroids over anemones (Fig. 6, Table 4). There were no significant differences in two- choice feeding for H. opalescens (Fig. 7, Table 4).

Haplotype Network Reconstruction

Seven haplotypes were identified for H. crassicornis and 13 haplotypes for H. opalescens from 57 COI sequences (Fig. 8). The most common haplogroup for H. crassicornis includes samples from Northern California, Oregon, Washington, British

Columbia, and Alaska. Most of the other H. crassicornis haplotypes differ from the most common haplogroup by one nucleotide substitution. The most common haplogroup for

H. opalescens includes samples from Baja California, Southern California, Northern

California, and Oregon. Samples with one nucleotide substitution from the most common haplogroup were from Baja California, Southern California, and Northern California.

There were additional substitutions for other samples from Northern and Southern

California and British Columbia. Based on this analysis, H. crassicornis occurs from

Point Reyes, California to Alaska, and H. opalescens occurs from Baja California to

Bamfield, British Columbia.

DISCUSSION

When two closely related taxa, likely filling similar ecological niches, overlap in portions of their ranges, there are numerous potential outcomes that can affect the persistence of these species over evolutionary time, as well as the overlapping populations over ecological time (Knowlton, 1993; Gaither and Rocha, 2013). For example, if closely related taxa can hybridize, this can cause selection against hybrids within hybrid zones (Barton, 1979; Harrison, 1993). Alternatively, these species may have evolved barriers inhibiting interbreeding thus driving competition between the species that are likely utilizing similar resources (Mayr, 1947; Howard et al., 1998).

Competition for these resources could persist as niche overlap (Wissinger, 1992), or alternatively, competition could be reduced through evolved resource partitioning whereby the species are utilizing different resources (Pianka, 1974; Sinopoli et al., 2017).

Historically, Hermissenda crassicornis life histories and ecology has been described similarly for populations along the Eastern Pacific (Beeman and Williams, 1980;

McDonald, 1983; Behrens, 1991). However, this species was recently determined to be a species complex with H. opalescens being a more southern species and H. crassicornis being a more northern species with some overlap in their ranges (Lindsay and Valdés,

2016). To examine biotic interactions between these two species when they overlap ranges, I focused on two interactions: the potential ability of these two species to interbreed and whether or not they consumed similar prey items. Hermissenda crassicornis and H. opalescens did not copulate with one another, suggesting assortative mating by species. Furthermore, H. crassicornis appeared to be more aggressive than its congener thus potentially giving it an advantage. In terms of diet, there was both overlap

in selectivity of certain prey items and patterns of partitioning of different prey utilization, although further studies are needed in order to confirm these results.

Since H. crassicornis and H. opalescens are not mating, there may be a prezygotic barrier that prevents the species from mating (Coyne and Orr, 2004). For example, there may be a chemical or physiological barrier that prevents these species from recognizing each other as potential mates (Paterson, 1980; Palumbi, 1994). Behavioral or sensory differences between species could also contribute to reproductive isolation (Palumbi,

1994; Coyne and Orr, 2004), as well as a mechanical barrier, such as differences in reproductive anatomy like those seen in Glaucus atlanticus and G. marginatus (Coyne and Orr, 2004; Churchill et al., 2013). The lack of mating between heterospecific pairs due to the presence of a prezygotic barrier suggests that the speciation process may be complete (Via, 2001; Coyne and Orr, 2004). These observations expand on previous studies of the antagonistic and mating behavior of Hermissenda (sensu lato) (Zack 1975;

Longley and Longley, 1982; Rutowski, 1983).

The differing diets of Hermissenda species in the Eastern Pacific may provide us with more information about how these closely related species occur in similar environments. Hermissenda crassicornis consumed hydroids and mussels over other prey options in both single- and two-choice experiments. Hermissenda opalescens consumed tunicates and mussels over other prey in single-choice experiments, whereas in two- choice experiments H. opalescens appeared to be more of a generalist. However, due to the limited number of animals available, hydroid single-choice trials were not conducted for H. opalescens, which could affect the hierarchy of prey choice for this species. This study, although only examining a few biotic interactions, suggests that H. crassicornis

may have a more specialized diet compared to H. opalescens, which may allow them to exist in similar environments. For example, Ventura et al. (2000) investigated differences in feeding preferences and niche overlap in three species of sea stars and found that prey partitioning occurred in the range overlap area. Astropecten brasiliensis had the most generalized diet in the overlap area compared to A. cingulatus and Luidia ludwigi, which had more specialized diets. This prey partitioning in a range overlap area could be a behavioral strategy that reduces competition between closely related species. A generalist diet could have additional benefits for H. opalescens, including sequestering the nematocysts for defense purposes while also having the ability to consume other prey for nutritional value (Megina et al., 2007). However, this would need to be further explored.

Contrary to what was reported by Merlo et al. (2018), Hermissenda opalescens was not occurring in abundance in British Columbia when the behavioral experiments were being conducted and needed to be shipped from California. Since California typically has a warmer ocean temperature than British Columbia, this could have caused issues with conducting the experiments due to differences in temperature tolerance between species (Armstrong et al., 2019). The H. opalescens animals may have behaved differently because they were in an environment that was not necessarily suited for them.

Future behavioral studies should be conducted within the range overlap and in a location where both species are occurring in abundance to ensure that the conditions are optimal.

The molecular data presented in this study further confirms the recruitment of H. opalescens into British Columbia partially due to Pacific warming patterns. Based on this study, Hermissenda crassicornis occurs from Point Reyes, California to Sitka, Alaska, and H. opalescens occurs from Baja California to Clayoquot Sound, British Columbia.

The region of sympatry between these species is from Point Reyes, California to

Clayoquot Sound, British Columbia. However, there have been reports of both

Hermissenda species occurring in abundance in Monterey Bay, California (iNaturalist; R.

Agarwal, personal communication, August 22, 2018), so further sampling is required to confirm the extent of the range overlap. In addition to an increase in ocean temperature during warming events, changes in ocean current patterns commonly associated with El

Niño Southern Oscillation (ENSO) events have also contributed to the range expansion of

H. opalescens. For example, the relaxation of the California Current and intensification of the counter current created ideal conditions for species to recruit in areas previously unavailable to them (Lluch-Belda et al., 2005). Climate change, El Niño Southern

Oscillation (ENSO) events, and the marine heatwave commonly known as The Blob have contributed to recruitment and northward range shifts of at least 52 heterobranch species in the Northeast Pacific Ocean, including H. opalescens (Merlo et al., 2018; Goddard et al., 2016; Goddard et al., 2018). Even after the warming events have ended, this poleward shift of Hermissenda opalescens appears to be persisting. However, this needs further confirmation. A review of Hermissenda photographs on iNaturalist (Available from https://www.inaturalist.org) showed that H. opalescens were occurring in British

Columbia as recently as July 2019, and there have also been reports of H. crassicornis occurring further south of their known range in Morro Bay, California in September 2019

(iNaturalist; R. Agarwal, personal communication, November 18, 2019). This southward movement of H. crassicornis could be due to intensification of the California Current in between ENSO events (Lluch-Belda et al., 2005). Goddard et al. (2018) predicts that heterobranch poleward shifts will become more permanent as oceans continue to warm.

Therefore, it is important to continue to monitor these changes in species ranges because it could have effects on the diversity observed in near-shore benthic communities

(Goddard et al., 2018; Schultz et al., 2011).

In addition to showing the persistence of H. opalescens in British Columbia, the molecular data in this study may also provide more information about the evolutionary history of Hermissenda. The haplotype network shows that H. opalescens has more genetic diversity in mtDNA than H. crassicornis. Most of the H. crassicornis samples have the same haplotype with few substitutions, whereas H. opalescens has many haplotypes and substitutions. Since H. crassicornis has lower genetic diversity and a high frequency of a single haplotype, this suggests that H. crassicornis may be displaying a leading edge effect. A leading edge effect occurs when a northern species with lower genetic diversity colonizes a previously unavailable geographic area, which leads to speciation (Hewitt, 2000). Leading edge effects have been observed in many marine species around the world (Edmands, 2001; Tolley et al., 2005; Tolley and Rosel, 2006;

Knutsen et al., 2013). For example, in the Eastern Pacific two recently diverged sea snail sister species of Nucella showed a leading edge effect because the northern species N. emarginata had lower genetic variability than the southern species N. ostrina (Marko,

1998). Despite a large range overlap, speciation was determined to be most likely caused by glaciation during the Pleistocene Period (Marko, 1998). Glacial cycles during the

Pleistocene have affected global diversity and significantly reduced global sea level, creating barriers to dispersal for marine taxa (Woodruff, 2010; Cabanne et al., 2016;

Craw et al., 2017; Ludt and Rocha, 2015). Based on this haplotype network, it is possible that Hermissenda diverged in a similar way. However, further studies of the population

structure and mechanism of speciation of Hermissenda are needed to determine when and how these sister species diverged.

Mechanisms of speciation in Eastern Pacific have been investigated in other recent heterobranch species complexes, including Doriopsilla, Diaulula, and

Melanochlamys (Hoover et al., 2015; Lindsay et al., 2016; Breslau et al., 2016).

However, the speciation mechanisms differ between genera, so there is no clear biogeographic pattern to compare with Hermissenda. For example, Diaulula sandiegensis and D. odonoghuei (Steinberg, 1963) diverged allopatrically due to glaciation because the split coincided with major cooling events during the Pleistocene (Lindsay et al., 2016).

The Melanochlamys species complex also showed differences in species diversity and population structure between the eastern and western Pacific, which also suggested a postglacial range expansion (Breslau et al., 2016). On the other hand, Doriopsilla albopunctata and D. fulva (MacFarland, 1905) diverged through ecological speciation due to significant differences in reproductive anatomy and a lack of obvious barriers to dispersal (Hoover et al., 2015). Based on the behavioral studies conducted and a lack of known dispersal barriers, it appears that H. crassicornis and H. opalescens may have diverged ecologically. However, the leading edge effect observed in the haplotype network could indicate a different mechanism. Morphological analyses of the reproductive anatomy and further molecular studies are needed to confirm how H. crassicornis and H. opalescens diverged.

In order to further investigate the mechanism of speciation of Hermissenda, molecular markers could be used to analyze small sections of the genome. The use of molecular markers, such as microsatellites and single nucleotide polymorphisms (SNPs),

in determining population structure has been used in other nudibranchs, including

Phidiana hiltoni (King, et al., 2019). More specifically, restriction site associated DNA

(RAD) tags are ideal for discovering SNPs because they allow for small sections of the genome to be sequenced, and the number of desired markers can be selected based on the

restriction enzymes that are used (Baird et al., 2008; Davey and Blaxter, 2010).

Investigating population structure using RAD sequencing (RADseq) may help us to

understand the mechanism of speciation for H. crassicornis and H. opalescens. I have

already extracted samples for RADseq across the range of H. crassicornis and H. opalescens in the Eastern Pacific. Due to a delay by the sequencing company, I was

unable to analyze the data in time to be included in this study. The data will be analyzed

at a later date.

Based on the biotic interactions and molecular data presented here, Hermissenda

crassicornis and H. opalescens likely fill similar niches within the large range overlap, but the mechanism of speciation is currently unclear. It is important to further investigate

the mechanism of speciation because Hermissenda crassicornis (sensu lato) has been

used for decades as a model organism in neuroscience (Alkon, 1974; Lederhendler, et al.,

1986; Richards and Farley, 1987; Epstein et al., 2000; Cavallo et al., 2014; Gunaratne et

al., 2014; Gunaratne and Katz, 2016; Webber et al., 2017). Depending on when the

species diverged, it is possible that these studies used a combination of the three species

of Hermissenda and must be reevaluated.

REFERENCES

Alkon DL (1974) Associative training of Hermissenda. The Journal of General Physiology,

64: 70–84. https://doi.org/10.1085/jgp.64.1.70.

Avila C, Arigue A, Tamse CT & Kuzirian AM (1994) Hermissenda crassicornis larvae

metamorphose in laboratory in response to artificial and natural inducers. The Biological

Bulletin, 187: 252–253. https://doi.org/10.1086/BBLv187n2p252.

Armstrong EJ, RL Tanner & JH Stillman (2019) High heat tolerance is negatively correlated

with heat tolerance plasticity in nudibranch mollusks. Physiological and Biochemical

Zoology 92: 430–44. https://doi.org/10.1086/704519.

Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, Lewis ZA, Selker EU, Cresko WA

& Johnson EA (2008) Rapid SNP discovery and genetic mapping using sequenced RAD

markers. PLoS One, 3: e3376.

Beeman RD & Williams GC (1980) Opisthobranchia and Pulmonata: The sea slugs and

allies. Pp 308-354 in Intertidal invertebrates of California (RH Morris, DP Abbott, and

EC Haderlie, eds.) Stanford University Press: Stanford, CA.

Behrens DW (1991) Pacific coast nudibranchs, 2nd ed. Sea Challengers, Monterey, California

97 pp.

Behrens DW & Hermosillo A (2005) Eastern Pacific nudibranchs: A guide to the

opisthobranchs from Alaska to Central America. Sea Challengers, Monterey, California

1-137 pp.

Bergh R (1879) On the nudibranchiate gastropod Mollusca of the North Pacific Ocean, with

special reference to those of Alaska, Part 1. Proceedings of the Academy of Natural

Sciences of Philadelphia 2: 71-132.

Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K, Ingram KK & Das I

(2007) Cryptic species as a window on diversity and conservation. Trends in Ecology and

Evolution 22: 148-155.

Bloom SA (1981) Specialization and noncompetitive resource partitioning among sponge-

eating dorid nudibranchs. Oecologia, 49: 305–15. https://doi.org/10.1007/BF00347590.

Booth DJ, Bond N & Macreadie P (2011) Detecting range shifts among Australian fishes in

response to climate change. Marine and Freshwater Research 62: 1027.

https://doi.org/10.1071/MF10270.

Cabanne GS, Caldern L, Arias NT, Flores P, Pessoa R, d’Horta FM & Miyaki CY (2016)

Effects of Pleistocene climate changes on species ranges and evolutionary processes in

the neotropical Atlantic forest. Biological Journal of the Linnean Society 119: 856–872.

https://doi.org/10.1111/bij.12844.

Cavallo JS, Hamilton BN & Farley J (2014) Behavioral and neural bases of extinction

learning in Hermissenda. Frontiers in Behavioral Neuroscience, 8: 277.

Charrette NA, Cleary DFR & Mooers AØ (2006) Range-restricted, specialist Bornean

butterflies are less likely to recover from ENSO-induced disturbance. Ecology 87: 2330–

2337. https://doi.org/10.1890/0012-9658(2006)87[2330:RSBBAL]2.0.CO;2.

Churchill CKC, Alejandrino A, Valdés A & Foighil DÓ (2013) Parallel changes in genital

morphology delineate cryptic diversification of planktonic nudibranchs. Proceedings of

the Royal Society, 280: 20131224. http://dx.doi.org/10.1098/rspb.2013.1224.

Clement M, Posada D & Crandall KA (2000) TCS: A computer program to estimate gene

genealogies. Molecular Ecology 9: 1657-1659.

Costello DP (1938) Notes on the breeding habits of the nudibranchs of Monterey Bay and

vicinity. Journal of Morphology 63: 319-343.

Coyne JA & HA Orr. “Species: Reality and Concepts.” In Speciation, 2004.

Craw D, Upton P, Waters J & Wallis G (2017) Biological memory of the first Pleistocene

glaciation in New Zealand. Geology 45: 595–598. https://doi.org/10.1130/G39115.1.

Cristescu ME, Adamowicz SJ, Vaillant JJ & Haffner DG (2010) Ancient lakes revisited:

From the ecology to the genetics of speciation. Molecular Ecology 19: 4837–4851.

https://doi.org/10.1111/j.1365-294X.2010.04832.x.

Davey JW & Blaxter ML (2010) RADseq: Next-generation population genetics. Briefings in

Functional Genomics, 9: 416-423.

Edmands S (2001) Phylogeography of the intertidal copepod Tigriopus californicus reveals

substantially reduced population differentiation at northern latitudes. Molecular Ecology

10: 1743–1750. https://doi.org/10.1046/j.0962-1083.2001.01306.x.

Epstein DA, Epstein HT, Child FM & Kuzirian AM (2000) Memory consolidation in

Hermissenda crassicornis. The Biological Bulletin, 199: 182-183.

Folmer I, Black M, Hoeh W, Lutz R & Vrijenhoek R (1994) DNA primers for amplification

of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

Molecular Marine Biology and Biotechnology 3: 294-299.

Goddard JHR, Treneman N, Pence WE, Mason DE, Dobry PM, Green B & Hoover C (2016)

Nudibranch range shifts associated with the 2014 warm anomaly in the Northeast Pacific.

Bulletin of the Southern California Academy of Sciences 115: 15–40.

https://doi.org/10.3160/soca-115-01-15-40.1.

Goddard JHR, Treneman N, Prestholdt T, Hoover C, Green B, Pence WE, Mason DE, Dobry

P, Sones JL, Sanford E, Agarwal R, McDonald GR, Johnson RF & Gosliner TM (2018)

Heterobranch sea slug range shifts in the Northeast Pacific Ocean associated with the

2015-16 El Niño. Proceedings of the California Academy of Sciences 65: 107-131.

Golestani H, Crocetta F, Padula V, Camacho-García Y, Langeneck J, Poursanidis D, Pola M,

Yokes MB, Cervera JL, Jung D, Gosliner TM, Araya JF, Hooker Y, Schrödl M & Valdés

A (2019) The little Aplysia coming of age: From one species to a complex of species

complexes in Aplysia parvula (Mollusca: Gastropoda: Heterobranchia). Zoological

Journal of the Linnean Society 187: 279–330. https://doi.org/10.1093/zoolinnean/zlz028.

Good JM, Hird S, Reid N, Demboski JR, Steppan SJ, Martin-Nims TR & Sullivan J (2008)

Ancient hybridization and mitochondrial capture between two species of chipmunks.

Molecular Ecology 17: 1313–1327. https://doi.org/10.1111/j.1365-294X.2007.03640.x.

Gunaratne CA & Katz PS (2016) Comparative mapping of GABA-immunoreactive neurons

in the buccal ganglia of nudipleura molluscs. The Journal of Comparative Neurology,

524: 1181-1192.

Gunaratne CA, Sakurai A & Katz PS (2014) Comparative mapping of GABA-

immunoreactive neurons in the central nervous systems of nudibranch molluscs. The

Journal of Comparative Neurology, 522: 794-810.

Harrigan JF & Alkon DL (1978) Larval rearing, metamorphosis, growth and reproduction of

the eolid nudibranch Hermissenda crassicornis (Eschscholtz, 1831) (Gastropoda:

Opisthobranchia). The Biological Bulletin, 154: 430–439.

https://doi.org/10.2307/1541069.

Hewitt GM (2000) The genetic legacy of the Quaternary ice ages. Nature, 405: 907-913.

Hoover RA, Armour R, Dow I & Purcell JE (2012) Nudibranch predation and dietary

preference for the polyps of Aurelia labiata (Cnidaria: Scyphozoa). Hydrobiologia, 690:

199–213. https://doi.org/10.1007/s10750-012-1044-x.

Hoover C, Lindsay T, Goddard JHR & Valdés Á (2015) Seeing double: pseudocryptic

diversity in the Doriopsilla albopunctata-Doriopsilla gemela species complex of the

North-Eastern Pacific. Zoologica Scripta 44: 612–631. https://doi.org/10.1111/zsc.12123.

Howard DJ & Berlocher SH (Eds.). (1998). Endless Forms: Species and Speciation. Oxford

University Press.

iNaturalist.org (2019). iNaturalist Research-grade Observations. Occurrence dataset

https://doi.org/10.15468/ab3s5x accessed via GBIF.org on 2019-11-18.

https://www.gbif.org/occurrence/2422902366.

Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A,

Markowitz S, Duran C & Thierer T (2012) Geneious Basic: An integrated and extendable

desktop software platform for the organization and analysis of sequence data.

Bioinformatics 28: 1647-1649.

King CJ, Ellingson RA, Goddard JHR, Johnson RF & Valdés Á (2019) Range expansion or

range shift? Population genetics and historic range data analyses of the predatory benthic

sea slug Phidiana hiltoni (Mollusca, Gastropoda, Nudibranchia). Bulletin of the Southern

California Academy of Sciences, 118: 1-20. https://doi.org/10.3160/0038-3872-118.1.1.

Knutsen H, Jorde PE, Gonzalez EB, Robalo J, Albretsen J & Almada V (2013) Climate

change and genetic structure of leading edge and rear end populations in a northwards

shifting marine fish species, the corkwing wrasse (Symphodus melops). PLoS ONE 8:

e67492. https://doi.org/10.1371/journal.pone.0067492.

Lederhendler II, Gart S, & Alkon DL (1986) Classical conditioning of Hermissenda: Origin

of a new response. Journal of Neuroscience, 6: 1325-1331.

Leigh JW & Bryant D (2015) POPART: Full-feature software for haplotype network

construction. Methods in Ecology and Evolution 6: 1110-1116.

Lindsay T, Kelly J, Chichvarkhin A, Craig S, Kajihara H, Mackie J & Valdés Á (2016)

Changing spots: Pseudocryptic speciation in the North Pacific dorid nudibranch Diaulula

sandiegensis (Cooper, 1863) (Gastropoda: Heterobranchia). Journal of Molluscan Studies

82: 564–574. https://doi.org/10.1093/mollus/eyw026.

Lindsay T and Valdés Á (2016) The model organism Hermissenda crassicornis (Gastropoda:

Heterobranchia) is a species complex. PLOS ONE, 11: e0154265.

https://doi.org/10.1371/journal.pone.0154265.

Lluch-Belda D, Lluch-Cota DB & Lluch-Cota SE (2005) Changes in marine faunal

distributions and ENSO events in the California Current. Fisheries Oceanography 14:

458–467. https://doi.org/10.1111/j.1365-2419.2005.00347.x.

Longley R & Longley A (1982) Hermissenda: Agonistic behaviour or mating behaviour? The

Veliger, 24:230-231.

Ludt WB & Rocha LA (2015) Shifting seas: The impacts of Pleistocene sea-level

fluctuations on the evolution of tropical marine taxa. Journal of Biogeography 42: 25–38.

https://doi.org/10.1111/jbi.12416.

MacFarland FM (1905) A preliminary account of the Dorididae of Monterey Bay, California,

and vicinity. Proceedings of the Biological Society of Washington, 18: 35-54.

Marko PB (1998) Historical allopatry and the biogeography of speciation in the prosobranch

snail genus Nucella. Evolution 52: 757–774. https://doi.org/10.1111/j.1558-

5646.1998.tb03700.x.

May RM & MacArthur RH (1972) Niche overlap as a function of environmental variability.

Proceedings of the National Academy of Sciences 69: 1109-1113.

McDonald GR (1983) A review of the nudibranchs of the California coast. Malacologia, 24:

114-276.

McDonald GR & Nybakken JW (1980) Guide to the nudibranchs of California. American

Malacologists, Inc., Melbourne, Florida, 77 pp.

Megina C, Gosliner T & Cervera JL (2007) The use of trophic resources by a generalist eolid

nudibranch: Hermissenda crassicornis (Mollusca: Gastropoda). Cahiers de Biologie

Marine, 48: 1-7.

Merlo EM, Milligan KA, Sheets NB, Neufeld CJ, Eastham TM, Estores-Pacheco ALK,

Steinke D, Hebert PDN, Valdés Á & Wyeth RC (2018) Range extension for the region of

sympatry between the nudibranchs Hermissenda opalescens and Hermissenda

crassicornis in the Northeastern Pacific. FACETS, 3: 764–776.

https://doi.org/10.1139/facets-2017-0060.

Mitsui Y, Nomura N, Isagi Y, Tobe H & Setoguchi H (2011) Ecological barriers to gene

flow between riparian and forest species of Ainsliaea (Asteraceae): Adaptive ecological

divergence in Ainsliaea.” Evolution 65: 335–349. https://doi.org/10.1111/j.1558-

5646.2010.01129.x.

Morris RH, Abbot DP, & Haderlie EC (Eds.). (1980). Intertidal invertebrates of California.

Stanford University Press, Stanford, CA.

Palumbi SR (1994) Genetic divergence, reproductive isolation, and marine speciation.

Annual Review of Ecology and Systematics, 2: 547–572.

Parmesan C (2006) Ecological and evolutionary responses to recent climate change. Annual

Review of Ecology, Evolution, and Systematics, 37: 637–69.

https://doi.org/10.1146/annurev.ecolsys.37.091305.110100.

Pascarella JB (2007) Mechanisms of prezygotic reproductive isolation between two

sympatric species, Gelsemium rankinii and G. sempervirens (Gelsemiaceae), in the

Southeastern United States. American Journal of Botany 94: 468–476.

https://doi.org/10.3732/ajb.94.3.468.

Paterson HE (1980) A comment on ‘Mate Recognition Systems.’ Evolution, 34: 330–331.

https://doi.org/10.1111/j.1558-5646.1980.tb04821.x.

Pianka ER (1974) Niche overlap and diffuse competition. Proceedings of the National

Academy of Sciences 71: 2141-2145.

R Core Team (2018) R: A language and environment for statistical computing. Vienna,

Austra: R Foundation for Statistical Computing [online]: Available from https://www.r-

project.org.

Richards WG and Farley J (1987) Motor correlates of phototaxis and associative learning in

Hermissenda crassicornis. Brain Research Bulletin, 19: 175-189.

Rogers RF, Fass DM & Matzel LD (1994) Current, voltage and pharmacological substrates

of a novel GABA receptor in the visual-vestibular system of Hermissenda. Brain

Research, 650: 93-106.

Rolán-Alvarez E, Erlandsson J, Johannesson K & Cruz R (1999) Mechanisms of incomplete

prezygotic reproductive isolation in an intertidal snail: Testing behavioural models in

wild populations. Journal of Evolutionary Biology, 12: 879-890.

https://doi.org/10.1046/j.1420-9101.1999.00086.x.

Rutowski RL (1983) Mating and egg mass production in the aeolid nudibranch Hermissenda

crassicornis (Gastropoda: Opisthobranchia). The Biological Bulletin, 165: 276–285.

https://doi.org/10.2307/1541369.

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S,

Rueden C, Saalfield S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K,

Tomancak P, Cardona A (2012) Fiji: An open-source platform for biological-image

analysis. Nature Methods, 9: 676-682. https://doi.org/10.1038/nmeth.2019.

Schultz ST, Goddard JHR, Gosliner TM, Mason DE, Pence WE, McDonald GR, Pearse VB

& Pearse JS (2011) Climate-index response profiling indicates larval transport is driving

population fluctuations in nudibranch gastropods from the Northeast Pacific Ocean.

Limnology and Oceanography 56: 749–763. https://doi.org/10.4319/lo.2011.56.2.0749.

Sinopoli M, Chemello R, Vaccaro A & Milazzo M (2017) Food resource partitioning

between two sympatric temperate wrasses. Marine and Freshwater Research 68: 2324-

2336.

Tanaisichuk R & Cooper C (2002) A northern extension of the range of the euphausiid

Nyctiphanes simplex into Canadian waters. Journal of Crustacean Biology, 22: 206-209.

Tolley KA, Groeneveld JC, Gopal K & Matthee CA (2005) Mitochondrial DNA panmixia in

spiny lobster Palinurus gilchristi suggests a population expansion. Marine Ecology

Progress Series 297: 225–231. https://doi.org/10.3354/meps297225.

Tolley KA & Rosel PE (2006) Population structure and historical demography of Eastern

North Atlantic harbour porpoises inferred through mtDNA sequences. Marine Ecology

Progress Series 327: 297–308. https://doi.org/10.3354/meps327297.

Tyndale E, Avila C & Kuzirian AM (1994) Food detection and preferences of the nudibranch

mollusc Hermissenda crassicornis: Experiments in a Y-maze. The Biological Bulletin,

187: 274–275. https://doi.org/10.1086/BBLv187n2p274.

Uribe RA, Seplveda F, Goddard JHR, & Valdés Á (2018) Integrative systematics of the

genus Limacia O. F. Müller, 1781 (Gastropoda, Heterobranchia, Nudibranchia,

Polyceridae) in the Eastern Pacific.” Marine Biodiversity, 48: 1815–1832.

https://doi.org/10.1007/s12526-017-0676-5.

Via S (2001) Sympatric speciation in animals: The Ugly Duckling grows up. Trends in

Ecology & Evolution, 16: 381–390. https://doi.org/10.1016/S0169-5347(01)02188-7.

Ventura CRR, Grillo MCG & Fernandes FC (2001) Feeding niche breadth and feeding niche

overlap of paxillosid starfishes (Echinodermata: Asteroidea) from a midshelf upwelling

region, Cabo Frio, Brazil. In M. Barker (Ed.), Echinoderms 2000 (pp. 227-233).

Rotterdam: AA Balkema.

Webber MP, Thomson JWS, Buckland-Nicks J, Croll RP & Wyeth RC (2017) GABA-,

histamine-, and FMRFamide-immunoreactivity in the visual, vestibular and central

nervous systems of Hermissenda crassicornis. The Journal of Comparative Neurology,

525: 3514-3528.

Wilson DS & Yoshimura J (1994) On the coexistence of generalists and specialists. The

American Naturalist 144: 692-707.

Wissinger SA (1992) Niche overlap and the potential for competition and intraguild

predation between size-structured populations. Ecology 73: 1431-1444.

Woodruff DS (2010) Biogeography and conservation in Southeast Asia: How 2.7 million

years of repeated environmental fluctuations affect today’s patterns and the future of the

remaining refugial-phase biodiversity. Biodiversity and Conservation 19: 919–941.

https://doi.org/10.1007/s10531-010-9783-3.

Yoshioka P (1986) Competitive co-existence of the dorid nudibranchs Doridella steinbergae

and Corambe pacifica. Marine Ecology Progress Series 33: 81–88.

https://doi.org/10.3354/meps033081.

Zack, S (1975) A description and analysis of agonistic behavior patterns in an Opisthobranch

mollusc, Hermissenda crassicornis. Behaviour, 53: 238–267.

TABLES

Table 1. Criteria of slug health used in feeding and mating experiments.

Criterion Description Healthy Rapid movement around tub Consumes prey, if present Spends significant time consuming prey Interacts with another slug with equal aggression, if present Cerata healthy Produces egg mass Oral tentacles healthy Able to quickly turn body over if falls onto dorsal side Questionable Moderate movement around tub Health May consume prey Spends little time consuming prey, if present Interacts with another slug, polarity of aggression by other slug may be noticeable Cerata healthy, but may show signs of degradation May produce egg mass Oral tentacles healthy Able to turn body over if falls onto dorsal side Sick Little to no movement around tub Does not eat prey, if present Does not interact with slug, or extreme polarity of aggression by the other slug, if present Cerata falling off of body Degradation of oral tentacles Not able to turn body over if falls onto dorsal side

Table 2. Criteria of slug behavior used during mating experiment video review.

Criterion Description Jousting Slug pairs touch and move oral tentacles Alignment After jousting slug pairs align feet with anterior end of one slug lining up with posterior end of other slug Copulation After alignment slug pairs create a wheel- like formation and raise cerata Aggression Antagonistic behavior between slug pairs that lasts for longer than 4 seconds, including but not limited to tail biting, biting of body, body slamming

Table 3. Summary of statistical significance separated by model for mating behavior in conspecific and heterospecific

Hermissenda pairings. Models used individuals as the null and tested for effect of pairing and individual and effect of

pairing by experiment and individual. There was a significant difference in jousting, aggression, and copulation behavior

based on pairing type. There was also a significant difference in aggressive behavior based on experiment. Abbreviations:

HOHO: conspecific H. opalescens pairs, HCHC: conspecific H. crassicornis pairs, HCHO: heterospecific pairs.

Model df AIC BIC logLik Deviance Chisq df P-value HOHO HCHC HCHO Aggression null (individuals 4 -181.03 -170.95 94.517 -189.03 - - - only)

35 pairing + ind 6 -186.41 -171.28 99.204 -198.41 9.3741 2 <0.001 a ab b Jousting null (individuals 4 -219.46 -209.38 113.73 -227.46 - - - only) pairing + ind 6 -228.73 -213.60 120.37 -240.73 13.2677 2 0.0013 a b b Copulation null (individuals 4 -306.72 -296.63 157.36 -314.72 - - - only) pairing + ind 6 -354.59 -339.46 183.30 -366.59 51.8757 2 <0.001 a b c