THE RETURN OF THE CLOWN: PSEUDOCRYPTIC SPECIATION IN THE

NORTH PACIFIC CLOWN , CATALINAE (COOPER,

1863) S.L. IDENTIFIED BY INTEGRATIVE TAXONOMIC APPROACHES

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In

Biological Sciences

By

Dae-Wui Jung

2019

SIGNATURE PAGE

THESIS: THE RETURN OF THE CLOWN: PSEUDOCRYPTIC SPECIATION IN THE NORTH PACIFIC CLOWN NUDIBRANCH, (COOPER, 1863) S.L. IDENTIFIED BY INTEGRATIVE TAXONOMIC APPROACHES

AUTHOR: Dae-Wui Jung

DATE SUBMITTED: Spring 2019

Department of Biological Sciences

Dr. Ángel Valdés Thesis Committee Chair Biological Sciences

Dr. Andrea Bonisoli-Alquati Biological Sciences

Dr. Jayson Smith Biological Sciences

ii

ACKNOWLEDGEMENTS

Since I arrived at California States Polytechnic University Pomona in 2016, I have received tremendous help from many people. I greatly appreciate all.

First of all, Muchas gracias, Ángel!! Prof. Valdés, my advisor, taught me repeatedly everything until I understood it well. I will continue to study nudibranchs in East Asia based on your teaching. I am grateful to Dr. Terry Gosliner, California Academy of Science, for allowing me to use valuable specimens. He always welcomed me with a bright and kind smile. Dr. Anton Chichvarkhin and I remember a tiny plane, smaller than the bus, that we took when flying to Rudnaya bay to collect Russian Triopha specimens. I hope you regain your health as soon as possible and dive with you soon. Many thanks to Craig Hoover for his advice and experience of diving in California and Baja California, Mexico. Visit Korea and dive together!! The trip to Bahía de los Ángeles was very pleasant and informative to me, because of with Dr. Hans Bertsch. I hope Dr. Hans and his Jeep (with the SOY BUZO plates) will travel long and healthy. I am grateful to Jongrak Lee and Inthesea Marine

Biodiversity Lab for support to study Korean nudibranchs. Dr. Carla Stout taught me kindly and easily about phylogenetic programs. Thanks!! And I would like to thank my committees, Prof. Jayson Smith and Prof. Andrea Bonisoli-Alquati for reviewing this thesis and valuable comments. Lastly, although I have away from home for a long time, I sincerely appreciate the continued support of my wife, Jae-Wan Kim. I love you!!

This study was supported by a grant from the National Institute of Biological Resources

(NIBR 201801104), funded by the Ministry of Environment (MOE) of the Republic of

Korea.

iii

ABSTRACT

The North Pacific nudibranch species Triopha catalinae (Cooper, 1863), also known as

the clown nudibranch, includes two distinct morphotypes based on photographic records,

the northern morphotype found from South Korea to Central California, and the southern

morphotype ranging from Alaska to Baja California. In this study, these two morphotypes

were found to be distinct species, based on integrative taxonomic approach, incorporating

both molecular and morphological analysis. Approaches included: (1) Bayesian inference and maximum-likelihood phylogenetic analyses based on concatenated gene sequences of

the mitochondrial 16S, COI and the nuclear Histone H3 genes, (2) haplotype network via

TCS based on COI sequences, (3) Automatic Barcode Gap Discovery (ABGD) based on

16s and COI, and (4) comparative internal and external morphological studies.

Phylogenetic analyses revealed that the Triopha catalinae species complex clearly clustered into two clades, one including specimens of the northern morphotype and the

other containing specimens of the southern morphotype. Two major groups as northern

morphotype and southern morphotype were also recovered from both TCS haplotype

network and ABGD analysis, which results supported the fact that T. catalinae is a species complex. Furthermore, these two clades display morphological differences in the dorsal tubercles and radula structure: the northern morphotype specimens possess relatively small and dendritic dorsal tubercles, two rows of arborescent tubercles on the dorso-lateral appendages of larger individuals, and the radular formulae are 19-24 × (9-12.5-10.R.5-

10.9-12). On the contrary, specimens of the southern morphotype have relatively large, conical or rounded dorsal tubercles, and 48-79 × (16-18.17-33.R.17-33.16-18) radular

iv formulae. The ABGD analysis confirmed that these two morphotypes constitute different species. A review of the literature (including original descriptions) and available type material, revealed that Triopha modesta Bergh, 1880 is the oldest available name for the northern morphotype, whereas T. catalinae is the valid name for the southern morphotype.

v

TABLE OF CONTENTS

SIGNATURE PAGE ...... ⅱ

ACKNOWLEDGMENTS ...... ⅲ

ABSTRACT ...... ⅳ

LIST OF TABLES ...... ⅶ

LIST OF FIGURES ...... ⅷ

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 3

RESULTS ...... 7

DISCUSSION AND CONCLUSIONS ...... 28

REFERENCES ...... 31

vi

LIST OF TABLES

Table 1. List of specimens sequenced for this study including locality, voucher numbers,

and GenBank accession numbers. Abbreviations: CASIZ: California Academy of

Science Invertebrate Zoology; CPIC: California State Polytechnic University

Invertebrate Collection; *denotes specimen sequences downloaded from GenBank;

**denotes outgroup...... 38

Table 2. Best partition scheme and corresponding models determined by PartitionFinder2.

...... 43

Table 3. List of species recovered in the ABGD analysis of mtDNA COI sequences of

specimens identified as using the Kimura 2-parameter model, including isolate and

GenBank accession numbers, missing data is indicated by dashes...... 44

Table 4. Mean p-distances within and between groups using the Kimura 2-parameter

model and 1,000 replicates bootstrap value...... 45

Table 5. Radular formulae of each specimen examined including voucher numbers. . 45

vii

LIST OF FIGURES

Figure 1. Bayesian phylogenetic tree of the Triopha catalinae species complex with

Triopha maculata as the outgroup, based on concatenated sequences of mitochondrial

16S, COI, and H3 genes. Posterior probabilities (PP > 0.9) are shown above the branches

and maximum-likelihood bootstrap (BS > 70) values shown below ...... 46

Figure 2. Automatic Barcode Gap Discovery (ABGD) results showing the distributions of

pairwise distances among 16S sequences for Triopha catalinae species complex using

the Kimura 2-parameter model ...... 47

Figure 3. Automatic Barcode Gap Discovery (ABGD) results showing the distributions of

pairwise distances among COI sequences for Triopha catalinae species complex using

the Kimura 2-parameter model ...... 47

Figure 4. TCS network of mtDNA COI sequences for Triopha catalinae species complex.

Circle size is proportional to the frequency of haplotypes detected. Haplotypes are

connected via lines with dashes representing mutational steps...... 48

viii

Figure 5. Living . A-B. Triopha catalinae (Cooper, 1863). A. Specimens from

Bahía de los Ángeles, Ensenada, Mexico (NUTC0002). B. Specimens from Bahía de los

Ángeles, Ensenada, Mexico (NUTC0003). C-D. Triopha modesta Bergh, 1880. C.

Specimen from Yangyang-gun, Gangwon-do, South Korea (NUTM0010). D. Specimen

from Rudnaya Pristan, Primorsky Krai, Russia (NUTM0014). A-B. Photos by Craig

Hoover, C-D. Photos by Dae-Wui Jung...... 49

Figure 6. Triopha catalinae, SEM micrographs of the radula. A. Rachidian plate. B.

Lateral teeth. C. Outermost teeth. D. Jaw elements. A-C. Specimens from Bahía de los

Ángeles, Ensenada, Mexico (NUTC0002). D. Specimen from Santa Rosa Island,

California (CASIZ071365). Scale bars: A-C. 200 µm D. 50 µm...... 50

Figure 7. Triopha catalinae, drawing of the reproductive system of a specimen from

Monterey County, California (CASIZ070643). Abbreviations: am, ampulla; bc, bursa

copulatrix; fg, female gland mass; gp, gonopore; pr, prostate; rs, receptaculum seminis;

v, vagina; vd, vas deferens...... 51

Figure 8. Penial hooks. A-B. Triopha catalinae. Specimens from San Mateo County,

California (CASIZ113461). C-D. Triopha modesta. Specimen from Yangyang-gun

Gangwon-do, South Korea (NUTM0007). A, C. Penial hooks at 1,000x magnification

under conventional fluorescence microscope. B, D. Drawings. Scale bars: A-D. 10 µm.

...... 52

ix

Figure 9. Triopha modesta, SEM micrographs of the radula. A. Rachidian plate. B. Lateral

teeth. C. Outermost teeth. D. Jaw elements. A-C. Specimens from Rudnaya Pristan,

Primorsky Krai, Russia (NUTM0015). D. Specimen from Vancouver Island, British

Columbia, Canada (CASIZ118667). Scale bars: A-C. 200 µm D. 50 µm...... 53

Figure 10. Triopha modesta, drawing of the reproductive system of a specimen from

Yangyang-gun, Gangwon-do, South Korea (NUTM0005). Abbreviations: am, ampulla;

bc, bursa copulatrix; fg, female gland mass; gp, gonopore; pr, prostate; rs, receptaculum

seminis; v, vagina; vd, vas deferens...... 54

x

INTRODUCTION

During the Pleistocene Period (2.6 Ma-11.7 Ka), biodiversity was affected globally

by more than 50 glacial cycles (Woodruff, 2010; Wang et al., 2013; Cabanne et al., 2016;

Craw et al., 2017). During this time, sea-level changes created barriers to dispersal, which resulted in speciation due to the interrupted gene flow between previously connected areas

(Ludt & Rocha, 2015). Several studies have reported on the connection between

Pleistocene refugia and speciation for various taxa, including Arthropoda (Schebeck et al.,

2019), Chordata (Wielstra et al., 2010), Echinodermata (Foltz et al., 2008), (Reid et al., 2012), and others. Furthermore, several studies on the role of Pleistocene refugia and glacial cycles in the formation of species complexes have been reported in the

Nudibranchia (Lindsay & Valdés, 2016; Lindsay et al., 2016; Hoover et al., 2017; Uribe et al., 2017).

There are other examples of nudibranch species with trans-Pacific ranges that could provide further evidence on the effects of climate change on speciation and the formation of cryptic diversity. For example, the genus Triopha comprises three valid species, which are only known from the North Pacific: Triopha catalinae (Cooper, 1863), Triopha maculata MacFarland, 1905, and Triopha occidentalis (Fewkes, 1889) (McDonald, 1983;

Goddard & Green, 2013). Both T. maculata and T. occidentalis are found only in the

Eastern Pacific (McDonald, 1983; Behrens & Hermosillo, 2005). However, Triopha

catalinae (Cooper, 1863), commonly known as the clown nudibranch, has a broad

distribution in the North Pacific from Korea, Japan, and Russia to Alaska, Canada,

California, and Baja California (Okutani, 2000; Behrens & Hermosillo, 2005; Jung et al.,

1

2013; Bertsch, 2014; Chichvarkhin, 2016). Triopha catalinae is clearly distinguished from other species of Triopha by differences in coloration, specifically the white body, with orange or vermilion red tips of both dorsal appendages and gill branches. However, a closer examination of the color variation of specimens of T. catalinae reveals a non-random pattern, specimens found across the North Pacific, northern morphotype, from Korea to

Mendocino County, California, typically have flat or arborescent dorsal tubercles. On the contrary, the southern morphotype, from Craig City, Alaska to Baja California, includes animals with relatively large, conical or rounded dorsal tubercles.

This study aims to investigate whether Triopha catalinae constitutes a species complex, using integrative taxonomic approaches involving molecular and morphological data. The main questions of this study are: (1) Are the two morphotypes of T. catalinae genetically distinct? (2) In addition to the external color differences, what other morphological characteristics distinguish the two morphotypes? (3) Are these color morphotypes distinct species? (4) If so, is there an overlap region in the range of these two species? To answer these questions, this study includes molecular phylogenetic trees, constructed using Bayesian (BI) and Maximum-likelihood (ML) analyses, based on two mitochondrial genes and a nuclear gene (16S ribosomal RNA, cytochrome c oxidase subunit 1, and Histone H3), as well as a TCS haplotype network using COI, and Automatic

Barcode Gap Discovery (ABGD) analysis for species delimitation, using 16S and COI and the Kimura 2-parameter genetic distance. Finally, a re-description of the morphological characters of this group is provided, including external features, radular teeth, and reproductive organs, and a comprehensive review of the literature is conducted to reveal the valid names for all the taxa involved.

2

MATERIALS AND METHODS

Specimen sampling

A total of 31 specimens were collected by SCUBA from February 2018 to September

2018 in Korea, Russia, California, and Baja California (Table 1). The collected individuals were anesthetized for 2-12 hours in 7% MgCl2 solution and fixed by 95% ethanol.

Additional 13 samples were obtained from the California State Polytechnic University

Invertebrate Collection (CPIC) and the Department of Invertebrate Zoology collection of

the California Academy of Sciences (CASIZ). Additional sequences were downloaded

from GenBank (Table 1). The specimens used in this study were deposited at the CPIC,

the National Institute of Biological Resources (NIBR), Incheon, Korea and DeNovo

Research Marine Biodiversity Lab, Daejeon, Korea. Museum specimen information was

obtained from the CASIZ, the University of Colorado Museum of Natural History, Boulder

(UCMNH), the Biology Department, Pomona College, Claremont, California, the Natural

History Museum of Denmark, Copenhagen (NHMD), the Natural History Museum of the

United Kingdom, London (NHMUK), the Royal British Columbia Museum, Victoria

(RBCM) and the Canadian Museum of Nature, Ottawa (CMN).

3

DNA extraction and assembly

DNA was extracted from the foot tissue of a total of 36 specimens, using a Qiagen

DNeasy blood and tissue kit (Qiagen, Germany) following the manufacturer’s instructions.

Purified genomic DNA was used as a template for PCR amplification of the partial genes for the mitochondrial 16s rRNA, COI (cytochrome c oxidase subunit I), and nuclear

Histone H3. The following primers were used to amplify: 16Sar-L, 16Sbr-H (Palumbi,

1996); LCO1490, HCO2198 (Folmer et al., 1994); HexAF, HexAR (Colgan et al., 1998).

The PCR conditions for the three genes amplification were: activation at 95 °C for 5 min, each PCR run consisted of 35 cycles: 1 min at 95 °C denaturation, 45 s at 42 °C annealing,

2 min at 72 °C elongation, and 10 min at 72 °C final elongation. The amplified products were analyzed by electrophoresis on 1.0 % agarose gel and stained with Midori Green

Advance (Nippon Genetics, Japan). The PCR product was purified using QIAquick PCR

Purification Kit (Qiagen, Germany). DNA concentrations (ng/µL) and purify (A260/A280) were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA).

And the purified PCR products were sequenced in both directions by 3730x1 DNA

Analyzer (Thermo Fisher Scientific, USA). Each primer sequences were removed, and each gene sequences were assembled and edited in Geneious Pro R11 (Kearse et al., 2012)

4

Phylogenetic analyses

The best-fit partitioning scheme and model of the concatenated dataset were selected

based on Corrected Akaike Information Criterion (AICc) and ‘greedy’ search algorithm

using PartitionFinder2 (Lanfear et al., 2016) on the CIPRES science gateway ver. 3.3

(Miller et al., 2010). Optimal partitioning schemes and model are indicated in Table 2.

Bayesian inference (BI) analysis was performed the following sampling 10 million

iterations, burn-in of 25%, every 1000 generations using MrBayes 3.2.6 on XSEDE

(Ronquist & Huelsenbeck, 2003). Maximum likelihood (ML) analysis was constructed by

performing bootstrapping on 1000 replicates using RAXML-HPC2 on XSEDE ver. 8.2.10

(Stamatakis, 2014). Both BI and ML analyses were performed at the CIPRES web portal

(Miller et al., 2010). Posterior probabilities (PP) ≥ 0.9 and Bootstrap (BS) values ≥ 70%

were treated as significant (Hillis & Bull, 1993; Huelsenbeck & Rannala, 2004).

Haplotype network reconstruction

Haplotype networks were generated by aligned COI sequences in the program

PopART 1.7 using TCS network analysis (Clement et al., 2000; Leigh & Bryant, 2015).

5

Species delimitation analysis

The relative genetic distance between the groups and within the group were calculated

using Kimura 2-parameter distances and 1,000 bootstraps replicated by MEGA 10.0.5

(Kumar et al., 2018). Candidate species were recovered using the COI and 16S genes by

applying the Automatic Barcode Gap Discovery (ABGD) method (Puillandre et al., 2012)

with default settings: P (prior intraspecific divergence) from 0.001 to 0.1, steps set to 10,

Nb bins (for distance distribution) set to 20, relative gap width (X = 1.5), and distance metrics (Kimura TS/TV 2.0).

Morphological analyses

Living animals were photographed using an underwater camera (Tough TG-4;

Olympus, Tokyo, Japan). A total of 37 preserved specimens were examined

morphologically and dissected under a stereoscopic microscope (Leica EZ4D; Leica

Microsystems, Wetzlar, Germany). The buccal mass was dissected and softened in 10%

NaOH solution for 30 minutes to 2 hours, after which the jaws and radular sac were

separated. The tissue around the jaws was removed manually and the jaws rinsed in DI

water. The tissue of the radular sac was further dissolved in 10% NaOH solution overnight,

the radula was washed with DI water, and debris removed using an ultrasonicator (Branson

1510; Branson, Danbury, Connecticut). The penis was extracted from vas deferens and

photographed with a Nikon D70 camera which was attached to a Nikon Eclipse E400 light microscope (Nikon, Tokyo, Japan). The jaws and radulae were mounted on an SEM stub,

sputter coated, and observed in a Scanning Electron Microscope (Jeol-6010; Jeol, Tokyo,

Japan).

6

RESULTS

Sequence data

The molecular dataset included newly generated sequences from 38 specimens and partial gene sequences of 6 individuals downloaded from GenBank (Table 1).

Concatenated gene alignments contained 1,442 base pairs (bp) for mitochondrial 16S and

COI partial gene and nuclear Histone H3 partial gene (16S: 456 bp, COI: 658 bp, H3: 328 bp). The species Triopha maculata MacFarland, 1905 was selected as the outgroup based on the similarities between T. maculata and T. catalinae (Ferreira, 1977), which suggest a close phylogenetic relationship.

Phylogenetic analyses

Bayesian and maximum-likelihood analyses of the dataset recovered two strongly supported clades within the Triopha catalinae species complex (Figure 1). One clade included specimens collected from South Korea to Oregon (northern morphotype) (PP=1,

BS=84), and the second clade included individuals from Oregon to Baja California

(southern morphotype) (PP=1, BS=98).

7

Haplotype network reconstruction

The TCS haplotype network identified 27 haplotypes in 41 COI sequences (Figure 4) and recovered two haplogroups correlated with a geographical origin of the samples: one haplogroup included samples from South Korea to Oregon (northern morphotype), and other specimens from Oregon to Baja California (southern morphotype). This result confirmed the genetic differences between northern and southern morphotypes, also recovered in the BI and ML phylogenetic trees.

Molecular species delimitation

The ABDG analysis of the mitochondrial 16S and COI sequences, based on the K2P model, recovered two candidate species (Figures 2-3), which correspond to the clades and haplogroups recovered in the phylogenetic and haplotype network analyses, respectively

(Table 3). These results also confirm that the two candidate species overlap in range in

Oregon. Furthermore, the genetic distance between the two candidate species was 2.7% in

16S and 9.5% in COI based on the K2P analysis (Table 4). The appropriate names for to these two candidate species are discussed in the Systematics and Discussion section below.

8

Morphological analyses

Consistent morphological differences were observed in the external features and radular teeth of the candidate species, providing further evidence to support the fact that

Triopha catalinae is a species complex. These species are formally re-described in the

systematics and discussion for each species below.

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SYSTEMATIC DESCRIPTIONS

Phylum Mollusca Linnaeus, 1758

Class Cuvier, 1795

Order Nudibranchia Cuvier, 1817

Family Alder & Hancock, 1845

Genus Triopha Bergh, 1880

Triopha Bergh, 1880: 112-113. Type species: Triopha modesta Bergh, 1880, by

subsequent designation by Ferreira (1977).

Cabrilla Fewkes, 1889: 139. Type species: Cabrilla occidentalis Fewkes, 1889, by

monotypy.

Diagnosis

Body limaciform, elongated. Dorsum relatively high, flattened towards head. Oral veil

slightly wider than body. Margin of oral veil with short, simple or arborescent appendages.

Rhinophores with perfoliate clavus, completely retractable into rhinophoral sheath.

Appendages on lateral edge of dorsum not uniform in number. Gill usually consists of 5-6 tripinnate branchial leaves. Anterior foot round and posterior foot pointed. Radula with 2-

4 rachidian plates; lateral teeth hook-shaped at apex; outer-lateral teeth flat, quadrangular.

Prostate gland large, penis armed with numerous falcate hooks.

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Remarks

Bergh (1880) introduced the new genus Triopha based on Triopha modesta Bergh, 1880

and Triopa carpenteri Stearns, 1873, and characterized by having dorsal appendages

“folded lengthwise and obtuse at the end” and “compressed cup-shaped or auriculate,” five gill plumes, two strong jaw plates, four false rachidian plates, and three or four lateral teeth

“with a wing-like process” and depressed hook. Ferreira (1977) subsequently designated

Triopha modesta Bergh, 1880 as the type species.

The genus Cabrilla Fewkes, 1889 was originally introduced without a description, when

Cabrilla occidentalis Fewkes, 1889 is described as a new species. Fewkes (1889) commented the genus Cabrilla was distinguishable from other genera by having translucent white colored rhinophores, five pairs of dorsal appendages, and a gill with

“stellate, bipinnate, consisting of primary arms and lateral branches.” While some authors regarded Cabrilla as a nomen dubium because of the absence of a full description of the radulae and internal traits (O’Donoghue, 1926; Steinberg, 1961), the original description of Cabrilla conforms with the requirements of the Code of Zoological Nomenclature

(ICZN, 1999).

McDonald (1983) considered that C. occidentalis and Triopha grandis are conspecific and the genus Cabrilla a junior synonym of genus Triopha, based on very similar external

characteristics, such as having a brownish body with light green spots and brownish distal ends of the rhinophores, as well as a sympatric range. This opinion is generally accepted and followed herein.

11

Triopha catalinae (Cooper, 1863)

(Figures 5A-B, 6-7, 8A-B)

Triopa catalinae Cooper, 1863: 59. Type locality: Santa Catalina Island, Los Angeles

County, California.

Triopa carpenteri Stearns,1873: 78, fig. 2. Type locality: Point Pinos, Monterey, California.

Triopha scrippsiana Cockerell, 1915: 228-229. Type locality: La Jolla, San Diego,

California.

Type material

Triopa catalinae Cooper, 1863 – type material presumably lost, not at CASIZ.

Triopa carpenteri Stearns,1873 – type material presumably lost, not at CASIZ.

Triopha scrippsiana Cockerell, 1915 – presumably lost, not at University of Colorado

Museum of Natural History (L. Elder, pers. com.), or at Pomona College (J. Wright, pers.

comm.).

12

Material examined

Monterey Bay, Monterey County, California, USA, 26 April 1972, 1 specimen, 57 mm

preserved length, leg. P. Clark (CASIZ 070643). Santa Rosa Island, California, USA, 22

October 1986, 1 specimen, 39 mm preserved length, leg. R. Van Syoc on R/V “Cormorant”

(CASIZ 071365). Pacific Coast of Roca Ben, Baja California, Mexico, 20 August 1987, 1 specimen, 9 mm preserved length, leg. T. M. Gosliner & R. J. Van Syoc (CASIZ 072121).

Moss Beach, San Mateo County, California, USA, 01 November 1963, 1 specimen, 28 mm preserved length, leg. W. Farmer (CASIZ 113461). San Miguel Island, Channel Islands,

California, USA, 18 February 2018, 1 specimen, 16 mm preserved length, leg. D.-W. Jung

(NUTC0001). Bahía de los Ángeles, Ensenada, Baja California, Mexico, 20 March 2018,

2 specimens, 8-11 mm preserved length, leg. D.-W. Jung (NUTC0002). 21 March 2018, 3 specimens, 6-13 mm preserved length, leg. D.-W. Jung (NUTC0003)

13

Description

Body shape limaciform. Head rounded, metapodium pointed (Figures 5A, 5B). Body color translucent white or opaque white. Frontal veil with small, dendritic tentacles on edge from end to end. Color of small tentacles orange or translucent white, some translucent white proximally, orange distally; orange colored tentacles on oral veil usually larger than

white tentacles. Rhinophores perfoliate; clavus orange or vermilion; stalk cylindrical,

translucent white; rhinophoral sheath covers stalk slightly; edge of rhinophoral sheath

smooth or bumpy. Gill with five tripinnate leaves; gill tips orange or vermilion, usually

same color as rhinophoral clavus. Rhinophoral clavus and gill leaves darker in color than

tubercles on body. Anus at center gills circlet. Metapodium short, posterior end blunt.

Small and opaque white colored glands scattered over whole body. Conical or round orange

tubercles present on dorsum, flat orange patches present on dorsum and both sides of body,

both tubercles and patches vary in size and number. Five to seven pairs of long club-shaped

appendages present on both sides of dorsum; few small nodules present on outside of

appendages; appendages slightly bent, leaning towards body. Oral tentacles auriculate.

Foot slightly narrower than body, anteriorly rounded, posteriorly pointed.

Radular formula 48-79 × (16-18.17-33.R.17-33.16-18) (Table 5). Rachidian plate

rudimentary (Figure 6A), consisting in central traces surrounded by wide inverted triangle

shape on each side. Lateral teeth elongate, gradually curved, taper abruptly, with slightly

blunt distal end (Figure 6B). Outermost teeth flat, rectangular, closely attached (Figure 6C).

Radula generally translucent, or amber to dark red in some large animals. Jaw with thin,

elongate, densely packed rodlets (Figure 6D).

14

Genital opening on right side of body, behind head, protected by three lobes.

Reproductive system triaulic (Figure 7). Ampulla long, convoluted; it branches connecting into female gland and prostate. Prostate connecting into seminal vesicle, granulate, large,

almost six times as large as seminal vesicle. Seminal vesicle ovoid, bulging, opening into

vas deferens. Vas deferens smooth, long. Penis armed with numerous hamate penial hooks

(Figures 8A, 8B). Vagina smooth, long, connecting to bursa copulatrix. Bursa copulatrix

large, about two-thirds of female gland. From bursa copulatrix leads another duct

connecting to receptaculum seminis and female gland.

Geographic range

Known from Oregon to Baja California (Sowell, 1949; Bertsch, 2014; present study).

15

Remarks

Cooper (1863) introduced the new species name Triopa catalinae Cooper, 1863 based

on four specimens from Santa Catalina Island, California, USA, without a description or illustrations of radulae. Cooper (1863) described the tubercles along with the frontal veil

as “14 ciliae, equally distributed around its margin”, and the rhinophores as “tentacles long,

conical, retractile.” Cooper (1863) indicated the presence of “two short ciliae close together

on the medium line, a little behind the branchiae.” The dorso-lateral appendages were described as “three pairs of short ciliae at equal distance apart between the tentacles and middle of the body, connected by the sharp edges of the dorsal surface.”

Stearns (1873) introduced the new species Triopa carpenteri Stearns,1873 apparently unaware of the earlier description of Triopa catalinae Cooper, 1863. Stearns (1873) examined several specimens collected in Monterey, California, and provided descriptions

of frontal veil papillae as “short tentacular processes in front of the head”, and the dorso-

lateral appendages as “six tentacular processes on each side, tipped with orange and 1-32

inch long”. Stearns (1873) also described the body color and dorsal tubercles as

“translucent white, covered with fine papillae of an orange color.”

It is difficult to determine the diagnostic characteristics of these species based on the

descriptions by Cooper (1863) and Stearns (1873), which did not provide detailed

descriptions of the body color and dorsal tubercles, as well as the shape of the dorso-lateral

appendages and the radula teeth. Additionally, the type material of both species is

unavailable.

Bergh (1880) introduced Triopha modesta Bergh, 1880 as a new species with a very

detailed description, including details of the external morphology, anatomical features, and

16

radulae formula (21 × 10-11.4-5.R.4-5.10-11). At this time, Bergh (1880) commented that

T. modesta could be the same as T. carpenteri Stearns 1873, but because the holotype was

lost this could not be verified. However, the radular formula of T. modesta (Table 5)

corresponds to that of the northern morphotype as described in this study. Therefore T.

carpenteri Stearns 1873 is suggested as a distinct species from T. modesta Bergh, 1880

according to the results of this study.

Bergh (1894) regarded T. carpenteri as a junior synonym of T. modesta without any

comments, although T. carpenteri is older than T. modesta. Regarding this situation,

O’Donoghue (1922) commented: “I cannot understand why he did this, for obviously if the

forms were identical, then the name of the species would have to be T. carpenteri, for this

name was applied in 1873 and, therefore, had priority.”

Cockerell (1915) proposed the new species name Triopha scrippsiana Cockerell,

1915, based on several individuals collected from La Jolla, California. Cockerell (1915)

described the color of T. scrippsiana as “white, marked with bright vermilion”, the

rhinophores as “pale yellowish olivaceous, not at all red,” the frontal veil as “wholly white”

and the dorsal tubercles as “very bright vermilion, but they are mere subpyramidal

eminences, not elongated structures as in carpenteri.” Cockerell (1915) indicated the

radular formula was 58 × (16.22.2.2.22.16).

Baba (1957) firstly reported T. carpenteri in the Western Pacific, examined two specimens in Shirikishinai, Hakodate and one specimen from Hirota, Miyagi, with radular formula 25 × (10-11.5-6.4.5-6.10-11) and 28 × (10-12.7-8.4.7-8.20-23). However, the specimens examined by Baba (1957) are here considered to be T. modesta, not T. carpenteri based on radular formula presented in this study (Table 5).

17

Ferreira (1977) considered Triopha catalinae (Cooper, 1863) as a senior synonym of

Triopa carpenteri Stearns, 1873, Triopha modesta Bergh, 1880, Triopha scrippsiana

Cockerell, 1915, and Triopha elioti O’Donoghue, 1921 based on external and internal

morphological similarities, including the dorsal tubercles, described as “moderately variable in size and thickness” and shaped as “blunt, rounded or cylindrical, somewhat granulated” (Ferreira, 1977). Ferreira (1977) concluded that the number of rows and teeth per row in the radula increase with the length, but cited some exceptions, such as the observation of more rows of teeth in one individual smaller than 10 mm than in adults of the same species, and commented “The problem of the radula of the Triopha catalinae gets still more complex.” Comparing Ferreira (1977) observations with the results of this study, indicate that he included in the description of T. catalinae specimens with the combined characteristics of the T. catalinae and T. modesta.

The results of the present study confirm that in T. catalinae there is a positive correlation between the number of rows of teeth and the length of the animal (Table 5).

This correlation, however, between body length and the number of rows, was not confirmed in T. modesta (Table 5). And a correlation between body length and teeth per

row, was found in neither T. catalinae nor T. modesta (Table 5).

Jung et al. (2013) reported 7 specimens of T. catalinae from Gangwon-do, South

Korea with an external morphological diagnosis, photographs of the living animal, and mtDNA COI partial sequences. But these specimens, examined in this study, were determined to belong to T. modesta based on the presence of flat and relatively numerous dorsal tubercles as well as the results of the ABDG analysis using the COI gene.

18

In this study, phylogenetic and ABGD analyses recovered specimens belonging to the southern morphotype of T. catalinae as a distinct species. All these animals possess an opaque white body color, conical or rounded dorsal tubercles, and radular formula 48-

79 × (16-18.17-33.R.17-33.16-18), which are consistent with the characteristics of the original description of T. catalinae. Therefore, the name T. catalinae is here proposed for the southern morphotype candidate species.

19

Triopha modesta Bergh, 1880

(Figures 5C-D, 8C-D, 9-10)

Triopha modesta Bergh, 1880: 113-117, pl. 14, figs. 17-20, pl. 15, figs. 1-10. Type locality:

Yukon Harbors, Shumagin Islands, Alaska.

Triopha elioti O'Donoghue, 1921: 165-167. Type locality: Several localities near Nanaimo,

Vancouver Island, British Columbia.

Type material

Triopha modesta Bergh, 1880 – holotype presumably lost, not at NHMD (K. Jensen, pers.

comm.).

Triopha elioti O’Donoghue, 1921 – two syntypes (NHMUK 1953.6.30.64-70); no

additional type material at RBCM (H. Gartner pers. com.) or at CMN (J.-M. Gagnon

pers. com.).

20

Material examined

Vancouver Island, British Columbia, Canada, 27 August 1966, 1 specimen, 19 mm

preserved length, leg. J. L. Page (CASIZ 118667). Akun Island, Aleutian Islands, Alaska,

USA, 28 May 2005, 1 specimen, 74 mm preserved length, leg. V.G. Smith aboard F/V

“Gladiator” (CASIZ 172972). Sanak Island, Aleutian Islands, Alaska, USA, 31 May 2005,

1 specimen, 44 mm preserved length, leg. V.G. Smith aboard F/V “Gladiator” (CASIZ

172971). Munamjin-ri, Jugwang-myeon, Goseong-gun, Gangwon-do, Republic of Korea,

30 April 2018, 1 specimen, 45 mm preserved length, leg. D. -W. Jung (NUTM0001).

Dongsan-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, Republic of Korea, 02 May

2018, 1 specimen, 51 mm preserved length, leg. D. -W. Jung (NUTM0002). Namae-ri,

Hyeonnam-myeon, Yangyang-gun, Gangwon-do, Republic of Korea, 12 May 2018, 1 specimen, 57 mm preserved length, leg. D. -W. Jung (NUTM0003); 13 May 2018, 2 specimens, 44-45 mm preserved length, leg. D. -W. Jung (NUTM0004); 14 May 2018, 2

specimens, 42-57 mm preserved length, leg. D. -W. Jung (NUTM0005); 15 May 2018, 2

specimens, 50-65 mm preserved length, leg. D. -W. Jung (NUTM0006); 16 May 2018, 1

specimen, 48 mm preserved length, leg. D. -W. Jung (NUTM0007); 17 May 2018, 1

specimen, 62 mm preserved length, leg. D. -W. Jung (NUTM0008); 18 May 2018, 1

specimen, 45 mm preserved length, leg. D. -W. Jung (NUTM0009); 19 May 2018, 1

specimen, 58 mm preserved length, leg. D. -W. Jung (NUTM0010); 20 May 2018, 1

specimen, 28 mm preserved length, leg. D. -W. Jung (NUTM0011). Munamjin-ri,

Jugwang-myeon, Goseong-gun, Gangwon-do, Republic of Korea, 24 July 2018, 1

specimen, 52 mm preserved length, leg. D. -W. Jung (NUTM0012); 25 July 2018, 2

specimens, 50-53 mm preserved length, leg. D. -W. Jung (NUTM0013). Rudnaya bay,

21

Rudnaya Pristan, Primorsky Krai, Russia, 02 September 2018, 4 specimens, 33-56 mm preserved length, leg. D. -W. Jung (NUTM0014). 03 September 2018, 3 specimens, 47-55 mm preserved length, leg. D. -W. Jung (NUTM0015).

22

Description

Body limaciform, slightly narrower towards front where it widens again; head round,

gradually becoming narrower from gill to metapodium (Figures 5C, 5D). Body color translucent white or yellowish white. Frontal veil with small, dendritic tentacles on edge

from end to end; small tentacles orange or translucent white, some translucent white

proximally, orange distally; orange colored tentacles on oral veil usually larger than white

tentacles. Rhinophores perfoliate; clavus orange or vermilion; stalk cylindrical, translucent white; rhinophoral sheath slightly covering stalk; edge of rhinophoral sheath smooth or tuberculate. Gill with five tripinnate leaves; leaf tips orange, usually same color as rhinophoral clavus. Anus at center of gill circlet. Metapodium short, distal end blunt. Small opaque white colored glands scattered on whole body. Small dendritic tubercles irregularly arranged on dorsum; usually 7-12 in front of gill, 1-2 behind gill. Numerous small-sized orange patches irregularly arranged on dorsum and body sides, except foot. Four to six pairs of long club-shaped appendages on both sides of dorsum; appendages rise and tilt slightly towards body; in larger individuals, top of appendages globularly shaped, yellow, stalk with two rows of long and arborescent tubercles. Oral tentacle auriculate. Foot slightly narrower than body, distal end round, and posterior end pointed. Rhinophoral clavus, gill

tips often lighter than dorsal tubercles.

Radular formula 19-24 × (9-12.5-10.R.5-10.9-12) (Table 5). Rachidian plate rudimentary, thin, composed of two ovoid-shaped structures in middle, with wide inverted triangular structures on either side (Figure 9A). Lateral teeth elongate, strongly hooked, narrowing from point of bending to pointed tips (Figure 9B). Outermost teeth square,

23

arranged in row (Figure 9C). Radula translucent, sometimes yellowish in large individuals.

Jaw with thin, elongate rodlets, densely packed (Figure 9D).

Genital opening on right side of body, in slightly recessed area behind head, covered with three lobes. Very small individuals lack recessed area. Reproductive system triaulic

(Figure 10). Ampulla long, convoluted; it connects to female gland and prostate. Prostate

granulate, large; almost four times as large as seminal vesicle, connecting into seminal

vesicle. Seminal vesicle convex ovoid, opening into vas deferens. Vas deferens smooth,

long. Penis armed with numerous hamate penial hooks (Figures 8C, 8D). Vagina smooth,

long, connecting to bursa copulatrix by wide duct. Bursa copulatrix large, almost same size

as female gland. From bursa copulatrix leads another duct connecting to receptaculum

seminis and female gland.

Geographic range

Known from East Sea (Sea of Japan), Alaska, British Columbia, Washington, and Oregon

(Baba, 1957; Okutani, 2000; Goddard & Foster, 2002; Shield, 2009; Marliave et al., 2011;

Jung et al., 2013),

24

Remarks

Bergh (1880) introduced the name Triopha modesta Bergh, 1880 based on a single

specimen collected by W. H. Dall in the Yukon Harbor, Alaska. According to Bergh (1880),

W. H. Dall described the color of the living animal as yellowish-white. Bergh (1880)

described the preserved holotype in great detail and indicated the color was of whitish, with the dorsal appendages, the gill and the rhinophores more yellowish. Bergh (1880) also provided numerous details of the internal anatomy of the specimen including the radular formula as 21 × (10-11.4-5.3.4-5.10-11). Efforts to locate the holotype have failed and therefore it is here considered presumably lost.

O’Donoghue (1921) introduced the name Triopha elioti O’Donoghue, 1921 based on several individuals collected near Nanaimo, British Columbia. Two of the syntypes are deposited at the NHMUK (1953.6.30.64-70), but the rest of the type series is presumably lost. O’Donoghue (1921) described the body color of T. elioti as translucent white or very pale yellowish white, 4-6 lateral teeth and 8 outermost teeth in radulae, and 5 numbers of

“knob-like ends” dorso-lateral appendages with “double series of small blunt papillae”.

O’Donoghue (1921) also indicated the dorsum and the body sides had a number of tiny brownish spots and some larger reddish spots. Whereas O’Donoghue (1921) provided detailed descriptions of the external coloration and morphology as well as of the radular teeth, the rest of the internal anatomy was not described. O’Donoghue (1921) compared T. elioti with Triopa carpenteri Stearns, 1873 but not with Triopha modesta of which he did not seem aware. These two rows of dendritic papillae on the appendages at the dorsal edge described by O’Donoghue (1921) are characteristics of large specimens of T. modesta.

However, these papillae are not evident in smaller animals. It is also difficult to observe

25

these papillae in the preserved specimens, even if the specimens were well preserved. The granular tubercles on the appendage at the dorsal edge are also found in T. catalinae, but

their granule size on the appendages is small and round compared to those of T. modesta.

O’Donoghue (1922) acknowledged that O’Donoghue (1921) was not aware of the

description of Triopha aurantiaca Cockerell, 1908, and considered that T. elioti was a

junior synonym of T. aurantiaca. Cockerell & Eliot (1905) collected a single individual described as “much contracted” and having “lost both its natural shape and colour” in San

Pedro, California, and identified it as Triopha sp. based on the external traits and the radular formula 25 × (8.4.R.4.8). According to Cockerell & Eliot (1905) the specimen of Triopha sp. did not match any of the previously described species in the genus Triopha, but

Cockerell & Eliot (1905) did not describe it as a new species because of the individual was considered immature and the quality of specimen was not perfect. Later, Cockerell (1908) reported that Triopha sp. sensu Cockerell & Eliot, 1905 have similar external characters to

T. carpenteri, but the body color is orange instead of white, and the color of appendage tips are vermilion. Ferreira (1977) synonymized T. aurantiaca with T. maculata on the basis of similarities in body color, external characters, radulae formula, and geographic range.

However, the information given in previous studies (Cockerell & Eliot, 1905 and Cockerell,

1908) is not sufficient to distinguish them accurately. The results of the present study suggest the possibility that T. aurantiaca may be a synonym of T. modesta which is characterized by a yellowish body color and radular formula 19-24 × (9-12.5-10.R.5-10.9-

12).

MacFarland (1966) considered that T. carpenteri and T. modesta were different species because they had different radular formulae [T. carpeteri: 33 × (9-18.9-18.R.9-

26

18.9-18); T. modesta: 21-28 × (10-13.4-7.R.4-7.10-13)]. However, Ferreira (1977) considered the radular formula discrepancies as intra-specific variation and suggested T. modesta was a junior synonym of T. catalinae.

In this study, the northern morphotype of T. catalinae was found to be genetically distinct from the southern morphotype, and species delimination analysis confirmed they

constitute distinct species. As mentioned above, the name T. catalinae is retained for the

southern morphotype. The characteristics of the northern morphotype specimens examined

match those in the original description of T. modesta.

The range in the radular formula of the northern morphotype identified by this study

is very similar to that of the original description of T. modesta (Bergh, 1880). Therefore, it

is proposed that the scientific name for the northern morphotype is T. modesta. The present

study also confirms that the dendritic papillae on the dorso-lateral appendages of large

individuals are characteristic of northern morphotype (T. modesta) based on examination

of living specimens and photographic records with geographical information (iNaturalist).

This confirms that T. elioti is a junior synonym of T. modesta.

Triopha modesta was reported in the Western Pacific; South Korea (Jung et al., 2013),

Japan (Baba, 1957; Okutani, 2000), and Russia (Martynov et al., 2015; Chichvarkhin,

2016).

According to the results of this study, T. modesta is distinguished from other species

of Triopha by the following characteristics: translucent white to yellow body color, flat or

small dendritic dorsal tubercles, arborescent tubercles present on dorsal appendages in

large individuals, color of rhinophoral clavus and gill tip is darker than dorsal tubercles and

radular formula 19-24 × (9-12.5-10.R.5-10.9-12).

27

DISCUSSION AND CONCLUSIONS

The molecular phylogenetic analyses presented in this study shows that the northern and southern morphotypes of Triopha catalinae constitute two different species. The name T. modesta is resurrected to the northern species based on the examination of samples collected from South Korea, Japan, Russia, Alaska, Canada, Washington, and Oregon and a re-evaluation of the literature. The name T. catalinae is retained as valid for the southern species based on the study of specimens collected from Oregon to Baja California and

comparison to the original description of this species.

These two species, T. catalinae and T. modesta, display consistent morphological

differences in the external and internal features. Triopha modesta is characterized by having a translucent white body color, relatively small and dendritic dorsal tubercles, two rows of arborescent tubercles on the dorso-lateral appendages in larger individuals, color of rhinophoral clavus and gill tip is often lighter than dorsal tubercles, and a radular formula

19-24 × (9-12.5-10.R.5-10.9-12). On the contrary, Triopha catalinae is distinguished by having an opaque white body color, large conical or rounded dorsal tubercles on dorsum, small nodules on the dorso-lateral appendages even in larger individuals, color of rhinophoral clavus and gill tip is darker than dorsal tubercles, and radular formula 48-79 ×

(16-18.17-33.R.17-33.16-18).

Despite the fact that these two species are readily distinguishable, before the availability of molecular data, were considered them to be synonymous. Triopa catalinae was first described by Cooper (1863) from Santa Catalina Island, California, and Triopha modesta was first introduced by Bergh (1880) from the Shumagin Islands, Alaska. O’Donoghue

28

(1921) noted that Triopha elioti, a junior synonym of T. modesta, had two rows of small and blunt tubercles present on the dorsal appendages and MacFarland (1966) described the differences in radular formula between T. catalinae and T. modesta. Ferreira (1977) suggested T. modesta was a junior synonym of T. catalinae based on morphological evidence including external morphological features and radular morphology, but failed to show a correlation between the size of individuals and radular formula using the “cold shock” technique (Ferreira, 1977). This is another example of a pseudocryptic species complex; whereas T. catalinae and T. modesta are morphologically distinct and those differences were already documented in the literature, the could not be formalized until molecular evidence confirmed their distinctiveness.

The consistent external differences between T. catalinae and T. modesta and a review of published illustrations of the two species (Okutani, 2000; Behrens & Hermosillo, 2005;

Jung et al., 2013; Bertsch, 2014; Martynov et al, 2015; Chichvarkhin, 2016), including photographs from iNaturalist (https://www.inaturalist.org), have made it possible to determine that the range of T. modesta includes South Korea, Japan, Russia, Alaska,

Canada, Washington, Oregon, and Central California, and T. catalinae ranges from Alaska to Baja California. The range of the two species is sympatric in a region from Craig City,

Alaska to Mendocino County, California. Further research on niche partitioning, including possible differences in bathymetric range, seasonal population variations, and prey preferences of the two species in the areas of range overlap is necessary to understand how the sympatry is maintained.

In addition to the Triopha catalinae species complex studied in this thesis, two other species of nudibranch were known to be distributed widely across the western and eastern

29

sides of the Pacific Ocean (the sandiegensis species complex: Lindsay et al., 2016

and the Hermissenda crassicornis species complex: Lindsay & Valdés, 2016). Sister taxa in these two species complexes were also observed to have sympatric ranges in the Eastern

Pacific: Diaulula sandiegensis was found from Barkely Sound, British Columbia to Fort

Bragg, California in conjunction with Diaulula odonoghuei (Lindsay et al., 2016), and both

Hermissenda crassicornis and Hermissenda opalescens were reported from Point Reyes,

California to Clayoquot Sounds, British Columbia (Lindsay & Valdés, 2016; Merlo et al.,

2018). According to Lindsay et al (2016), allopatric speciation in the D. sandiegensis

species complex during the Calabrian age of the Pleistocene, followed by a range

expansion of D. odonoghuei eastward, resulted in the formation of the sympatric range. I

hypothesize that the ranges of Triopha catalinae and T. modesta could have resulted from

a similar process. All these biogeographical studies (Diaulula, Hermissenda and Triopha)

reveal the existence of range overlapping regions for nudibranch sister taxa, from British

Columbia to California. This consistent pattern is suggestive of a shared evolutionary

history for all these different species complexes and needs to be studied in more detail. In

future studies, this information should be coupled with the examination of ecological

factors that contribute to the maintenance of the sympatric range (e.g., niche displacement)

to have a better understanding of the role of glacial driven radiations during the Pleistocene

period, and provide insights to understanding the underlying evolutionary processes.

30

REFERENCES

Baba K (1957) A revised list of the species of Opisthobranchia from the northern part of

Japan, with some additional descriptions. Journal of the Faculty of Science Hokkaido

University Series ⅤⅠ. Zoology, 13: 8-14.

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 (1880) On the nudibranchiate gasteropod Mollusca of the north Pacific Ocean,

with special reference to those of Alaska. Part II. Proceedings of the Academy of Natural

Sciences of Philadelphia, 32: 40-127.

Bergh LSR (1894) XIII. Die Opisthobranchien. Reports on the dredging operations off the

west coast of Central America to the Galapagos, to the west coast of Mexico, and in the

Gulf of California, in charge of Alexander Agassiz, carried on by the U. S. Fish

commission steamer “Albatross”, during 1891, Lieut. Commander Z. L. Tanner, U.S.N.,

commanding. Bulletin of the Museum of Comparative Zoology, 25: 125-233.

Bertsch H (2014) Biodiversity in La Reserva de la Biósfera Bahía de los Ángeles y Canales

de Ballenas y Salsipuedes: naming of a new genus, range extensions and new records,

and species list of (Mollusca: Gastropoda), with comments on

biodiversity conservation within marine reserves. The Festivus, 46: 158-177.

Cabanne GS, Calderón L, Trujillo-Arias N, Flores P, Pessoa R, d'Horta FM & Miyaki CY

(2016) Effects of Pleistocene climate changes on species ranges and evolutionary

31

processes in the Neotropical Atlantic Forest. Biological Journal of the Linnean Society,

119: 856-872.

Chichvarkhin A (2016) Shallow water sea slugs (Gastropoda: Heterobranchia) from the

northwestern coast of the Sea of Japan, north of Peter the Great Bay, Russia. PeerJ, 4:

e2774.

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

genealogies. Molecular Ecology, 9: 1657-1659.

Cockerell TDA (1908) Mollusca of La Jolla, California. The Nautilus, 21: 106-107.

Cockerell TDA (1915) The nudibranch-genus Triopha in California. Journal of

Entomology & Zoology, 7: 228-229.

Cockerell TDA & Eliot C (1905) Notes on a collection of Californian nudibranchs. Journal

of Malacology, 12: 31-53.

Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J,

Cassis G & Gray MR (1998) Histone H3 and U2 snRNA DNA sequences and arthropod

molecular evolution. Australian Journal of Zoology, 46: 419-437.

Cooper JG (1863) On new or rare Mollusca inhabiting the coast of California. No. II.

Proceedings of the California Academy of Natural Sciences, 3: 56-60.

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

glaciation in New Zealand. Geology, 45: 595-598.

Ferreira AJ (1977) Review of genus Triopha (Mollusca-Nudibranchia). The Veliger, 19:

387-402.

Fewkes JW (1889) New invertebrata from the coast of California. Bulletin of the Essex

Institute, 21: 99-146.

32

Folmer O, 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.

Foltz DW, Nguyen AT, Kiger JR & Mah CL (2008) Pleistocene speciation of sister taxa in

a North Pacific clade of brooding sea stars (Leptasterias). Marine Biology, 154: 593-

602.

Goddard JHR & Foster NR (2002) Range extensions of sacoglossan and nudibranch

mollusks (Gastropoda: Opisthobranchia) to Alaska. The Veliger, 45: 331-336.

Goddard JHR & Green B (2013) Developmental mode in opisthobranch molluscs from the

northeast Pacific Ocean: additional species from southern California and supplemental

data. Bulletin, Southern California Academy of Sciences, 112: 49-63.

Hillis DM & Bull JJ (1993) An empirical test of bootstrapping as a method for assessing

confidence in phylogenetic analysis. Systematic Biology, 42: 182-192.

Hoover CA, Padula V, Schrödl M, Hooker Y & Valdés Á (2017) Integrative of

the Felimare californiensis and F. ghiselini species complex (Nudibranchia:

Chromodorididae), with description of a new species from Peru. Journal of Molluscan

Studies, 83: 461-475.

Huelsenbeck JP, Rannala B (2004) Frequentist properties of Bayesian posterior

probabilities of phylogenetic trees under simple and complex substitution models.

Systematic Biology, 53: 904-913.

Jung D, Lee J & Kim C-B (2013) A report on species of phyllidiid and polycerid

nudibranch including two species new to Korea. Journal of Species Research, 2: 7-14.

33

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.

Kumar S, Stecher G, Li M, Knyaz C & Tamura K (2018) MEGA X: Molecular

evolutionary genetics analysis across computing platforms. Molecular Biology and

Evolution, 35: 1547-1549.

Lanfear R, Frandsen PB, Wright AM, Senfeld T & Calcott B (2016) PartitionFinder 2: New

methods for selecting partitioned models of evolution for molecular and morphological

phylogenetic analyses. Molecular Biology and Evolution, 34: 772-773.

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

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

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

Heterobranchia) is a species complex. PloS one, 11: e0154265.

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.

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.

MacFarland FM (1966) Studies of opisthobranchiate mollusks of the Pacific coast of North

America. Memoirs of the California Academy of Sciences, 6: 1-546 pp.

34

Marliave JB, Gibbs CJ, Gibbs DM, Lamb AO & Young SJF (2011) Biodiversity stability

of shallow marine benthos in Strait of Georgia, British Columbia, Canada through

climate regimes, overfishing and ocean acidification. In: Grillo O & Verona G, eds.

Biodiversity loss in a changing planet. IntechOpen, Rijeka, Croatia. 49-74 pp.

Martynov AV, Sanamyan NP & Korshunova T (2015) Review of the opisthobranch

mollusc fauna of Russian Far Eastern Seas: Pleurobranchomorpha, Doridida and

Nudibranchia. Bulletin of Kamchatka State Technical University. 34: 62-87. [in Russian]

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

24: 114-276.

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

Steinke D, Hebert PD, 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.

Miller MA, Pfeiffer W & Schwartz T (2010) Creating the CIPRES Science Gateway for

inference of large phylogenetic trees. Proceedings of the Gateway Computing

Environments Workshop (GCE), 1-8.

O’Donoghue CH (1921) Nudibranchiate Mollusca from the Vancouver Island region.

Transactions of the Royal Canadian Institute, 13: 147-209.

O’Donoghue CH (1922) Notes on the taxonomy of nudibranchiate Mollusca from the

Pacific coast of North America. Journal of Molluscan Studies, 15: 133-150.

O’Donoghue CH (1926) A list of the nudibranchiate Mollusca recorded from the Pacific

coast of North America, with notes on their distribution. Transactions of the Royal

Canadian Institute, 15: 199-247.

35

Okutani T (2000) Marine mollusks in Japan. University of Tokyo Press, Tokyo, Japan. 1-

1173 pp.

Palumbi SR (1996) Nucleic Acids II: The polymerase chain reaction. In: Hillis DM, Moritz

C & Mable BK, eds. Molecular Systematics Second Edition. Sinauer, Sunderland, USA.

205-247 pp.

Puillandre N, Lambert A, Brouillet S & Achaz G (2012) ABGD, Automatic Barcode Gap

Discovery for primary species delimitation. Molecular Ecology, 21: 1864-1877.

Reid DG, Dyal P & Williams ST (2012) A global molecular phylogeny of 147 periwinkle

species (Gastropoda, Littorininae). Zoologica Scripta, 41: 125-136.

Ronquist F & Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under

mixed models. Bioinformatics, 19: 1572-1574.

Schebeck M, Schuler H, Einramhof B, Avtzis DN, Dowle EJ, Faccoli M, Battisti A,

Ragland GJ, Stauffer C & Bertheau B (2019) The Apennines as a cryptic Pleistocene

refugium of the bark beetle Pityogenes chalcographus (Coleoptera: Curculionidae).

Biological Journal of the Linnean Society, 127: 24-33.

Shields C (2009) Nudibranchs of the Ross Sea, Antarctica: phylogeny, diversity, and

divergence. Master thesis, Clemson University, South Carolina, USA. 1-82pp.

Sowell RR (1949) Taxonomy and ecology of the nudibranchiate Mollusca of the Coos Bay,

Oregon region. Master thesis, Oregon college, Oregon, USA. 1-54pp.

Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis

of large phylogenies. Bioinformatics, 30: 1312-1313.

36

Stearns REC (1873) Descriptions of a new genus and two new species of nudibranchiate

mollusks from the coast of California. Proceedings of California Academy of Sciences,

5: 77-78.

Steinberg JE (1961) Notes on the opisthobranchs of the west coast of North America. I.

nomenclatural changes in the order Nudibranchia (Southern California). The Veliger, 4:

57-63.

Uribe RA, Sepúlveda F, Goddard JH & Valdés Á (2018). Integrative systematics of the

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

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

Wang Q, Abbott RJ, Yu Q-S, Lin K & Liu J-Q (2013) Pleistocene climate change and the

origin of two desert plant species, Pugionium cornutum and Pugionium dolabratum

(Brassicaceae), in northwest China. New Phytologist, 199: 277-287.

Wielstra B, Themudo GE, Güçlü Ö, Olgun K, Poyarkov NA & Arntzen JW (2010) Cryptic

crested newt diversity at the Eurasian transition: the mitochondrial DNA

phylogeography of Near Eastern Triturus newts. Molecular Phylogenetics and Evolution,

56: 888-896.

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

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

the remaining refugial‐phase biodiversity. Biodiversity and Conservation, 19: 919-941.

37

TABLES

Table 1. List of specimens sequenced for this study including locality, voucher numbers, and GenBank accession numbers.

Abbreviations: CASIZ: California Academy of Science Invertebrate Zoology; CPIC: California State Polytechnic University

Invertebrate Collection; *denotes specimen sequences downloaded from GenBank; **denotes outgroup.

Collection Date GenBank Accession Number Species Locality Voucher Isolate (YYYY. MM. DD) 16S COI H3

San Francisco bay, San Francisco,

38 CASIZ170648 TCCAL01 2004. 07. 15 *HM162600 *HM162690 *HM162506 California, USA San Miguel Island, Channel Islands, NUTC0001 TCCAL02 2018. 02. 18 Pending Pending Pending California, USA San Miguel Island, Channel Islands, NUTC0001 TCCAL03 2018. 02. 18 — Pending Pending California, USA Bahía de los Ángeles, Ensenada, NUTC0002 TCMEX01 2018. 03. 20 Pending Pending Pending T. catalinae Baja California, Mexico Bahía de los Ángeles, Ensenada, NUTC0002 TCMEX02 2018. 03. 20 Pending Pending Pending Baja California, Mexico Bahía de los Ángeles, Ensenada, NUTC0003 TCMEX03 2018. 03. 21 Pending Pending Pending Baja California, Mexico Bahía de los Ángeles, Ensenada, NUTC0003 TCMEX04 2018. 03. 21 Pending Pending Pending Baja California, Mexico Continued on next page

Collection Date GenBank Accession Number Species Locality Voucher Isolate (YYYY. MM. DD) 16S COI H3

Bahía de los Ángeles, Ensenada, Baja NUTC0003 TCMEX05 2018. 03. 21 — Pending Pending California, Mexico

— KM2F4 KM2F4 — — Pending Pending T. catalinae Oregon, USA MC002 MC002 — Pending Pending —

— DQ026830 DQ026830 — — *DQ026830 —

39 Akun Island, Aleutian Islands, CASIZ172972 TMALK01 2005. 03. 28 — Pending Pending Alaska, USA

Sanak Island, Aleutian Islands, CASIZ172971 TMALK02 2005. 03. 31 — Pending Pending Alaska, USA

11BIOAK- Kachemak Bay, Alaska, USA KF643788 2011. 05. 15 — *KF643788 — 0565 T. modesta 11BIOAK- Kachemak Bay, Alaska, USA KF643916 2011. 05. 15 — *KF643916 — 0566

San Juan Island, Washington, USA BMBM-0114 MH243010 2016. 06. 01 — *MH243010 —

Munamjin-ri, Jugwang-myeon, Goseong-gun, Gangwon-do, NUTM0001 TMKOR01 2018. 04. 30 Pending Pending Pending Republic of Korea Continued on next page

Collection Date GenBank Accession Number Species Locality Voucher Isolate (YYYY. MM. DD) 16S COI H3

Dongsan-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0002 TMKOR02 2018. 05. 02 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0003 TMKOR03 2018. 05. 12 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0004 TMKOR04 2018. 05. 13 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0004 TMKOR05 2018. 05. 13 Pending Pending Pending

40 Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0005 TMKOR06 2018. 05. 14 Pending Pending Pending Republic of Korea T. modesta Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0005 TMKOR07 2018. 05. 14 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0006 TMKOR08 2018. 05. 15 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0006 TMKOR09 2018. 05. 15 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0007 TMKOR10 2018. 05. 16 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0008 TMKOR11 2018. 05. 17 Pending Pending Pending Republic of Korea Continued on next page

Collection Date GenBank Accession Number Species Locality Voucher Isolate (YYYY. MM. DD) 16S COI H3

Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0009 TMKOR12 2018. 05. 18 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0010 TMKOR13 2018. 05. 19 Pending Pending Pending Republic of Korea Namae-ri, Hyeonnam-myeon, Yangyang-gun, Gangwon-do, NUTM0011 TMKOR14 2018. 05. 20 Pending Pending Pending Republic of Korea Munamjin-ri, Jugwang-myeon, Goseong-gun, Gangwon-do, NUTM0012 TMKOR15 2018. 07. 24 Pending Pending Pending

41 Republic of Korea Munamjin-ri, Jugwang-myeon, Goseong-gun, Gangwon-do, NUTM0013 TMKOR16 2018. 07. 25 Pending Pending Pending T. modesta Republic of Korea Munamjin-ri, Jugwang-myeon, Goseong-gun, Gangwon-do, NUTM0013 TMKOR17 2018. 07. 25 Pending Pending Pending Republic of Korea Rudnaya Bay, Rudnaya Pristan, NUTM0014 TMRUS01 2018. 09. 02 Pending Pending Pending Primorsky Krai, Russia

Rudnaya Bay, Rudnaya Pristan, NUTM0014 TMRUS02 2018. 09. 02 Pending Pending Pending Primorsky Krai, Russia

Rudnaya Bay, Rudnaya Pristan, NUTM0014 TMRUS03 2018. 09. 02 Pending Pending Pending Primorsky Krai, Russia

Rudnaya Bay, Rudnaya Pristan, NUTM0015 TMRUS04 2018. 09. 03 Pending Pending Pending Primorsky Krai, Russia Continued on next page

Collection Date GenBank Accession Number Species Locality Voucher Isolate (YYYY. MM. DD) 16S COI H3

Oregon, USA MC001 MC001 — Pending Pending —

Rudnaya Bay, Rudnaya Pristan, MC003 MC003 — Pending Pending — Primorsky Krai, Russia

Rudnaya Bay, Rudnaya Pristan, T. modesta MC004 MC004 — Pending Pending — Primorsky Krai, Russia

Rudnaya Bay, Rudnaya Pristan, MC005 MC005 — Pending — — Primorsky Krai, Russia

42 Washington, USA GQ292040 GQ292040 — — *GQ292040 —

Santa Barbara, California, USA CASIZ184514 CASIZ184514 2010. 11. 05 Pending Pending — **T. maculata Santa Barbara, California, USA CASIZ184515 CASIZ184515 2010. 11. 05 Pending Pending —

Table 2. Best partition scheme and corresponding models determined by

PartitionFinder2.

Gene Partition # Sites Best Model

16S 1 1-456 HKY + I

1 457-1114 GTR + G

COI 2 458-1114 GTR + I

3 459-1114 F81 + I

H3 1 1115-1442 GTR + I + G

43

Table 3. List of species recovered in the ABGD analysis of mtDNA COI sequences of

specimens identified as using the Kimura 2-parameter model, including isolate and

GenBank accession numbers, missing data is indicated by dashes.

Species 16S COI

—, HM162600, —, DQ026830, —, HM162690,

—, MC002, TCCAL02, KM2F4, MC002, TCCAL02, T. catalinae —, TCMEX01, TCMEX02, TCCAL03, TCMEX01, TCMEX02,

TCMEX03, TCMEX04, — TCMEX03, TCMEX04, TCMEX05

—, —, —, GQ292040, KF643788, KF643916,

MC001, MC003, MC004, MC001, MC003, MC004,

MC005, —, TMALK01, —, MH243010, TMALK01,

TMALK02, TMKOR01, TMKOR02, TMALK02, TMKOR01, TMKOR02,

TMKOR03, TMKOR04, TMKOR05, TMKOR03, TMKOR04, TMKOR05,

T. modesta TMKOR06, TMKOR07, TMKOR08, TMKOR06, TMKOR07, TMKOR08,

TMKOR09, TMKOR10, TMKOR11, TMKOR09, TMKOR10, TMKOR11,

TMKOR12, TMKOR13, TMKOR14, TMKOR12, TMKOR13, TMKOR14,

TMKOR15, TMKOR16, TMKOR17, TMKOR15, TMKOR16, TMKOR17,

TMRUS01, TMRUS02, TMRUS03, TMRUS01, TMRUS02, TMRUS03,

TMRUS04 TMRUS04

44

Table 4. Mean p-distances within and between groups using the Kimura 2-parameter

model and 1,000 replicates bootstrap value.

Mean Gene T. catalinae T. modesta p-distances

16s 0.25% 0.38% Within species COI 1.41% 0.78%

16s 2.72% Between species COI 8.98%

Table 5. Radular formulae of each specimen examined including voucher numbers.

Species Voucher Length of specimen Radular formula (mm) CASIZ070643 57 63 × (16.17.R.17.16)

T. catalinae CASIZ071365 39 79 × (18.33.R.33.8)

NUTC0002 11 48 × (18.20.R.20.18)

CASIZ118667 19 19 × (10.7.R.7.10)

CASIZ172971 44 22 × (9.5.R.5.9)

CASIZ172972 74 23 × (10.8.R.8.10)

NUTM0008 62 22 × (12.8.R.8.12) T. modesta NUTM0009 45 23 × (11.7.R.7.11)

NUTM0012 52 24 × (11.10.R.10.11)

NUTM0013 53 23 × (11.9.R.9.11)

NUTM0015 55 22 × (11.7.R.7.11)

45

FIGURES

Figure 1. Bayesian phylogenetic tree of the Triopha catalinae species complex with

Triopha maculata as the outgroup, based on concatenated sequences of mitochondrial

16S, COI, and H3 genes. Posterior probabilities (PP > 0.9) are shown above the branches

and maximum-likelihood bootstrap (BS > 70) values shown below.

46

nbr

125

112

100

87

75

62

50

37

25

12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ......

00 00 00 01 01 01 01 01 01 02 02 02 02 02 02 03 03 03 03 03 Dist. value

Figure 2. Automatic Barcode Gap Discovery (ABGD) results showing the distributions

of pairwise distances among 16S sequences for Triopha catalinae species complex

using the Kimura 2-parameter model.

nbr

224

201

179

156

134

112

89

67

44

22 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ......

00 00 01 01 02 02 03 03 04 04 05 05 06 06 07 07 08 08 09 Dist09 . value

Figure 3. Automatic Barcode Gap Discovery (ABGD) results showing the distributions

of pairwise distances among COI sequences for Triopha catalinae species complex

using the Kimura 2-parameter model.

47

Figure 4. TCS network of mtDNA COI sequences for Triopha catalinae species complex.

Circle size is proportional to the frequency of haplotypes detected. Haplotypes are

connected via lines with dashes representing mutational steps.

48

Figure 5. Living animals. A-B. Triopha catalinae (Cooper, 1863). A. Specimens from

Bahía de los Ángeles, Ensenada, Mexico (NUTC0002). B. Specimens from Bahía de los

Ángeles, Ensenada, Mexico (NUTC0003). C-D. Triopha modesta Bergh, 1880. C.

Specimen from Yangyang-gun, Gangwon-do, South Korea (NUTM0010). D. Specimen

from Rudnaya Pristan, Primorsky Krai, Russia (NUTM0014). A-B. Photos by Craig

Hoover, C-D. Photos by Dae-Wui Jung.

49

Figure 6. Triopha catalinae, SEM micrographs of the radula. A. Rachidian plate. B.

Lateral teeth. C. Outermost teeth. D. Jaw elements. A-C. Specimens from Bahía de los

Ángeles, Ensenada, Mexico (NUTC0002). D. Specimen from Santa Rosa Island,

California (CASIZ071365). Scale bars: A-C. 200 µm D. 50 µm.

50

Figure 7. Triopha catalinae, drawing of the reproductive system of a specimen from

Monterey County, California (CASIZ070643). Abbreviations: am, ampulla; bc, bursa

copulatrix; fg, female gland mass; gp, gonopore; pr, prostate; rs, receptaculum seminis;

v, vagina; vd, vas deferens. Scale bar: 1 mm.

51

Figure 8. Penial hooks. A-B. Triopha catalinae. Specimens from San Mateo County,

California (CASIZ113461). C-D. Triopha modesta. Specimen from Yangyang-gun

Gangwon-do, South Korea (NUTM0007). A, C. Penial hooks at 1,000x magnification

under conventional fluorescence microscope. B, D. Drawings. Scale bars: A-D. 10 µm.

52

Figure 9. Triopha modesta, SEM micrographs of the radula. A. Rachidian plate. B. Lateral

teeth. C. Outermost teeth. D. Jaw elements. A-C. Specimens from Rudnaya Pristan,

Primorsky Krai, Russia (NUTM0015). D. Specimen from Vancouver Island, British

Columbia, Canada (CASIZ118667). Scale bars: A-C. 200 µm D. 50 µm.

53

Figure 10. Triopha modesta, drawing of the reproductive system of a specimen from

Yangyang-gun, Gangwon-do, South Korea (NUTM0005). Abbreviations: am, ampulla;

bc, bursa copulatrix; fg, female gland mass; gp, gonopore; pr, prostate; rs, receptaculum

seminis; v, vagina; vd, vas deferens. Scale bar: 1 mm.

54