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PHYLOGENETIC AND PHYLOGEOGRAPHIC ANALYSES REVEAL A COMPLEX IN THE ESTUARINE ADSPERSA

Amanda Sobel University of New Hampshire, Durham

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PHYLOGENETIC AND PHYLOGEOGRAPHIC ANALYSES REVEAL A SPECIES COMPLEX IN THE ESTUARINE NUDIBRANCH TENELLIA ADSPERSA

BY

AMANDA J. SOBEL

B.A., SAINT ANSELM COLLEGE, 2011

THESIS

Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of

Master of Science In Biological Sciences, Marine Biology

December, 2017

This thesis has been examined and approved in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences by:

Thesis Director, Dr. Larry G. Harris, Professor of Zoology

Dr. Terrence M. Gosliner, Senior Curator of Zoology and Geology Department, California Academy of Sciences, San Francisco CA.

Dr. Alan M. Kuzirian, Professor Emeritus/ Senior Scientist, Marine Biological Laboratory, Woods Hole, MA

Dr. Walter J. Lambert, Professor of Zoology, Framingham State University, Framingham MA.

Dr. David C. Plachetzki, Associate Professor, Genetics

On July 31st, 2017

Original approval signatures are on file with the University of New Hampshire Graduate School

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ACKNOWLEDGEMENTS

The old adage says, “nothing is ever done in a vacuum,” and that’s certainly true in science. There are so many individuals that made this work possible and for that, I am grateful.

First, thank you especially to my thesis advisor, Larry Harris, who took a big risk in bringing me into the lab. You have handled my work with the utmost patience, care, and a bit of push when needed. I am grateful to have been able to choose my own research topic and have learned so much from you. I am appreciative of the chance you took on me. I will miss our talks of nudibranchs, frustrations, and other marine wonders.

To my Graduate Thesis Committee, thank you for your constant mentorship and for agreeing to be a part of this journey. I am continuously in awe of the magnitude of accomplishments you collectively achieve and am truly lucky to have been able to work with you all. Thank you especially to David Plachetzki, who allowed me to use his laboratory space and resources while teaching me, patiently, molecular biology and phylogenetics.

Thank you to Nadine Rorem, a colleague who collected numerous specimens used in this study, and to Juan Lucas Cervera who allowed me to utilize two museum specimens. To

Kristen (Roberts) Cella, thank you for answering my questions about your work. To Brian

Penney, thank you for your continued advisement and support, and for first exposing me to nudibranchs!

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To my fellow graduate students Anna Chase, Sara Edquist, and Seth Goodnight, thank you for your guidance, support, and friendship. To the undergraduates who assisted me, thank you from the bottom of my . It was a great honor teaching you what I know, and in return learning what you knew. Kendall Young, Dalton Ryan, Jasmin Buteau, and Molly

Hartley, especially were instrumental in many tedious aspects of my research.

Thank you to the following marinas and other locations where I was allowed to conduct sampling: Great Bay Marine (Newington, NH), Hampton Marina (Hampton, NH), Liberty

Marina (Danvers, MA), Wentworth Marina (Newcastle, NH), the Coastal Marine Lab

(Newcastle, NH), Jackson Estuarine Lab (Durham, NH), York Harbor Marine (York, ME),

Marston’s Marina (Saco, ME), Dover Marina (Dover, NH), Robinhood Marine Center

(Georgetown, ME), Great Island Boat Yard (Harpswell, ME), Yarmouth Boat Yard

(Yarmouth, ME), Eddy Yacht Sales (Edgecomb, ME), Schooner’s Landing (Damariscotta,

ME), Kennebec Tavern (Bath, ME), Eastport Commercial Pier (Eastport, ME), Robbinston

Boat Launch (Robbinston, ME), Calais Town Dock (Calais, ME). A special thank you to Lisa and Don Lee, my Aunt and Uncle, who provided me a home point while I was hunting nudibranchs throughout the coast of Maine, and allowing me to sample at Neil’s Point

(Harpswell, ME).

Sources of funding for this project included the University of New Hampshire College of

Life Sciences and Agriculture (COLSA) Department of Biological Sciences (DBS), the New

England Estuarine Research Society (NEERS), the UNH Graduate School, the UNH School of

Marine Sciences and Engineering (SMSOE), and the Lerner Gray Memorial Fund of the American Museum of Natural History,

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To my family, I know you never understood my passion for nudibranchs but you stood by my side and supported my decisions nonetheless. Thank you for your emotional, physical, and financial support throughout my studies. Thank you especially to Mildred, my grandmother, who supported my undergraduate summer experience at Duke Marine Lab, during which I actually collected Tenellia adspersa, along with other nudibranchs. Thank you my sister, Allison Gifford, whose natural curiosity and willingness to assist with collections and sorting pushed me to become a better researcher. I know you will go far and will carry that appreciation and wonderment for the natural world forever.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS…………………………………………………………………………………………..………ii

LIST OF TABLES………………………………………………………………………………………………………….....vi

LIST OF FIGURES…………………………………………………………………………………………………………..vii

ABSTRACT…………………………………………………………………………………………………………………..viii

INTRODUCTION………..…………………………………………………………………………………………………….1 MATERIALS AND METHODS...... ………………………………………………………………...8 Specimen Collection…………………………………………………………………………………………………….8 Radulae analysis…………………………………………………………………………………………………………9 DNA Extraction, Amplification, and Sequencing……………………………………………………...…...10 DNA Barcoding Analyses and Haplotype Network………………………………………..……………...12 Phylogenetic Analyses……………………………………………………………………………………………….14 RESULTS………………………………………………………………………………………………………………………16 Morphological Results……………………………………………………………………………………………….16 Molecular Analyses…....……………………………………………………………………………………………...17 DISCUSSION………………………………………………………………………………………………………………...21 Distribution……………………………………………………………………………………………………………...21 Morphology………...……………………………………………………………………………………………………23 Molecular Data…………………………………………………………………………………………………………24 CONCLUSIONS………………………………………………………………………………………………………...…...28

LITERATURE CITED…………………………………………………………………………………………..………….51

APPENDIX…………………………………………………………………………………………………………………….59

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LIST OF TABLES

1 Summary of historical referenced sightings and collections of Tenellia spp.…………………..30

2 Sampling sites for T. adspersa collection……………………………………………………………………...31

3 List of attempted collection sites within the Gulf of Maine watershed ………………………….32

4 Polymerase chain reaction (PCR) primers………………………………………………………………...... 33

5 List of outgroup taxa and additional T. adspersa taxa for the TASC phylogenetic hypothesis……………………………………………………………………………………………………………………34

6 Average percent divergence among specimens in the TASC for COI sequences……..……….34

7 Results of partition analysis by Automatic Barcode Gap Discover (ABGD)…………………….35

8 List of specimens used for phylogenetic analyses of the family ………..……………..36

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LIST OF FIGURES

1 Photo of adult Tenellia adspersa specimen collected from the Squamscott River near the Exeter Town Dock in Exeter, NH……………………………………………………………………………....40

2 Photos depicting both morphotypes of T. adspersa………………………………………...…………41

3 Photos of select Fionid specimens collected for study…………………………...…………………..42

4 Geographical locations of collection sites within the Gulf of Maine watershed…………...43

5 Scanning electron micrographs of Tenellia spp. radulae………………………………………….....44

6 Scatter plot comparison of number of denticular teeth in radular vs. size of specimen.45

7 Automatic partition of the CO1 sequence data set from the ABGD analysis………………...46

8 Haplotype network based on CO1 data from thirty-seven specimens of T. adseprsa…...47

9 Phylogenetic hypothesis for Tenellia spp. based on CO1 gene sequence data, inferred by Maximum Likelihood……………………………………………………………………………………………….48

10 Maximum Likelihood (ML) phylogenetic analysis of the nudibranch family Fionidae....49

11 Phylogenetic hypothesis for Fionidae based on posterior probabilities (PP) from Bayesian inference…………………………………………………..………………………………………...... 50

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ABSTRACT

PHYLOGENENTIC AND PHYLOGEOGRAPHIC ANALYSES REVEAL A SPECIES COMPLEX IN THE ESTUARINE NUDIBRANCH TENELLIA ADSPERSA

By

Amanda Sobel

University of New Hampshire, December, 2017

Until recently, the nudibranch Tenellia (Nudibranchia: Fionidae) was thought to include a single or group of species restricted to temperate estuarine waters. Given the addition of numerous other species from recent studies, the genus now encompasses species from polar, temperate, and tropical from oceanic to estuarine salinities. One such fionid, Tenellia adspersa, is found in temperate estuarine waters globally and its presence is ecologically important as its congeners are capable of decimating colonies of their hydroid prey within a single generation (approx. 20-60 days). The literature is historically vague and conflicted on the morphology, taxonomy, and geographic distribution of the various estuarine species of Tenellia, that includes: T. adspersa

(Nordmann, 1844), T. fuscata (Gould, 1870), T. pallida (Alder & Hancock, 1842), T. mediterannea (Costa, 1866), T. ventilabrum (Dalyell, 1853), and fuscata

(Chambers, 1934). While Tenellia adspersa has been affirmed by several authors, others have proposed the taxon is comprised of at least two species.

This study represents an extensive sampling of T. adspersa, and the first to incorporate more than two genetic sequences within a phylogenetic study. Specimens of T. adspersa

viii were collected from global localities to produce a more current and detailed phylogenetic analysis and systematic review of the genus. Ninety-five specimens were collected from

Eastern Pacific, Western Atlantic, and Eastern Atlantic localities. From these, forty-five specimens were sequenced for the genes cytochrome c oxidase subunit I (CO1), 16S ribosomal RNA, or the histone protein H3. While morphological analyses were informative but not definitive in separating species, molecular analyses revealed a species complex; with multiple groups. Species delimitation by the Automatic Barcode Gap Discovery tool, coupled with a TCS haplotype network analysis and Maximum Likelihood were conducted.

They revealed several distinct within the T. adspersa species complex (TASC). Eleven new sequences of T. adspersa fit into the larger Fionidae phylogeny, in addition to two previous specimens from past studies. There is 100% support for the monophyletic status of a T. adspersa species complex. Equally supported are four distinct clades, two primary and two secondary, within the species complex based upon geographic localities. These molecular analyses elucidate both natural distributions and introductions for this cryptic nudibranch.

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INTRODUCTION

Under the biological species concept, a species is defined as a group of individuals that can interbreed in nature and produce fertile offspring (Mayr, 1942). This concept often leads to conflicts when determining biological species, especially when trying to apply terms created by taxonomists to sort and organize organisms based solely upon morphological structures. Mayer’s species concept does not aptly apply to bacteria, and the definition becomes even less appropriate when dealing with asexually reproducing organisms as well as cryptic species and hybrids.

Taxonomic groups are generally organized into cladograms or phylogenetic trees, based on shared, or derived features. These cladograms are used to help understand inter- relationships among groups, evolutionary traits, and biogeography. Historically, scientists classified taxonomic groups based on morphological, physiological, geographical, behavioral, and ecological traits. With the advent of molecular phylogenetics, focus now centers heavily on genetic elements to compare groups based upon the premise that closely related organisms have significant genetic similarities or molecular structures, while those that are distantly related will demonstrate a greater genetic dissimilarity.

There are specific DNA genomic sequences (i.e., 16S, 18S, CO1, H3) that are highly conserved across all taxa that also exhibit slow mutation rates and are therefore used to detect genetic divergences and to estimate probable evolutionary patterns. Mitochondrial

DNA (CO1) is one such highly conserved sequence. Therefore, DNA sequencing and comparisons have become the most reliable technique used to analyze evolutionary 1 relationships, since the effects of evolution are ultimately reflected in the transformed genetic sequences (Yang & Rannala, 2012). Comparing homologous gene sequences using computer sequence alignment programs can reveal similarities between species while DNA barcoding techniques can identify species based on small sections of mitochondrial or chloroplast DNA (Hebert et al, 2003).

Molecular approaches can be used to detect species-level differences, which are important when dealing with cryptic and potential sibling species; species that are difficult or impossible to distinguish based on morphological (phenotypic) characteristics

(Puillandre et al, 2012). Molecular phylogenies have also been useful in identifying cosmopolitan species; organisms with ranges that extend across the world in similar habitats. In marine systems, there are numerous historical examples of suspected cosmopolitan species that are thought to have been introduced via ballast water and ship fouling (Carlton, 1985; Knowlton, 1993). Many marine cosmopolitan species have been found to actually be complexes of cryptic, sibling, or undescribed species; such as the moon jelly Aurelia aurita (Dawson & Jacobs, 2001), the fire worm Eurythoe complanata (Barroso et al, 2010), the polychaete Capitella capitata (Grassle & Grassle, 1976), and the gastrotrich

Xenotrichula intermedia (Todaro et al, 1996).

Nudibranchs (: : Heterbranchia) more commonly referred to as sea slugs. They are carnivorous marine, nearly or completely shell-less, and soft-bodied with well-defined heads and posteriorly located gill structures unlike other groups of non- heterobranch gastropods whose respiratory organs are situated anteriorly.

Nudibranchs are found worldwide in every of marine ecosystem, although intertidal and sub-tidal habitats support the greatest species numbers (i.e., Clark, 1975;

2

Bleakney, 1996; Carmona et al, 2013). Taxonomically, these are broadly split into two major groups: the dorids and the aeolids, and among these are four described

‘suborders’: Dendronotacea (now Dendronotida), , Arminacea (now Arminida), and Aeolidacea (now ) (Thompson & Brown, 1984; Goodheart et al, 2015a).

Dendronotida, Arminida, and Aeolidida are groups within the large . Of these groups, the Dendronotida and Arminida have been shown to by polyphyletic (Pola &

Gosliner, 2010). Aeolid nudibranchs are distinguished by having long, tapering bodies that bear dorso-lateral clusters and/or rows of , while dorid nudibranchs are generally dome-shaped and possess a circle of gills around the dorsally situated anus. Cerata in aeolids function in respiration, absorption of dissolved organics, and defense. They also contain branches of the digestive gland and filled with nematocysts in their distal tips. Nematocysts are the specialized stinging organelles sequestered by the nudibranchs from their cnidarian prey and used for their own defense.

Nudibranchs inhabiting fouling communities or pelagic areas are subject to anthropogenic-mediated introductions via ballast water and ship hull fouling communities during inter-regional and international transport. This has led to many species being listed as having widespread, or cosmopolitan distributions (e.g., Edmunds, 1997; Malaquias et al,

2009). Prior to molecular phylogenetics, it was unclear if these species had a true cosmopolitan distribution, as this natural distribution status is rare. There are now several species-level phylogenetic studies that reveal anthropogenic cryptic speciation events (e.g.,

Trickey, 2012; Carmona et al, 2013; Churchill et al, 2013; Kienberger et al, 2016). Examples of such species complexes include papillosa (Kienberger et al, 2016), spp.

(Churchill et al, 2013; Churchill et al, 2014), pinnata (Trickey, 2012), Northern and

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Arctic nudibranchs of the genus (Ekimova et al, 2015), and ianthina (Wilson & Burghardt, 2015).

Individuals of Tenellia adspersa appear to exhibit a cosmopolitan distribution

(Roginskaya, 1970) in temperate estuaries. They are relatively small animals (4-7mm as adults) and are generalists preying on a variety of calyptoblastic and gymnopblastic hydroid species such as , Laomedea, Bougainvillea, Gonothyraea, Ectopleura,

Eudendrium, Hydractinia, and Cordylophora (Clark, 1975; Eyster, 1979; Chester 1996b;

Caine, 1998; Wintzer et al, 2011). These colonial hydroids are seasonally abundant in temperate estuaries worldwide and grow on piers, floating docks, eel grass blades and other stable structures.

Tenellia adspersa (Fig. 1) has ≥6 rows of cerata (as opposed to single ceras, as seen in

Tergipes ) along the upper sides of their bodies arranged in groupings of ≥ 3 cerata/row. They have thin, short tapering laterally or posteriorly directed oral tentacles around a dome-shaped anterior oral veil.

The dorsal are smooth, and cylindrical. The uniserate tapering radulae contains up to 45 tooth rows, with each tooth bearing a variable number of lateral denticles on either side of the median cusp (Chambers, 1934; Eyster, 1979; Thompson & Brown,

1984; Blezard, 1992; Chester, 1996a; Present study).

Tenellia adspersa is a sub-annual species with an average generation time of 20 days from to egg when raised under optimal conditions (20°C and 30ppt salinity) on

Cordylophora spp. (Chester, 1996b; reported as T. fuscata). Animals >1mm in size can decimate a colony of Cordylophora spp. within a single generation by feeding at the rate of

~6 polyps//day (Chester, 1996b). Under culture conditions, individuals produce 3-5

4 spawn per day with 25-50 per egg mass (Harris et al, 1980). T. adspersa exhibit developmental plasticity, or poecilogony, meaning eggs from the same spawn can produce that hatch as pelagic lecithotrophic larvae that metamorphose, or undergo direct

(complete) development within the egg capsule (Rassmussen, 1944; Eyster, 1979; Chester,

1996a, Chia et al, 1996). This bet-hedging strategy is dependent on egg size; eggs < 125μm exhibit lecithotrophic larvae, while eggs > 125μm will develop directly to miniature adults

(Chester, 1996a). Egg size is determined by the adult nutritional state with capsular metamorphic development occurring only under well-fed conditions (Chester, 1996a).

While adult nutritional state does not affect the overall growth and survival of juveniles, juveniles produced from underfed adults are smaller (Chester, 1996b).

Developmental plasticity allows T. adspersa to thrive in temporally and spatially unpredictable environments with many environmental stressors (Chester, 1996a). Juvenile or adult animals occur in salinities as low as 8ppt, and as high as 35ppt (Eyster, 1979;

Roginskaya, 1970; personal observation). The species is found most frequently at salinities

>15ppt, T. adspersa. At salinities <15ppt the species is unable to produce viable offspring

(Harris et al, 1980). Given its unique ability to thrive under euryhaline conditions and on a generalist diet, they are well adapted to survive in global temperate estuaries. The genus has been reported in estuaries in Japan (Baba & Hamatani, 1963), California (Carlton,

1979), New Hampshire (Harris et al, 1980), Maine (Cella et al, 2016), South Carolina

(Eyster, 1979), Great Britain (Schmekel & Portmann, 1982), France (Poddubetskaia, 2006),

Russia (Nordmann, 1844), Sweden (Evertsen et al, 2004), India (Dhanya et al, 2017), and

Brazil (Marcus, 1955) (Table 1). It is unclear whether this is true cosmopolitan distribution, or the occurrence of several species complexes resulting from distribution through

5 anthropogenic facilitation or from conflicting and confusing species descriptions of

Tenellia.

Based on morphology, the species identification of T. adpsersa has been vague and conflicting since it was first described from the Black Sea (Nordmann, 1844). There have been numerous synonyms of T. adspersa, including Eolis ventilabrum (Dalyell, 1853),

Embletonia pallida (Alder & Hancock, 1854), Tenellia mediterranea (type species for genus

Tenellia, Costa, 1866), Embletonia fuscata (Gould, 1870), Tergipes lacinulatus (Schultze,

1849), Embletonia grayi (Kent, 1869), Tenellia pallida (Thompson & Brown, 1984), and

Embletonia mediterranea (Vannucci & Hosoe, 1953). According to Chambers (1934),

Embletonia (=Tenellia) fuscata lacks a penial stylet although it has a hermaphroditic valve

(stylets have been easily missed or overlooked as a taxonomic character). According to

Brown (1980), Tenellia adspersa has an apical stylet while Bergh (1886) notes that the genus Embletonia (=Tenellia) lacks a penial stylet; E. pallida is the same species as E. fuscata. Bleakney (1996) writes that Tenellia fuscata occurs in the Western Atlantic, while a similar species, Tenellia pallida is found in European locales. Thompson & Brown (1984) described Tenellia adspersa as the only member of the genus to inhabit British waters, noting that T. mediterranea and T. pallida are synonyms. In 2014, Harris observed that there were two distinct morphotypes of T. adspersa (Fig. 2) within the Gulf of Maine watershed, and that they could potentially be two different species, as the colors were so strikingly different and that is not commonly observed in this species (L.G. Harris, 2014, personal correspondence).

The inconsistency in morphological traits and nomenclature pertaining to each supposed species supports the need to conduct molecular comparisons to delimitate

6 species of Tenellia. Molecular studies on Tenellia adspersa are few, although a recent molecular study has placed it into the family Fionida (Cella et al, 2016). Although the family

Fionidae is now resolved with 100% support, data is still lacking on some taxa, including

Tenellia adspersa which was represented by only two specimens within the Cella et al, 2016 revision.

This study primarily comprises a phylogeographic and secondarily, a morphological examination of a geographically diverse collection of T. adspersa specimens, obtained from

California, New England, and France. It addresses the following questions: Is there more than one species of Tenellia in the Gulf of Maine ecosystem, and globally? Are there distinguishable morphological differences among widely geographically collected T. adspersa specimens, or are they only molecular? Since a true, natural cosmopolitan distribution is rare at a species level in marine , is a T. adspersa species complex with a global distribution present? Additionally, while T. adspersa has been shown with strong support to be monophyletic within the nudibranch family Fionidae based upon only two specimens (Cella et al, 2016), more taxa are needed to determine whether that redirects the currently accepted phylogeny of the species.

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MATERIALS AND METHODS

Specimen Collection

Ninety nudibranchs of the genus Tenellia were collected from 09/2006 - 08/2015 from

nine different localities throughout New England (Fig. 4), California, and France (Table 2).

Three additional fionid specimens were collected (Table 5), and 55 sequences were also

used from GenBank, a free, world-wide genetic sequence database supported by the

National Center for Biotechnology Information (NCBI). GenBank sequences are well

documented and frequently appear in peer-reviewed publications. Identifications of

specimens collected were confirmed according to published descriptions (Picton &

Morrow, 1994; Bleakney, 1996; Pollock, 1998). When in question, specimen identifications

were additionally confirmed by Dr. Larry Harris (University of New Hampshire). Two

museum specimens (F1, F2) collected at Le Domain de Certes-Graveyron in Audenge,

France were generously provided by Dr. Juan Lucas Cervera (Universidad de Cádiz). All

specimens from California localities (M, CW, and SC) were graciously collected and

provided by Dr. Nadine Rorem (Wheaton College). nana and concinna

samples used as outgroups were provided by Dr. Larry Harris and Sara Edquist (University

of New Hampshire, Department of Biological Sciences). Cuthonella concinna was collected

in Eastport, ME, USA, and was collected from Schoodic Point, Winter Harbor,

ME, USA.

Numerous localities within the Gulf of Maine were sampled, but only five sites yielded

Tenellia specimens (Table 2 and 3). Collections occurred on floating docks in estuaries,

8 except in the case of the samples from the Squamscott River, Exeter, NH. Nudibranchs there were found floating on a blade of eelgrass covered by Cordylophora sp. Specimens collected from the Suisun City location were also found among Cordylophora sp., (Nadine Rorem, personal communication). There was no Cordylophora sp. at the Coastal Marine Lab

(Newcastle, NH), Robbinston Boat Launch (Robbinston, ME), Great Bay Marine (Newington,

NH), or Marina del Rey, (Marina del Rey, CA) but rather Obelia spp., Eudendrium spp., and

Hydractinia spp.

All specimens were fixed in 70 to 100% ethanol. When possible, specimens were first anesthetized with 8% MgCl2 in sea water to prevent curling (nudibranchs tend to contract their bodies in ETOH, curling into a ball). Non-contracted specimens allowed a more precise

DNA extraction and description of external morphology. All specimens were photographed and gross external morphology described (Appendix).

Analysis of Radulae

In addition to molecular analyses (Ekimova et al, 2015), nudibranch radulae can and have been used to distinguish between cryptic and sibling species. However, radulae are not always a defining character as demonstrated in the nudibranch family (Carmona et al, 2013). Radulae were collected from T. adspersa specimens when possible. Some specimens were too small (<2mm) or degraded, and thus were not used. To obtain radulae, the cephalic (head) region was decapitated from live specimens using a thin blade and immediately placed in 95% ETOH, then later transferred to a small, clear plastic petri dish and placed in 10% NaOH overnight (approx. 12 hours). Radulae were teased from the surrounding odontophore muscles, then gently rinsed in dH20 and immediately placed on carbon tape-covered SEM stainless steel stubs and allowed to air dry. After sputter coating

9 with Au/Pd (20nm thickness; Anatech Hummer V Sputter Coater), they were viewed with a

Lyra 3 GMU TESCAN scanning electron microscope.

Analysis of six morphological parameters of jaws and radulae (based on Bloom, 1976) were originally planned, but most specimens were too degraded or insufficiently prepared, so a mathematical analysis could not be completed. Some size measurements were able to be obtained and were digitized using the Lyra 3 GMU TESCAN software. A total of 7 radulae were analyzed, representing specimens from 7 different localities. The average number of denticular teeth per row were quantified for each specimen, and compared.

DNA Extraction, Amplification, and Sequencing

Total genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen,

Maryland, USA) according to the manufacturer’s instructions. Approximately 10mg of tissue was taken from the posterior end of the foot of each specimen, although for smaller specimens (<4mm) it was necessary to use the entire animal. Cerata were removed prior to

DNA extraction to diminish contamination from possible foodstuffs in the digestive tract.

Polymerase chain reactions (PCR) were carried out with 1μL of genomic extract using

12.5μL of OneTaq (New England Biolabs, NEB, Ipswich, MA) and 10.5μl of deionized water

(dH20). All PCR amplifications were run in 25μL reactions using an Eppendorf Nexus

MasterCycler.

Mitochondrial DNA is essential in resolving species-level phylogenies. The mitochondrial gene, cytochrome c odidase (CO1) has been frequently used in phylogenetic studies of gastropods (e.g., Malaquias & Reid, 2008, Pola & Gosliner, 2010; Carmona et al, 2013;

Hanson et al, 2013, Ekimova et al, 2015). Cytochrome c oxidase is an enzyme that assists in electron transport. The CO1 gene evolves slowly compared to other protein coding mtDNA

10 and can be used as a ‘barcode’ for invertebrate identification (Puillandre et al, 2012; Layton et al, 2014).

All T. adspersa were sequenced for the gene CO1. Some specimens also had 16S and H3

genes sequenced. The three partial gene fragments were amplified using the following

primers (Table 4): Approx. 658bp of CO1 amplified with universal primers HCO2198 and

LC01490 (Folmer et al, 1994), ~430bp of the mitochondrial gene16S amplified with

universal primers 16Sar and 16Sbr (Palumbi et al, 1991) and ~328bp of the nuclear gene

histone 3 (H3) amplified with primers H3AD and H3BD (Colgan et al, 1998). These three

gene regions are commonly used in phylogenetic studies of marine gastropods. All PCR

amplifications were carried out in 25μL reactions containing 1μL of genomic DNA template,

1μL of primer template (forward and reverse primer mix at 10mM), 10.5μL of dH20, and

12.5μL of OneTaq Hot Start 2X Master Mix with Standard Buffer (New England Biolabs,

Ipswich, MA). All reactions were run on an Eppendorf Nexus Mastercycler thermocycler.

CO1 fragments were amplified using the following parameters: initial denaturation at

94°C for 3 minutes, 40 cycles of 94°C for 30 seconds, 50-56°C for 30 seconds, and 72°C for 1

minute, followed by a final extension at 72°C for 5 minutes. 16S fragments were amplified

using the following parameters: initial denaturation at 94°C for 3 minutes, 39 cycles of 94°C

for 30 seconds, 50-55°C for 30 seconds and 72°C for 1 minute, followed by a final extension

at 72°C for 5 minutes. H3 fragments were amplified using the following parameters: initial

denaturation at 94°C for 3 minutes, 35 cycles of 94°C for 35 seconds, 50-55°C for 1 minute

and 72°C for 1 minute 15 seconds, followed by a final extension at 72°C for 2 minutes.

PCR products were viewed using gel electrophoresis on a 1% agarose gel stained with

ethidium bromide in 0.5% TAE Buffer. PCR amplicons were purified with 2μL of ExoSap-IT

11

(USB, Affymetirx, Fremont, CA) mixed with 5μL of PCR product. Samples were incubated at

37°C for 15 minutes, followed by an inactivation step at 80°C for 15 minutes. Samples were

then analyzed using a DS-11 Spectrophotometer (DeNovix).

Samples were diluted and prepared as a mixture of 8μL of DNA template and 4μL of

primer. Ultimately, samples were sent overnight to Eurofins MWG Operon LLC (Louisville,

KY) for commercial sequencing.

DNA Barcoding Analyses and Haplotype Network

The method of DNA barcoding uses a short (approx. 600 bp) sequence of the CO1 gene which provides a quick, useful species-level identification tool to suggest new species and to explore unknown groups. It should be noted that DNA barcoding analyses are not definitive proof of the existence of new species and the results must also be combined with other characters such as analysis of other phylogenetically indicative genes (i.e., 16S and H3), morphological, geographical or ecological data; researchers have described this amalgamation as an integrative framework (Hebert et al, 2003; Puillandre et al, 2012).

In this study, molecular putative species limits in T. adspersa were explored with the

Automatic Barcode Gap Discovery (ABGD) (Puillandre et al, 2012). This method uses a range of prior intraspecific divergence algorithms to infer from the supplied data a model- based (Jukes Cantor JC69, Kimura K80, or Simple Distance) one-sided confidence limit for intraspecific divergence. It works by detecting the barcode gap, or the value that exists whenever the divergence among specimens of the same species is smaller than the divergence among specimens from different species. In general, ABGD is a distance-based method that detects this barcode gap in the distribution of pairwise distances within a CO1 alignment.

12

A nucleotide sequence alignment was first uploaded and analyzed for pair-wise distances between T. adspersa specimens to MEGA7 (Sudhir et al, 2015). The resulting dataset was uploaded as a fasta file to http://www.abi.snv.jussieu.fr/public/abgd/html and ABGD was run with the default parameters: [Pmin = 0.001, Pmax=0.1, Steps = 10, X (relative gap width)

= 1.5, Nb bins = 20) and with all three distance measures: Jukes-Cantor (JC69), Kimura

(K80) TS/TV, and Simple Distance. Outgroups were not used in these distance based analyses.

In addition to ABGD, genetic distances were calculated using the Kimura two-parameter model (K2P, Kimura, 1980). The K2P model works particularly well in datasets in which distances are assumed to be low (Hebert et al, 2003). Pair-wise distances within T. adspersa were calculated in MEGA7 (Sudhir et al, 2015).

An unrooted statistical parsimony network (Haplotype network) based on unique CO1 haplotypes in T. adspersa was generated using a TCS network analysis (Clement et al, 2002) in popART: Population Analysis with Reticulate Trees (Leigh & Bryant, 2015). The haplotype network was also confirmed using MEGA7 (Sudhir et al, 2015). Fourteen sequences were excluded from the haplotype network analysis due to missing or uninformative data. Its inclusion would have skewed statistical parsimony analysis because all variation outside of the incomplete sequence data available for these samples would have been ignored. The sequences excluded were: M3, W1, S1, S4, S7, G4, G6, C1, CW1, CW3, CW7,

SC1, SC6, and SC7. In total, 37 sequences were analyzed with popART (it should be noted that not all of the 90 specimens collected were utilized for genetic sequencing as some were too degraded or they weren’t sequencing correctly). This particular haplotype network analysis focused on the inheritance of a cluster of single nucleotide polymorphisms (SNPs),

13 which are variations at single positions in the DNA sequence among individuals. While haplotypes suggest genetic diversity, they do not necessarily provide species level differentiation.

Phylogenetic Analyses

DNA sequences were viewed and edited using 4Peaks v.1.7.1 software (Griekspoor &

Groothius, 2005). All the sequences were checked for contamination with the Basic Local

Alignment Search Tool (BLAST) software program (Altschul et al, 1990) implemented in the

Genbank database. Sequence alignment was performed initially using Seaview v4.6.1 (Guoy et al, 2010), and again with MAFFT (Katoh & Standley, 2013) prior to analyses. The alignments were checked by eye using Seaview v4.6.1. Three taxa ( exiguus,

Tergipes tergipes, and Tenellia gymnota) were chosen for the outgroup in the Tenellia adspersa phylogenetic hypothesis. The outgroup used for the Fionidae phylogenetic hypotheses included specimens of the genera , , Dendronotus, ,

Phylodesmium, and . Outgroup gene sequences and data were obtained from

GenBank (Table 5) and sequenced specifically for this study. They were selected based on comparable studies (Pola & Gosliner, 2010; Cámara et al, 2014; Goodheart et al, 2015b) and their close relatedness to Tenellia adspersa (Cella et al, 2016).

The GTR+I+G model was selected for all genes, and used to analyze each gene sequence

individually, then a concatenated sequence for the family phylogenetic analysis

(CO1+16S+H3). The program seqCat.pl was used to concatenate sequences.

Maximum likelihood (ML) analyses were performed using the software RAxML v8

(Stamatakis, 2014). Node support was assessed with non-parametric bootstrapping (BS)

with 5,000 replicates, 20 random starting trees, and parameters estimated from each data

14 set under the model selected for the original data set. Bayesian inference analyses (BI) were conducted using PhyloBayes v4.1 (Lartillot et al, 2009) under the CAT model of evolution (Lartillot & Phillipe, 2004). All trees were viewed and edited using FigTree v.1.4.0

(Rambaut, 2014).

15

RESULTS

Morphological Results

Overall there were six specimens of Tenellia adspersa collected from the Coastal Marine

Lab (Newcastle, NH), one specimen from Robbinston Boat Launch (Robbinston, ME), nine specimens from Cutting’s Wharf (Napa, CA), two specimens from Domaines des Certes

Graveyrons (Audenge, France), thirteen specimens from Great Bay Marine (Newington,

NH), five specimens from Palawan Way (Marina del Rey, CA), thirteen specimens from the

Squamscott river (Exeter, NH), ten specimens from Suisun City Marina (Suisun City, CA), and two specimens from Wentworth Marina (Newcastle, NH) (Table 2). Three fionid specimens were also collected for this study: Cuthona nana, Cuthonella concinna, and

Eubranchus exiguus (Table 5) and used as outgroups.

Two distinct morphotypes were found among all the Tenellia adspersa species complex

(TASC) samples collected: a pale cream/white/ivory body with dark speckles and spots, and a dark brown/black body with dense speckling (Fig. 1 and 2). The darker morphotype was only found in the Piscataqua River (Great Bay Marina, Newington NH; G11, G12, G13) in June and in the Squamscott River (Exeter Town Docks, Exeter NH; S13) in October

(Appendix). Larry Harris (UNH) photographed specimens of the dark morphotype in Great

Bay, and Gould (1870) described Embletonia (=Tenellia) fuscata as being a “smutty slate color” from the Charles River in Boston, MA. All specimens from this study are described in

Appendix.

16

There were no quantifiable differences in gross morphology (Fig. 5). Only seven samples were usable, as many specimens were too small (<2mm) or there were issues in transferring radulae to SEM stubs. Among the seven samples, size analyses could not be performed due to poor specimen preparation and degradation. Overall, each specimen shows a uniserate radula with one rachidian tooth per row. A linear regression was done to test the hypothesis that the number of denticles on the radula increases with the age and size of the animal (R2 = 0.2692, P= 0.233, Fig. 6). The effect of size of the animal on the number of denticles was not statistically significant. Radulae from this study were similar to the radula described by Thompson & Brown (1984). There were not enough samples to test whether the specimens showed differences in radula morphology.

Molecular Analyses

The Automatic Barcoding Gap Discovery (ABGD) analysis indicates the existence of cryptic diversity within T. adspersa, likely due to a species complex. Analyses resulted in separate groups with P-values ranging from 0.001 to 0.0215 (Fig. 7, Table 7). The initial partition determined that the number of groups ranged from 2 (when P=0.001) to 1 (when

P=0.0215). The first group included specimens from California and Northern Atlantic localities: CW6, SC2, SC3, SC4, SC5, SC8, and SWE. The second group included all other specimens, including the rest of the CW specimens (CW2, 4, 5, 8, and 9). The recursive partition determined that the number of groups ranged from 25 (when P=0.001) to 3

(when P=0.0017-0.0215). The 3 groups are similar to the initial partition but this time the analysis separates SWE in its own group. The grouping results were independent of the distance method used (JC69 vs. K80 vs. Simple Distances).

17

The CO1 haplotype network generated in popART using the TCS algorithm (Clement et al, 2002) also supports three groups within the Tenellia adspersa species complex (TASC), similar to the ABGD analysis. The separate groups included CW6, SC2, SC3, SC4, SC5, and

SC8 (CW6 and SC24 haplotypes), the SWE sample, and the rest of the haplotypes. One of the haplotypes (S13) sampled from the Squamscott River was prevalent as it was found in five out of thirty-seven specimens sampled (Fig. 8). The analysis found 34 parsimony informative sites (the number of sites containing at least two states that occur in at least two sequences each), 59 segregating sites (the number of sites that differ among sequences), and nucleotide diversity π=147.725 (average number of nucleotide differences per site between DNA sequences). Nineteen distinct haplotypes were determined across thirty-seven specimens. There were two node splits with missing haplotypes in the haplotype network, occurring between the M2 haplotype and the S13, and one that split the

SWE and CW6/SC2 haplotypes. Missing haplotypes (nodes) can indicate missing information such as unsampled haplotypes, or extinct sequences.

The data set from the CO1 gene yielded a sequence alignment of 615 positions (base pairs) for T. adspersa, and a combined dataset of 1460 positions (CO1+16S+H3) for the family Fionidae. Some taxa did not have all three gene sequences available (Table 8). For the species phylogeny, only CO1 provided resolution as opposed the combined tree

(CO1+16S+H3) and as opposed to just the 16S and H3 trees alone (not shown). Although many studies on nudibranch phylogenies have shown better resolution using a combined dataset than a single gene dataset (e.g., Malaquias & Reid, 2008; Carmona et al, 2013;

Camacho-Garcia et al, 2014; Oskars et al, 2015), only five 16S and H3 sequences were successfully sequenced for T. adspersa, and these sequences proved to be completely

18 uninformative in determining the species phylogeny (similar to Kienberger et al, 2016).

The combined tree (CO1+16S+H3) in the family phylogeny provided greater resolution than the individual trees (individual trees not shown) alone, similar to Cella et al, 2016.

A phylogenetic hypothesis tree of T. adspersa (Fig. 9) for the CO1 gene was generated based on Maximum Likelihood (ML) using RAxML. Values at nodes indicate Boot Strap (BS) values, and there is strong support for the TASC being monophyletic (BS=100), as well as four distinct clades. The first two clades (BS=100), which can be considered primary clades, are split into a group of specimens. Specimens from Suisun Slough (SC2, SC3, SC4, SC5, and

SC8), one specimen from Cutting’s Wharf (CW6), and the specimen from Sweden (SWE) are herein named Clade A (BS=93), while all of the remaining specimens, are herein named

Clade B. There is also strong support for two additional (secondary) clades. The third includes a secondary clade from a small group of specimens from the Gulf of Maine watershed (T. adspersa_NH, G9, and R1), herein called clade C (BS=89), and the fourth, also a secondary clade herein called Clade D, includes the rest of the California specimens (CW2,

CW4, CW5, CW8, CW9), the remainder of the Gulf of Maine samples and the France specimens (C2, C3, C5, F1, F2, G1, G7, G8, G10, G11, G12, G13, S2, S3, S5, S6, S8, S9, S10, S11,

S12, S13, and W2) (BS=93).

Overall, the ML analysis reveals multiple polytomies indicating a species complex. The phylogenetic hypothesis results are congruent with results of the other molecular analyses conducted in this study (Fig. 7, 8, Table 6, and Table 7).

The results also support Fionidae as a monophyletic group (Fig. 10 and 11: BS=100,

PP=.99) and are congruent with Cella et al, 2016. Within the family, there is strong support for the monophyletic status of the TASC (BS=100, PP=.99) with the inclusion of two

19 publicly available sequences and the ones collected specifically for this study. Within the family, there is 99% support for Clade A (Fig. 10), which is in agreeance with the species phylogeny (Fig. 9). Further, there is strong support (BS=91) for Clade B.

K2P pairwise distance measurements within the TASC showed an overall average of

1.96%. Intraspecific percent divergence among geographic groups are as follows: Eastern

Atlantic (F1, F2, and SWE) is 3.16%, Western Atlantic (Gulf of Maine localities) is 0.83%,

Marina del Rey (MDR) is 0.67%, and San Francisco Bay Estuary (SC and CW localities,

SFBE) is 2.86%. Interspecific percent divergence among geographic groups is as follows:

Eastern Atlantic versus Western Atlantic = 1.97%, Eastern Atlantic versus MDR = 2.14%,

Eastern Atlantic versus SFBE = 3.06%, Western Atlantic versus MDR = 1.19%, Western

Atlantic versus SFBE = 2.99%, and MDR versus SFBE = 2.90%.

20

DISCUSSION

The goal of this study was to use morphological and molecular data to establish a phylogeny of the Tenellia adspersa species complex and to detect population level differences.

Distribution As a result of this study, the occurrence of Tenellia adspersa was documented for the first time from Marina del Rey, CA (Palawan Way, Basin D), and Robbinston ME. Marina del

Rey is a man-made harbor exposed to open ocean and full strength salinities (31-35 PSU, on average; www.SCCOOS.org) with depths of 4.6-6.4m. Samples collected on 06/21/2015 were collected under 32.6 PSU and 22.5°C (Nadine Rorem, personal communication). It’s been historically polluted since 1998, due to very little water circulation and high Bacterial

Total Maximum Load (TMDL). Additionally, copper pollution from anti-fouling hull paint is prevalent. Nearly 5,000 boats dock in Marina del Rey annually and it sees high boat traffic

(LAwaterkeeper.org), increasing the potential for ship fouling.

Robbinston, ME exhibits estuarine and open ocean conditions, ranging from 24-34 PSU, and depths of 0-8.8m in the center of the St. Croix River (Fife et al, 2015). The specimen R1 was the only T. adspersa confirmed from the samples, but it took several days to bring it back to the lab to confirm, so if there were any other specimens they could have perished in travel. There were several specimens of collected here as well, and hydroids of the genus Obelia.

21

Thirty-eight specimens from the Cutting’s Wharf (CW) locality in the Napa River, CA were collected, though not all of them were sequenced. Cutting’s Wharf is an estuarine environment experiencing salinities as low as 3 PSU (www.napa-sonoma-marsh.org) and was at 21.6 PSU at the time of collection, 06/28/2015. The depth of the Napa River in this area is 3.9-6.4m and does experience small boat traffic. Napa River experiences a large tidal differential (8.44 HAT, -1.14 LAT), while water temperature ranges from 9-26°C and was

23.3°C on the date of collection.

Ten specimens from the Suisun City Marina, CA locality were collected, at 6.7 PSU and

24.5°C. This site experiences estuarine conditions and is 1.5-2.7m in depth. The Marina has over 160 boats docked on average as well as a public dock and boat launches, lending it a high traffic area of the Slough (Suisun City Marina). All specimens were found feeding on

Cordylophora spp. Out of the ten specimens collected, only SC2, 3, 4, 5, and 8 were sequenced successfully, which were the largest of the specimens (3-4mm). The SC specimens group separately from the rest of the samples in every molecular analysis, along with one specimen from CW (CW6) with a 17 point mutation in between the nearest relative (Fig. 8). Assuming that all specimens were labeled correctly at the time of collection, during sorting in the lab, and during the preparation and subsequent sequencing of genes, then CW6 had the same haplotype and genetic structure of the CO1 gene as the SC specimens. However, it is significantly different than the rest of the CW samples, thus indicating the possibility gene flow.

Sampling sites from this study confirmed previously documented seasonality of T. adspersa in the Gulf of Maine watershed (Table 1 and 2) and in the San Francisco Bay

Estuary. For example, Chester (1996a) recorded presence of T. adspersa in the Great Bay

22

Estuary, NH in 1993 during the months of June, July, and August only. Bleakney (1996) recorded the presence of T. fuscata (=adspersa) in summer months only as well in Nova

Scotia (Minas Basin). Wintzer et al, (2011, personal communication) recorded T. adspersa in several areas within the Greater San Francisco Bay Estuary including the Boynton Slough in October, the Petaluma River in July, and the Montezuma Slough July through September,

2008. Wasson et al, (2001) recorded T. adspersa in the Elkhorn Slough, CA, in July, 2008.

Morphology

The two distinct morphotypes documented only in the Western Atlantic (G11, G12, G13,

S13) can most likely be explained as intraspecific color variability, especially given that the two morphotypes have little or no genetic differences. Samples G11 and S13 share the same haplotype, but G12 and G13 were not included in the haplotype analysis due to poor sequencing but all 4 grouped out to the same clade in the phylogenetic analysis, Fig 8.

Nudibranch coloration can be phylogenetically informative, such as in spp.

(Carmona et al, 2014), spp. (Hoover et al, 2015), spp. (Padula et al,

2016), spp. (Lindsay & Valdes, 2016), and spp. (Uribe et al, 2017) but that does not appear to be the case here. The darker morphotype in T. adspersa could be due to an increased ingestion of a darker colored hydroid, such as Cordylophora spp. Most specimens (Squamscott, Great Bay Marine, Wentworth Marina, Suisun City) were found among Cordylophora, so it’s inconclusive if this is the case. Carmona et al, (2014) found that only color seemed to be a speciation marker for Berghia spp., and Kienberger et al, (2016) found that shape and some color patterns in Aeolidia spp. were indicators of speciation (i.e., warty rather than smooth rhinophores of A. loui).

23

An in-depth analysis of morphological characters was not conducted (i.e., anatomical descriptions, spawn), only general appearance of specimens was recorded (Appendix).

While the presence/absence of the penial stylet has been determined to be an important factor in species determination in historical texts, that analysis was not conducted for this study. I felt it would be too difficult to conduct detailed anatomical analyses for this study due to the large number of preserved specimens (preserved in ethanol, which induces tissue artifacts over time; approximately 45 specimens were preserved in ethanol before conducting genetic analyses) and because of the lack of accordance in species among historical references (i.e., between species descriptions of T. adspersa vs. T. pallida vs. T. fuscata in Bergh, 1886, Chambers, 1934, Brown, 1980, Thompson & Brown 1984, and

Bleakney, 1996). Additionally, a separate clade within the TASC was found in Suisun City,

Suisun Slough and at Cutting’s Wharf, CA, based on molecular analyses. Although it would be best to collect live specimens from these locales, it was impossible to do for this study.

As a result of these factors, molecular data was postulated to be determinant within the

TASC.

Molecular Data

The results indicate that T. adspersa is an amphiatlantic species, since the same species is found throughout the sampled Northern Atlantic localities. However, additional collections and morphological and molecular analyses are needed to confirm that T. adspersa is the only species of the TASC in the Northern Atlantic. A sample from Sweden (from NCBI) was included in the phylogenetic hypothesis which grouped more closely with Clade A than other Eastern Atlantic samples found in Clade B. Additional suggested localities to be sampled for analyses to help resolve this issue include the North Inlet (South Carolina.),

24

Newport River and North River (North Carolina), Indian River Lagoon (Florida, based on

Mikkelsen et al, 1995), Chesapeake Bay (Maryland), Sea of Azov (Russia/Ukraine), and the

Fleet at Dorset (Great Britain). T. adspersa also occurs in the Pacific (Baba & Hamatani,

1963; Carlton, 1979; Wasson et al, 2001; WWFHK, 2006; and Wintzer et al, 2011) and was confirmed by this study (Table 1). Additional sampling is recommended within estuaries along the California coast, to include numerous sites within the San Francisco Bay estuary.

Japan and Hong Kong samples are needed to determine if there are more than two clades within the TASC or potentially more inclusive species. Comparing TASC gene sequences from the Western and Eastern Pacific may elucidate the population’s expansion and sources of gene flow. T. adspersa was recently identified in Cochin Harbor, India (Dhanya et al, 2017). Molecular analyses of specimens from additional localities of the TASC are needed to determine whether the complex exhibits a true cosmopolitan distribution; i.e., a species with widespread naturally occurring distributions versus anthropogenic introductions. According to this study, T. adspersa has been introduced into California.

Phylogenetic analysis of mitochondrial COI sequence data, along with ABGD and haplotype network analyses revealed the presence of at least four distinct clades within the

TASC. Given that the ancestral haplotype is found in the Atlantic, this data suggests there were multiple introductions of the Atlantic clade of T. adspersa into the San Francisco Bay

Estuary. It remains unclear whether or not T. adspersa originated from the Western or

Eastern Atlantic because currently, there aren’t enough sequences available from Eastern localities. S13 is the ancestral haplotype based solely on this limited analysis, but because there are unsampled/missing nodes, additional specimens must be sampled. As for Clade A,

25 it is unclear whether it originated in the Eastern Pacific, if it diverged from a population of

Clade B taxa, or if it was introduced from a population from the Western Pacific.

Since there is only one haplotype network, this indicates the presence of only one species. This hypothesis is also supported by the Maximum Likelihood tree. There is not enough evidence to suggest that the genus is comprised of more than one species globally.

There is some evidence, based on the ML analysis, that the clade of CW2, 4, 5, 8, and 9 could represent a separate clade. However, this is only weakly supported by the haplotype analysis and not supported by the ABGD analysis.

As currently constructed, the phylogenetic tree includes many polytomies that indicates incomplete information. Thus, the phylogenetic tree can only be used to confirm a strong likelihood that there are four distinct clades within the TASC. The ABGD and haplotype network analyses show two distinct groups, but not necessarily separate species, within

TASC.

The biggest challenge with this is study is the quality of the sequences. Many specimens were quite small, and were combined with those bearing foot mucus with attached debris and prey tissue, thus sequencing resulted in many uninformative sequences and missing data. CO1 should have been the easiest gene to sequence, but many sequences were unusable due to missing data (e.g., W1, S1, S7, and SC1). Evidenced from sequence alignments prior to analyses indicated there were missing nucleotides even among the usable sequences. While large, broad phylogenetic analyses (maximum likelihood, Bayesian inference) can sort through these missing or incorrect sites when sufficient taxa are included, other analyses are not as sound (haplotype network, genetic difference %). The polytomies depicted in the phylogenetic tree indicate that more taxa are needed to

26 complete the phylogeny of the TASC. Despite this situation, the ABGD analysis did reveal the existence of one or two distinct groups, Clade A and B, while ML analysis confirmed four clades were present which was also indicated by the haplotype analysis; i.e., four groups. The family Fionidae ML analysis showed two distinct clades (Clade A and B) as well as the monophyletic status of the TASC, and the Phylobayes tree only indicated with very strong support that the family Fionidae and TASC is monophyletic.

This is the first study to include more than two specimens of Tenellia adspersa in a molecular phylogeny. Cella et al, 2016 places T. adspersa within the family Fionidae with strong support (BS=100, PP=1) from two specimens of T. adspersa, one from Sweden, and the other from NH (also sampled in this study). The molecular analyses done in this study confirm the monophyletic status of the TASC within the family Fionidae with strong support (BS=100, PP=.99) and did not disagree with the proposed phylogeny.

27

Conclusions

From this molecular analysis study, it can be concluded that there are at least two distinct color morphotypes of Tenellia adspersa in Gulf of Maine waterways, but not separate species. This further implies that external morphological parameters alone may not be sufficient in detecting species-level differences within the TASC, but analyses should be broadened to include in-depth studies of reproductive organs and radulae. There is strong molecular sequence evidence that supports four taxonomic clades within the TASC, which is reinforced by direct genetic sequence evidence. Separate clades within a phylogeny are not necessarily indicative of separate species but as demonstrated in this case seem to represent divergence within one species, lending to a species complex: a common occurrence among nudibranchs (i.e., Ekimova et al, 2015; Hoover et al, 2015;

Wilson & Burghardt, 2015).

For the TASC, gene flow is most likely unidirectional, originating from an ancestral location in the Atlantic, followed by geographic dispersal and transport from shipping and ballast water vectors. Members of the TASC are extremely resilient to salinity and temperature changes. They are also eurytrophic generalists and exhibit poecilogony. These combined characteristics enables them to be successful invasive species whose range spreads readily. Collection data indicates that T. adspersa is not only an amphi-Atlantic species, but is also a potential cosmopolitan species. This is confirmed by its presence

Marina Del Rey (CA), Napa River Valley (CA), India, Japan, and throughout the Gulf of Maine and the North Eastern Atlantic.

28

Results of this study suggest additional research is needed to complete the analysis of the TASC. More extensive species collections are especially needed from Japan, Hong Kong,

Brazil, India, Southwest Atlantic regions, the Northeast Atlantic, and Eastern Pacific localities to see if they support additional clades being added to the TASC. Additional studies should also include interclade breeding experiments between populations and supported by further molecular analyses.

29

Table 1. Historical referenced sightings and collections of Tenellia spp.

Species Reported Locality Reference T. adspersa Black Sea, Russia Nordmann, 1844 adspersa Upper Thames River, UK Kent, 1869 (as Embletonia grayi) adspersa Le Canal de Caen, France Naville, 1926 adspersa Elkhorn Slough, CA Carlton, 1979; Wasson et al., 2001 adspersa Fleet in Dorset; Bristol Channel GB Schmekel & Portmann,1982; Thompson & Brown, 1984. adspersa Great Bay, NH (various) Chester, 1996a Jackson Estuarine Lab, NH Blezard, 1992 adspersa Uto Archipelago, Finland Evertsen et al, 2004 adspersa Mai Po Reserve, Hong Kong WWF Hong Kong, 2006 adspersa San Francisco Estuary, CA Wintzer et al, 2011 adspersa Cochin Harbor, India Dhanya et al, 2017 adspersa Australia (unspecified) Hewitt & Martin, 1996

30 T. fuscata Boston, MA Gould, 1870

fuscata Woods Hole, MA Bergh, 1886 fuscata Barnegat Bay, NJ Chambers, 1934 fuscata Mystic River, CT Clark, 1975 fuscata Thames River, CT Clark, 1975 fuscata Great Bay, NH Harris et al, 1980 T. pallida Birkenhead, England Alder & Hancock, 1854 pallida Copenhagen, Denmark Rasmussen, 1944 pallida Isenfjord, Denmark Rasmussen, 1944 pallida Brazil (unspecified) Marcus, 1955 pallida Mukashima, Japan Baba & Hamatani, 1963 pallida Azov Sea, Russia Roginskaya, 1970 pallida Georgetown County, SC Eyster, 1979 pallida Newport River, NC Caine, 1998

Table 2. Sampling sites for T. adspersa collection. Site abbreviations used throughout study appear in parentheses.

Number of Locality & Group Identification Coordinates Specimens Collected Date of Collection 43°04'18.7"N Coastal Marine Lab, Newcastle, NH (C) 70°42'42.7"W 6 6/30/14 7/22/14 38°13'33.7"N 6 Cuttings Wharf Rd, Napa, CA (CW) 122°18'27.5"W 38 6/28/15 43°06'58.2"N Great Bay Marine, Newington, NH (G) 70°50'09.2"W 13 6/26/14 Domaines des certes Graveyrons, 44°41'00.5"N Audenge, France (F) 1°02'27.6"W 2 9/1/06 14000 Palawan Way, Marina Del Rey, CA 33°58'51.8"N (M) 118°27'19.1"W 5 6/21/15 Boat Launch, 607 S River Rd, Robbinston, 45°04'59.8"N ME (R) 67°06'31.7"W 1 8/7/14 Exeter town dock, Squamscott River, 42°58'58.5"N

Exeter, NH (S) 70°56'53.8"W 13 10/16/14 31

38°14'14.4"N 800 Kellogg St, Suisun City, CA (SC) 122°02'18.6"W 10 6/28/15 Wentworth by the Sea Marina, E Dock, 43°03'31.0"N Newcastle, NH (W) 70°43'39.4"W 2 7/14/15

Table 3. List of attempted collection localities for Tenellia adspersa within the Gulf of Maine watershed area.

Locality Coordinates Date Tenellia collected Other Notes Calais Town Dock, ME 45°11’25.4"N 67°16'36.7"W 8/7/14 No Very low salinity

Eastport Commercial 44°54’22.9"N 66°59’02.8"W 8/6/14 No High salinity Pier, ME Eddy Yacht Sales, ME 43°59’26.5"N 69°39’00.6"W 8/22/14 No No hydroids Great Island Boat Yard, 43°49’47.7"N 69°54’56.3"W 8/20/14 No Few hydroids ME Hampton Marina, NH 42°54’01.5"N 70°49’08.2"W 10/5/14 No Few hydroids

Jackson Estuarine Lab, 43°05’31.7"N 70°51’50.9"W 10/2014 No No hydroids NH Kennebec Tavern, ME 43°54’56.0"N 69°48’45.4"W 8/22/14 No Very low salinity

Liberty Marina, MA 42°32’49.4"N 70°55’03.8"W 7/20/14 No High boat traffic Marston’s Marina, ME 43°28’50.4"N 8/23/14 No Few hydroids

70°24’45.3"W4'46.0"W 32

Neil’s Point, ME 43°47’58.6"N 69°58’17.5"W 8/20/14 No Few hydroids Portsmouth Commercial 43°04’33.5"N 70°44’58.3"W 8/27/14 No only Ectopleura sp. Pier, NH Robinhood Marine 43°51’13.2"N 69°44’00.4"W 8/20/14 No Hydroids plentiful, egg Center, ME masses Schooner Landing, ME 44°01’56.1"N 69°32’01.1W 8/22/14 No No hydroids

Yarmouth Boat Yard, ME 43°47’48.6"N 70°10’34.4"W 8/22/14 No Few hydroids

York Harbor Marina, ME 43°07’49.9"N 70°38’49.2"W 8/19/14, No Hydroids plentiful, egg 8/23/14 masses present

Table 4. Polymerase chain reaction (PCR) primers used.

Name 5' --> 3' References

LCO1490 5'-GGTCAAATCATAAAGATATTGG-3' Folmer et al, 1994 HCO2198 5'-TAAACTTCAGGGTGACCAAAAATCA-3' Folmer et al, 1994 16S ar-L 5'-CGCCTGTTTATCAAAAACAT-3' Palumbi et al, 1991 16S br-H 5'-CCGGTCTGAACTCAGATCACGT-3' Palumbi et al, 1991 H3AD5'3' 5'-ATGGCTCGTACCAAGCAGACVGC-3' Colgan et al, 1998 H3BD5'3' 5'-ATATCCTTR GGCATRATRGTGAC-3' Colgan et al, 1998

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Table 5. List of outgroup taxa and additional T. adspersa taxa for the TASC phylogenetic hypothesis.

GenBank Accession Number Species Locality Voucher no. CO1

Eubranchus exiguous* Newcastle, NH, USA - Not yet available Tenellia adspersa NH, USA CASIZ 184191 KY129085 Tenellia adspersa Sweden GnM 9011 KY129084 Tenellia gymnota NH, USA CASIZ 184188 KY128908 NH, USA CASIZ 184192 KJ434079.1

*Collected for this study

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Table 6. Between and within group mean distance (percent divergence) among specimens in the Tenellia adspersa species complex for CO1 sequences. COI sequences calculated with the Kimura (1980) 2-parameter model of sequence evolution. Eastern Atlantic includes F1, F2, and Sweden. Western Atlantic includes all taxa found within the Gulf of Maine watershed. Marina del Rey includes all tax found at this location. San Francisco Bay includes taxa from the Suisun City and Cutting’s Wharf localities. Average percent divergences were calculated from pre-aligned sequence data with MEGAv.7.0.20.

Eastern Atlantic Western Atlantic Marina del Rey San Francisco Bay

Eastern Atlantic 3.16%

Western Atlantic 1.97% 0.83%

Marina del Rey 2.14% 1.19% 0.67%

San Francisco Bay 3.06% 2.99% 2.90% 2.86%

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Table 7. Results of partition analysis by Automatic Barcode Gap Discover (ABGD). Number of species (Ns) in data set for this analysis is 43.

Number of Groups (Ng)

Prior Intraspecific Initial Recursive Divergence (P)

0.001 2 25 0.0017 2 3 0.0028 2 3 0.0046 2 3 0.0077 2 3 0.0129 2 3 0.0215 1

36

Table 8. List of specimens used for Fionidae family molecular phylogeny analyses. Museum codes: CASIZ=California Academy of Sciences, CPIC= California State Polytechnic University Invertebrate Collection, GnM=Germanisches Nationalmuseum, MNCN=Museo Nacional de Ciencias Naturales, ZMMU= Zoological Museum of Moscow, ZSM=Zoologische Staatssammlung Munchen. *Specimens collected by author for this study, not yet submitted to Genbank.

Species Locality Voucher Genbank Accession Numbers CO1 16S H3 Abronica abronia California, USA CASIZ 174485 KY128917 KY128712 KY128504 Abronica purpureoanulata Philippines CASIZ 181296 KY128971 KY128763 KY128558 Bornella stellifer Hawaii, USA CASIZ 167989 HM162703 HM162623 HM162529 glaucoides Portugal MNCN 406 HG810893 HG810887

37 Portugal MNCN 407 HG810894 HG810888

Calma gobioophaga Croatia MNCN 409 HG810896 HG810890 Calmella cavolini Italy MNCN15.05/53688 HQ616772 HQ616737 pilata Mass, USA CASIZ 184187 - KY128709 KY128502 Cuthona nana California, USA JQ699569 JQ699479 JQ699391 Cuthona nana* Maine, USA Cuthona nana New Hampshire, USA CASIZ 182700 KY128961 KY128754 KY128548 Cuthonella cocoachroma California, USA CASIZ 185193 KY128931 KY128726 KY128519 Cuthonella concinna* Maine, USA Cuthonella concinna California, USA CASIZ 179469 KY127824 KY128719 KY128512 Cuthonella soboli Sea of Japan WS3453 KY129024 KY128815 KY128609 Cuthonella sp. A NW Pacific WS3450 KY129019 KY128810 KY128594 Barents Sea ZMMU Op298 KM396985 KM397067 KM397095 Dendronotus robustus Barents Sea ZMMU OP343 KM397002 KM397084 KM397106 Eubranchus exiguus Germany AF249792 AF249246 Eubranchus exiguus New Hampshire, USA Eubranchus rupium Maine, USA CASIZ 183925 KY129034 KY128825 KY128620 Eubranchus farrani Sweden GnM 9093 KY129028 KY128819 KY128614

Fiona pinnata Morocco MNCN/ADN51997 JX087558 JX087492 JX087628 Vanautu CASIZ 179238 KY129047 KY129938 KY128486 Flabellina ischitana Morocco MNCN 15.05/53700 HQ616756 HQ616719 HQ616785 Flabellina pedata Spain MNCN 15.05/53702 HQ616758 HQ616721 HQ616787 Flabellina trilineata Antarctica GQ292024 Murmania antiqua Kara Sea WS3455 KY129066 KY128857 KY128651 longicirrum Philippines JQ699634 JQ699559 JQ699471 Phyllodesmium parangatum Philippines JQ699635 JQ699560 JQ699472 Rubramoena amoena Great Britain GnM 9098 KY128491 KY128904 KY12869696 Rubramoena rubescens Great Britain GnM 9102 KY1284503 KY128916 KY128710 Tenellia melanobrachia Hawaii, USA PmPO1-Tc DQ417274 DQ417228 Tenellia minor Ngel Channel, Palau Pmi2P4-PI DQ417313 DQ417263 Tenellia minor Pago Bay, Guam PmiG1-Pa DQ417304 DQ417256 Tenellia sibogae Western Channel, Palau PsP4-Pcy DQ417297 DQ417251 38 Tenellia adpsersa New Hampshire, USA CASIZ 184191 KY129085 KY128876 KY128668 Tenellia adspersa Sweden GnM 9011 KY129084 KY128875 - Tenellia adspersa F1* Audenge, France Tenellia adspersa F2* Audenge, France Tenellia adspersa G10* New Hampshire, USA Tenellia adspersa G9* New Hampshire, USA Tenellia adspersa S9* New Hampshire, USA Tenellia adspersa SC2* California, USA Tenellia adspersa W2* New Hampshire, USA Tenellia columbiana California, USA CASIZ 185195 KY128906 KY128698 KY128493 Tenellia foliata Ireland GnM 9100 KY128912 KY128704 KY128499 Tenellia gymnota New Hampshire, USA CASIZ 184188 KY128908 KY128700 KY128495 Tenellia gymnota New Hampshire, USA CASIZ 184182 KY128907 KY128699 KY128494 Tenellia gymnota Väderö Islands, Sweden GnM 8948 - KY128707 KY128500 Tenellia cf. maua Sao Tome, Africa CASIZ 179403 KY128905 KY128697 KY128492

Tenellia poritophages Philippines CASIZ 177737 KY128968 KY128759 KY128554 Tenellia pustulata Maine, USA CASIZ 183930 KY128972 KY128764 KY128559 Tenellia sp. D Hawaii, USA CASIZ 185133 KY128909 KY128701 KY128496 Tenellia sp. D Hawaii, USA CASIZ 185139 KY128910 KY128702 KY128497 Tenellia sp. E Philippines CASIZ 181298 KY1284593 KY129006 KY128798 Tenellia sp. G Hawaii, USA CASIZ 176796 KY128979 KY128771 KY128566 Tenellia speciosa South Africa CASIZ 176954 KY128998 KY128790 KY128585 Tergipes tergipes New Hampshire, USA CASIZ 184192 KJ434079 KJ434066 KJ434097 Tergipes tergipes Maine, USA CASIZ 183940 KJ434078 - JK434096 Tergipes tergipes E Schheldt, Netherlands MNCN 15.05/67227 KJ434071 - KJ434087 Tergipes tergipes Swansea, Wales, UK MNCN 15.05/67235 KJ434067 - KJ434082 Tergipes tergipes Trieste, Italy MNCN 15.05/67224 KJ434075 - KJ434093 Tritonia festiva Washington, USA CASIZ 186478 KP153291 KP153258 KP153324 Tritonia nilsodhneri False Bay, South Africa CASIZ 176222 KP871653 KP871702 KP871677

39

Figure 1. Tenellia adspera: adult specimen collected by N. Rorem, Squamscott River at the Exeter Town Dock, Exeter, NH (October 2014).

40

A B

D C

Figure 2. Morphotypes of Tenellia adspersa. B and D were taken by Larry Harris; adults, Great Bay, NH. A and C were taken by Amanda Sobel; adults found in Newcastle and Great Bay, NH respectively. Each animal approximately 4mm in length.

A B

C D

E F

Figure 3. Photos of select Fionid specimens. A. Cuthonella concinna, Eastport, Maine; B. Eubranchus sp., Newcastle, NH; C. Tenellia adspersa, Squamscott River, Exeter, NH; D. Eubranchus exiguus., Newcastle, NH; E. Tenellia gymnota, Newcastle, NH; F. Tenellia adspersa (dark morphotype with egg capsules); H, Newington, NH.

Figure 4. Geographical locations of sampling locations for Tenellia adspersa within the Gulf of Maine watershed. Map created with Google Earth, 2017.

A B B

5μm 10μm C D

5μm 10μm

E F

10μm 5μm

G H

10μm 10μm

Figure 5. Scanning electron micrographs of Tenellia adspersa radulae. A: specimen M1 (Marina Del Rey, CA. B: specimen S9 (Squamscott River, NH). C: specimen CW1 (Cutting’s Wharf, CA. D: specimen W2 (Newcastle, NH). E and F: specimen C5 (CML, NH). G: specimen G13 (Great Bay, NH). H: specimen R1 (Robbinston, ME).

10

G13 9

8 y = 0.4763x + 4.578 R² = 0.2692 7 CW1 6 M1 5

4

3 Number Number of denticles observed 2

1

0 0 1 2 3 4 5 6 Size of Specimen (mm)

Figure 6. Scatter plot comparison of number of denticular tooth rows in radulae versus specimen size (mm). R2 value = 0.269, P = 0.233. Each data point accounts for one specimen. The grouping of four points represents specimens C5, S9, W2, and R1 which are each 4mm long and have 6 denticles. CW = Cutting’s Wharf, Napa River CA; M = Marina Del Rey, CA; S = Squamscott River, Exeter NH; C = Coastal Marine Lab, Newcastle, NH; R = Robbinston Town Dock, Robbinston, ME; and G = Great Bay Marine, Newington, NH.

26 24 22 20 18 16 14 12

46 10 No. of Groups of No. 8 6 4 2 0 0 0.003 0.006 0.009 0.012 0.015 0.018 0.021 0.024 Prior Intraspecific Divergence (P)

Figure 7. Automatic partition of the CO1 sequence data set from Automatic Barcode Gap Discovery (ABGD) analysis. The number of groups inside the partitions (initial and recursive) reported as a function of prior limit between intra and interspecies divergence. Circles represent initial partition, and triangles represent the recursive partition analysis.

Figure 8. Haplotype network based on thirty-seven Tenellia spp. mitochondrial CO1 sequence data, using TCS analysis. Each line connecting the haplotypes represents a single nucleotide mutation, the dots on the lines represent additional mutations. Following taxa have haplotype ‘S13’: S13, F2, G7, G8, and G11. Haplotype ‘CW4’: CW4, CW5, CW8, and CW9. Haplotype ‘M2’: M2, M1, M4, and M5. Haplotype ‘C3’: C3, S11 and S3. Haplotype’W2’: W2 and S5. Haplotype ‘SC4’: SC4 and SC8. Haplotype ‘CW6’: CW6, SC2, SC3, and SC5. Haplotype ‘R1’: R1 and T. adspersa__NH. Created using software popART (Population Analysis with Reticulate Trees) v. 3.0. Circle size related to specimens numbers. Junctions with no specimens are an unsampled or missing intermediates. Specimen ID’s are found in Table 2.

47

Figure 9. Phylogenetic hypothesis for TASC based on CO1, and inferred by ML. Numbers at nodes represent bootstrap values from ML analysis, only values >80 are presented. Highlighted clades represent Eastern Pacific localities. Eastern Atlantic specimens include F1, F2, and SWE. Specimen ID’s found in Table 2.

48

Figure 10. Molecular phylogeny of the nudibranch family Fionidae with outgroups based on the combined dataset (CO1+16S+H3) inferred by ML using RAxML. Values at nodes indicate Bootstrap (BS) support values. The TASC is highlighted.

49

Figure 11. Molecular phylogeny of the nudibranch family Fionidae based the combined dataset (CO1+16S+H3) using posterior probabilities (PP) inferred by Bayesian Inference (BI). Values at nodes represent BI support values. Only values >0.8 are shown. Constructed using PhyloBayes 4.1. The TASC is highlighted.

50

LITERATURE CITED

Alder, J., and A. Hancock. 1842. Eolis. The Annals and Magazine of Natural history, including Zoology, Botany, and Geology. 9:35-36.

Alder, J., and A. Hancock. 1845-1855. A Monograph of the British Nudibranchiate Mollusca. Ray Society, London Parts I-VIII:438pp.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and D.J. Lipman. 1990. Basic local alignment search tool. Journal of Molecular Biology. 215:403-410.

Baba, K., and I. Hamatani. 1963. A short account of the species Tenellia pallida (A. & H.) taken from Mukashima, Japan (Nudibranchia: Eolidoidea). Publications of the Seto Marine Biological Laboratory. 11:167-168.

Bandelt, H., P. Forster, and A. Röhl. 1999. Median-joining networks for inferring intraspecific phylogenies. Molecular Biological Evolution. 16(1):37–48.

Barroso, R., M. Klautau, A.M. Sole-Cava, and P.C. Paiva. 2010. Eurythoe complanata (Polychaeta: Amphinomidae), the ‘cosmopolitan’ fireworm, consist of at least three cryptic species. Marine Biology. 157:69-80.

Bergh, R. 1886. Beitrage zur kenntnis der Aeolidiaden, VIII. K. k. zool.-botan. Gesell, Wien, XXXV, pp.1-60, 7 plates.

Bleakney, J.S. 1996. Sea Slugs of Atlantic Canada and the Gulf of Maine. Nimbus Publishing and the Nova Scotia Museum. Halifax, Nova Scotia. pp.216.

Blezard, D.J. 1992. Salinity as a refuge from in a nudibranch-hydroid relationship within the Great Bay Estuary system (Master’s Thesis). UNH library database. Accession number: unh.b2209585.

Bloom, S.A. 1976. Morphological correlations between dorid nudibranch predators and prey. . 18(33):289-301.

Brown, G.H. 1980. The British species of the aeolidacean family (Gastropoda: ) with a discussion of the genera. Zoological Journal of the Linnean Society. 69: 225-255.

Caine, E.A. 1998. First case of Caprellid amphipod-hydrozoan mutualism. Journal of Crustacean Biology. 18(2):317-320.

Camacho-Garcia, Y.E., E. Ornelas-Gatdula, T.M. Gosliner, and A. Valdes. 2014. Phylogeny of

51

the family (Pilsbry, 1895) (Heterbranchia: ) inferred from mtDNA and nDNA. Molecular Phylogenetics and Evolution. 71:113-126.

Cámara, S., L. Carmona, K. Cella, I. Ekimova, A. Martynov, and J.L. Cervera. 2014. Tergipes tergipes (förskal, 1775) (Gastropoda:Nudibranchia) is an amphiatlantic species. Journal of Molluscan Studies. 80(5):473-474.

Carlton, J.T. 1979. History, biogeography, and ecology of the introduced marine and estuarine invertebrates of the Pacific coast of North America. Ph. D. Dissertation, University of California, Davis.

Carlton, J.T. 1985. Transoceanic and interoceanic dispersal of coastal marine organisms: the biology of ballast water. Ocean Marine Biology Annual Review. 23:313-374.

Carmona, L., M. Pola, T. Gosliner, and J.L. Cervera. 2013. A tale that morphology fails to tell: A molecular phylogeny of Aeolidiidae (Aeolidida, Nudibranchia, Gastropoda). PLoS ONE. 8(5):1-13.

Carmona, L., M. Pola, T.M. Gosliner, and J.L. Cervera. 2014. The Atlantic-Mediterranean genus Berghia Trinchese, 1877 (Nudibranchia: Aeolidiidae): taxonomic review and phylogenetic analysis. Journal of Molluscan Studies. 80:482-498.

Cella, K., L. Carmona, I. Ekimova, A. Chichvarkhin, D. Schepetov, and T. M. Gosliner. 2016. A radical solution: the phylogeny of the nudibranch family Fionidae. PLoS ONE. 11(12):1- 32.

Chambers, L.A. 1934. Studies on the organs of reproduction in the nudibranchiate mollusks, with special reference to Embletonia fuscata Gould. Bulletin of the American Museum of Natural History. 66(6):599-641.

Chester, C.M. 1996a. Life history and reproductive biology of the estuarine nudibranch Tenellia adspersa (Nordmann, 1844) (Doctoral dissertation). Retrieved from UNH Library database. Accession Number: unh.b2659733.

Chester, C.M. 1996b. The effect of adult nutrition on the reproduction and development of the estuarine nudibranch, Tenellia adspersa (Nordmann, 1844). Journal of Experimental Marine Biology and Ecology. 198(1):113-130.

Chia, F.S., G. Gibson, and P.Qian. 1996. Poecilogony as a reproductive strategy of . Oceanologica Acta. 19(3-4):203-208.

Churchill, C.K.C., A. Alejandrino, A. Valdes, and D.O. Foighil. 2013. Parallel changes in genital morphology delineate cryptic diversification of planktonic nudibranchs. Proceedings of the Royal Society. 280(1765):1471-2954.

Churchill, C.K.C., A. Valdes, and D. Foighil. 2014. Afro-Eurasia and the Americas present 52

barriers to gene flow for the cosmopolitan neustonic nudibranch . Marine Biology. 161(4):899-910.

Clark, K.B. 1975. Nudibranch lifestyles in the North-West Atlantic and their relationship to the ecology of fouling communities. Helgolander Wissenschaftiches Meeresuntersuchungenuchungen. 27: 28-69.

Clement, M., D. Posada, and K.A. Crandall. 2002. TCS: a computer program to estimate gene genealogies. Molecular Ecology. 9(10):1657-1659.

Colgan, D., J. Macaranas, G. Cassis, and M.R. Gray. 1998. Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology. 46:419- 437.

Costa, A. 1866. Saggio sui molluschi eolididei del Golfo di Napoli. Annals of Molluscan Zoology. Napoli. 3:59-60.

Dalyell, J. G. 1853. The powers of the creator displayed in the creation. London, Van Voorst. 2, Pp. 359.

Dawson, M.N., and D.K. Jacobs. 2001. Molecular evidence for cryptic species of Aurelia aurita (, Scyphozoa). Biological Bulletin. 200:92-96.

Dhanya, A.M., R. Jeyabaskaran, D. Prema, S. Chinnadurai, K.S. Abhilash, K.K. Sajikumar, and V. Kripa. 2017. Non-indigenous Tenellia adspersa in the southeast coast of the Arabian Sea, India. Indian Academy of Sciences Current Sciences. 113(1):24-26.

Edmunds, M. 1977. Larval development, oceanic currents, and origins of the Opisthobranch fauna of Ghana. Journal of Molluscan Studies. 43:301-308.

Ekimova, I., T. Korshunova, D. Schepetov, T. Neretina, N. Sanamyan, and A. Martynov. 2015. Integrative systematics of northern and Arctic nudibranchs of the genus Dendronotus (Mollusca, Gastropoda), with descriptions of three new species. Zoological Journal of the Linnean Society. 173:841-886.

Evertsen, J., T. Bakken, and S. Green. 2004. Rediscovery of Tenellia adspersa (Nudibranchia) from the Finnish archipelago. Sarsia North Atlantic Marine Science. 89:362-365.

Eyster, L. S. 1979. Reproduction and developmental variability in the opisthobranch Tenellia pallida. Marine Biology. 51: 133-140.

Fife, F.J., R.L.M. Goreham, and F.H. Page. 2015. A century of monitoring station Prince 6 in the St. Croix River estuary of Passamaquoddy Bay. Bay of Fundy Estuarine Partnership. Pp: 1-13.

53

Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrigenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 3:294-299.

Goodheart, J.A., A.L. Bazinet, A.G. Collins, and M.P. Cummings. 2015a. Relationships within Cladobranchia (Gastropoda: Nudibranchia) based on RNA-seq data: an initial investigation. Royal Society Open Science. 2(9):1-13.

Goodheart, J.A., A.L. Bazinet, A.G. Collins, and M.P. Cummings. 2015b. Phylogeny of Cladobranchia (Gastropoda: Nudibranchia): a total evidence analysis using DNA sequence data from public databases. Digital Repository at the University of Maryland (Drum).

Gould, A. A. 1870. Report on the invertebrata of Massachusetts. Cambridge, Massachusetts. Pp. 1-524.

Grassle, J.P., and J.F. Grassle. 1976. Sibling species in the marine pollution indicator Capitella (Polychaeta). Science. 192:567-569.

Griekspoor, A., and T. Groothuis. 2005. 4Peaks, version 1.7.1. http://www.mekentosj.com/4peaks/.

Guoy, M., Guindon, S., and O. Gascuel. 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Molecular Biology and Evolution. 27(2): 221-224.

Hanson, D., Y. Hirano, and A. Valdes. 2013. Population genetics of Haminoea (Haloa) Japonica Pilsbry, 1895, a widespread non-indigenous sea slug (Mollusca: Opisthobranchia) in North America and Europe. Biological Invasions. 15:395-406.

Harris, L.G., M. Powers, and J. Ryan. 1980. Life history studies of the estuarine nudibranch Tenellia fuscata (Gould 1870). The Veliger. 23:70-74.

Hebert, P.D.N., S. Ratnasingham, and J.R. deWaard. 2003. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London. 270: S96-S99.

Hewitt, C. L., and R. B. Martin. 1996. Centre for research on introduced marine pests, Technical Report No. 4. CSIRO Marine Research, Hobart, Australia.

Hoover, C., T. Lindsay, J. H. R. Goddard, and A. Valdes. 2015. Seeing double: pseudocryptic diversity in the -Doriopsilla gemela species complex of the north-eastern Pacific. Royal Swedish Academy of Sciences. 44(6):612-631.

Katoh, K., and D.M. Standley. 2013. MAF multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and 54

Evolution. 30(4):772-780.

Kent, W.S. 1869. On a new British nudibranch (Embletonia grayi). Proceedings of the Zoological Society of London. Pp. 109-111.

Kienberger, K., L. Carmona, M. Pola, V. Padula, T.M. Gosliner, and J.L. Cervera. 2016. (Linnaeus, 1761), single species or a cryptic species complex? A morphological and molecular study. Zoological Journal of the Linnaean Society. 177:481-506.

Kimura, M. 1980. A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution. 16:111-120.

Knowlton, N. 1993. Sibling species in the sea. Annual Review of Ecological Systematics. 24:189-216.

LA Water Keeper. Pollution prevention: Marina del Rey toxic TMDLs. Retrieved from https://lawaterkeeper.org/pollution-prevention/.

Lartillot, N., and H. Philippe. 2004. A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process. Molecular Biology Evolution. 21: 1095–1109.

Lartillot, N., Lepage, T., and S. Blanquart. 2009. PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating. Bioinformatics 25: 2286–2288.

Layton, K.S., A.L. Martel, and P.D.N. Hebert. 2014. Patterns of DNA barcode variation in Canadian marine molluscs. PLoS ONE. 9(4): e95003.

Leigh, J.W., and D. Bryant. 2015. Popart: full-feature software for haplotype network construction. Methods of Ecology & Evolution. 6:1110-1116.

Lindsay, T., and A. Valdes. 2016. The model organism (Gastropoda: ) is a species complex. PLoS ONE. 11(4).

Malaquias, M.A.E., and D.G. Reid. 2008. Functional morphology of the gonoduct of the gastropod Bulla strata Bruguiere, 1972 (Opisthobranchia: Cephalaspidea): evidence for a monaulic system. Acta Zoology. 89:205-210.

Malaquias, M.A.E., G.P. Calado, V. Padula, G. Villani, and J.L. Cervera. 2009. Molluscan diversity in the North Atlantic Ocean: new records of opistobranch gastropods from the Archipelago of Azores. Marine Records. 2:1-9.

Marcus, E. 1955. Opisthobranchia from Brazil (1). Bolm Far. Fil. Cienc. University Sao Paulo, Zoology. 164:165-203.

55

Mayr, E. 1942. Systematics and the origin of species, from the viewpoint of a zoologist. Columbia University Press, New York. 334pp.

Mikkelson, P.M., P.S. Mikkelsen, and D.J. Karlen. 1995. Molluscan biodiversity in the Indian River Lagoon, Florida. Bulletin of Marine Science. 57(1):94-127.

Napa Sonoma Marsh Restoration Project. 2015. www.napa-sonoma-marsh.org.

Naville, A. 1926. Notes sur les eolidiens. Un eolidien d’eau saumatre. Origine des nematocysts. Zooxanthelles et homochromie. Reviews of Suisse Zoology. 33:251-289.

Nordmann, A. 1844. Versuch einer Natur und Entwicklungsgeschichte des Tergipes edwardsii. Bulletin de la Classe Physico-Mathematique de l’Academie Imperiale des Sciences de Saint Petersbourg. 3(16-17): 270.

Oskars, T.R., P. Bouchet, and M.A.E. Malaquias. 2015. A new phylogeny of the Cephalaspidea (Gastropoda: Heterobranchia) based on expanded taxon sampling and gene markers. Molecular Phylogenetics and Evolution. 89:130-150

Padula, V., J. Bahia, I. Stoger, Y. Camacho-Garcia, M.A.E. Malaquias, J.L. Cervera, and M. Schrodl. 2016. A test of color-based taxonomy in nudibranchs: molecular phylogeny and species delimitation of the Felimida clenchi (Mollusca: ) species complex. Molecular Phylogenetics and Evolution. 103:215-229.

Palumbi, S., A. Martin, S. Romano, W.O. McMillan, L. Stice, and G. Grabowski. 1991. The simple fool’s guide to PCR, version 2. Honolulu: Department of Zoology, University of Hawaii.

Picton, B.E., and C. Morrow. 1994. A field guide to the nudibranchs of the British Isles. Immel Publishing.

Poddubetskaia, M. 2006. Tenellia adspersa. Sea Slug Forum. http://www.seaslugforum.net/find/teneadsp.

Pola, M., and T.M. Gosliner. 2010. The first molecular phylogeny of cladobranchian opisthobranchs (Mollusca, Gastropoda, Nudibranchia). Molecular Phylogenetics and Evolution. 56:931-941.

Pollock, L.W. 1998. A practical guide to the marine animals of Northeastern North America (Rutgers University Press).

Puillandre, N., A. Lambert, S. Brouillet, and G. Achaz. 2012. ABGD, Automatic barcode gap discovery for primary species delimitation. Molecular Ecology. 21(8):1864-1877.

Rambaut, A. 2014. FigTree: Tree figure drawing tool. http://tree.bio.ed.ac.uk/software/figtree. 56

Roginskaya, I.S. 1970. Tenellia adspersa, a nudibranch new to the Asov Sea, with notes on its taxonomy and ecology. Malacological Review. 3: 167-174.

Schmekel, L., and A. Portmann. 1982. Opisthobranchia des Mittelmeeres Nudibranchia and Saccoglossa. Berlin, Springer-Verlag. Pp. 1-410.

Southern California Coastal Ocean Observing System (SCCOOS): A science based-decision support system. 2015-2017. Automated Shore Stations, Santa Monica Pier. Retrieved from www.sccoos.org/data/autoss/.

Stamatakis, A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. Doi:10.1093/bioinformatics/btu033.

Sudhir, K., G. Stecher, and K. Tamura. 2015. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution. 33:1870- 1874.

Suisun City Marina. 2017. https://www.suisun.com/departments/recreation-community- services/marina/.

Thompson, T.E., and G.H. Brown. 1984. Biology of opisthobranch mollucs, Volume II. Department of Zoology, University of Bristol. 229pp.

Todaro, M.A., J.W. Fleeger, Y. P. Hu, A. W. Hrincevich, and D.W. Foltz. 1996. Are meiofaunal species cosmopolitan? Morphological and molecular analysis of Xenotrichula intermedia (Gastrotricha: Chaetonotida). Marine Biology. 125:735-742.

Trickey, J.S. 2012. Phylogeography and molecular systematics of the rafting aeolid nudibranch Fiona pinnata (Eschscholtz, 1831) (Master’s Thesis). University of Otago.

Uribe, R.A., F. Sepulveda, J.H.R. Goddard, and A. Valdes. 2017. Integrative systematics of the genus Limacia O. F. Muller, 1781 (Gastropoda, Heterobranchia, Nudibranchia, ) in the Eastern Pacific. Marine Biodiversity. Online: 1-18.

Vannucci, M. and E.K. Hosoe. 1953. Sobre Embletonia mediterranea (Costa) nudibranchquio de regiao lagunar de Cananeia. Bol. Institute Oceanografique. 4:103-120.

Wasson, K., C.J. Zabin, L. Bedinger, M.C. Diaz, and J.S. Pearse. 2001. Biological invasions of estuaries without international shipping: the importance of intraregional transport. Biological conservation. 102:143-153.

Wilson, N.G., and I. Burghardt. 2015. Here Be Dragons – Phylogeography of (Angas, 1864) reveals multiple species of photosynthetic nudibranchs (Aeolidina: Nudibranchia). Zoological Journal of the Linnean Society. 175:119-133.

57

Wintzer, A.P., M.H. Meek, P.B. Moyle, and B. May. 2011. Ecological insights into the polyp stage of non-native hydrozoans in the San Francisco Estuary. Aquatic Ecology. 45(2):151-161.

WWFHK. 2006. An extension to the existing boardwalk and new mudflat bird-watching hide at Mai Po Nature Reserve for education and conservation purposes. WWF Hong Kong Project Report. Pp. 81.

Yang, Z., and B. Rannala. 2012. Molecular phylogenetics: principles and practice. Nature Reviews, Genetics. 13:303-314.

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APPENDIX

Appendix: General gross morphology of each specimen used in this study.

Date Specimen Collected Location Morphology Collected By Coastal Marine Lab, Pale body, some black freckles dorsally and cerata; C1 6/30/14 Newcastle NH juvenile <1mm Amanda Sobel Coastal Marine Lab, Pale/cream body, many black freckles. Dark cerata; C2 6/30/14 Newcastle NH juvenile 1.5mm Amanda Sobel Coastal Marine Lab, Pale/cream body, many black freckles. Dark cerata; C3 6/30/14 Newcastle NH juvenile, 1.5mm Amanda Sobel Coastal Marine Lab, Pale/cream body, many black freckles. Dark cerata; C4 6/30/14 Newcastle NH 2mm Amanda Sobel Coastal Marine Lab, Cream body. Few black freckles throughout entire body. C5 7/22/14 Newcastle NH Orange/brown cerata, 12 cerata. 4mm Amanda Sobel Coastal Marine Lab, Tannish body. Many black freckles. Bloated cerata, 12.

59 C6 7/22/14 Newcastle NH 4mm Amanda Sobel

Robbinston Boat Cream body color. Many black freckles throughout; R1 8/7/14 Launch, ME including cerata, 12. 4mm Amanda Sobel 6 Cutting's Wharf Rd, Dr. Nadine CW1 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm Rorem 6 Cutting's Wharf Rd, Dr. Nadine CW2 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm Rorem 6 Cutting's Wharf Rd, Dr. Nadine CW3 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm. Rorem 6 Cutting's Wharf Rd, Dr. Nadine CW4 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm Rorem 6 Cutting's Wharf Rd, Dr. Nadine CW5 6/28/15 Napa CA Pale/cream body. Few black freckles. 2mm Rorem 6 Cutting's Wharf Rd, Dr. Nadine CW6 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm. Rorem 6 Cutting's Wharf Rd, Dr. Nadine CW7 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm Rorem

6 Cutting's Wharf Rd, Dr. Nadine CW8 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm Rorem 6 Cutting's Wharf Rd, Dr. Nadine CW9 6/28/15 Napa CA Pale body. Few black freckles. Junevile. 1.5mm Rorem Preserved specimen. Light cream color, few freckles. provided by Juan F1 Sep-06 Audenge, France Juvenile. 8 cerata, 2mm. Lucas Cervera Preserved specimen. Pale/cream body. Few freckles. provided by Juan F2 Sep-06 Audenge, France Juvenile. 1.1mm. Lucas Cervera Great Bay Marine, Cream body. Few black freckles along body and oral G1 6/26/14 Newington NH hood, dark brown cerata, 6. 1mm Amanda Sobel Great Bay Marine, Pale body. Few Black freckles throughout. Tannish G2 6/26/14 Newington NH cerata, 10. 2mm Amanda Sobel Great Bay Marine, Pale body. Few Black freckles throughout. Tannish G3 6/26/14 Newington NH cerata, 12. 2mm Amanda Sobel Great Bay Marine, Pale body. Heavy black freckles throughout. Dark brown G4 6/26/14 Newington NH cerata, white tips, 12. 2mm Amanda Sobel Great Bay Marine, Pale/cream body. Heavy black freckles throughout. G5 6/26/14 Newington NH Dark brown bloated cerata, 12. 1.5mm Amanda Sobel Great Bay Marine, G6 6/26/14 Newington NH Pale body. Few black freckles. Pale cerata, 10. 1mm Amanda Sobel

60 Great Bay Marine, Pale/cream body. Few black freckles throughout but

G7 6/26/14 Newington NH light. Cerata same color as body, 12. 2mm Amanda Sobel Great Bay Marine, Pale/cream body. Few black freckles, many towards G8 6/26/14 Newington NH tail. Cerata same color as body, 12. 2mm Amanda Sobel Great Bay Marine, Pale/cream body. Few black freckles, heavy towards G9 6/26/14 Newington NH tail. Dark brown cerata, 12, 2mm Amanda Sobel Great Bay Marine, G10 6/26/14 Newington NH Pale body. Few black freckles. Pale cerata, 10. 1.5mm Amanda Sobel Great Bay Marine, Mottled slate body. Heavy black freckles throughout. G11 6/26/14 Newington NH Dark brown cerata, 14. 3.5mm Amanda Sobel Great Bay Marine, Mottled slate body. Heavy black freckles throughout. G12 6/26/14 Newington NH Dark brown cerata, 14. >5mm Amanda Sobel Great Bay Marine, Tannish body. Heavy black freckles throughout. Dark G13 6/26/14 Newington NH brown cerata, 15. 5mm Amanda Sobel

Palawan Way, Marina Pale/cream body. Heavy black freckles throughout. Pale Dr. Nadine M1 6/21/15 Del Rey CA cerata with freckles. 2-3mm Rorem Palawan Way, Marina Dr. Nadine M2 6/21/15 Del Rey CA Pale/cream body. Few black freckles. Juvenile. 1mm Rorem Palawan Way, Marina Dr. Nadine M3 6/21/15 Del Rey CA Pale/cream body. Few black freckles. Juvenile. 1mm Rorem Palawan Way, Marina Dr. Nadine M4 6/21/15 Del Rey CA Pale/cream body. Few black freckles. Juvenile. 1mm Rorem Palawan Way, Marina Dr. Nadine M5 6/21/15 Del Rey CA Pale/cream body. Few black freckles. Juvenile. 1mm Rorem Exeter Town Dock, Pale/cream body. Few black freckles throughout. Cerata Dr. Nadine S1 10/16/14 Exeter NH same, 10. (4.5mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S2 10/16/14 Exeter NH (4.5mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S3 10/16/14 Exeter NH (5.1mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S4 10/16/14 Exeter NH (3.5mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S5 10/16/14 Exeter NH (4.9mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S6 10/16/14 Exeter NH (4mm) Rorem 61 Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S7 10/16/14 Exeter NH (5mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S8 10/16/14 Exeter NH (4mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S9 10/16/14 Exeter NH (4mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles. Cerata same, 10. Dr. Nadine S10 10/16/14 Exeter NH (4mm) Rorem Exeter Town Dock, Pale/cream body. Few black freckles, cerata same. Long Dr. Nadine S11 10/16/14 Exeter NH rhinophores. 12 cerata. (4.9mm) Rorem Exeter Town Dock, Cream body. Many black freckles, cerata same. 12 Dr. Nadine S12 10/16/14 Exeter NH cerata. (4.2mm) Rorem

Exeter Town Dock, Mottled slate body with lighter cream tail, dark cerata. Dr. Nadine S13 10/16/14 Exeter NH 12 cerata. (6mm) Rorem Suisun City Marina, Dr. Nadine SC1 6/28/15 Suisun City CA Pale body. Few black freckles, cerata same. (4mm). Rorem Suisun City Marina, Pale/cream body. Heavy black freckles dorsally, cerata Dr. Nadine SC2 6/28/15 Suisun City CA same. (3mm) Rorem Suisun City Marina, Pale/cream body. Few black freckles throughout, cerata Dr. Nadine SC3 6/28/15 Suisun City CA same. (3mm) Rorem Pale/cream body. Few black freckles throughout, Suisun City Marina, cerata same. Dr. Nadine SC4 6/28/15 Suisun City CA (3.5mm) Rorem Suisun City Marina, Pale/cream body. Few black freckles throughout, Dr. Nadine SC5 6/28/15 Suisun City CA cerata same. (3mm) Rorem

Suisun City Marina, Pale body. Many black freckles, cerata same. 6 cerata. Dr. Nadine SC6 6/28/15 Suisun City CA (1mm) Rorem

62 Suisun City Marina, Pale body. Many black freckles, cerata same. 6 cerata. Dr. Nadine

SC7 6/28/15 Suisun City CA (1mm) Rorem Suisun City Marina, Pale body. Heavy black freckles dorsally, cerata few Dr. Nadine SC8 6/28/15 Suisun City CA freckles. (4mm) Rorem Suisun City Marina, Pale body. Few black freckles, cerata same. 6 cerata. Dr. Nadine SC9 6/28/15 Suisun City CA (1mm) Rorem Suisun City Marina, Pale body. Few black freckles, cerata same. 6 cerata. Dr. Nadine SC10 6/28/15 Suisun City CA (1mm) Rorem Wentworth Marina, Pale/cream body. Black freckles throughout, many on W1 7/14/15 Newcastle NH cerata. 3.5mm Amanda Sobel Wentworth Marina, Pale/cream body. Black freckles throughout, cerata W2 7/14/15 Newcastle NH same. 10 cerata. (4mm) Amanda Sobel