Distribution, Genetic Differentiation, and Assortative Mating

DISTRIBUTION, GENETIC DIFFERENTIATION, AND ASSORTATIVE MATING

OF DISTINCT MORPHOTYPES OF DIAULULA SANDIEGENSIS, A NUDIBRANCH

WITH HIGH DISPERSAL POTENTIAL

Julie Anne Kelly

A Thesis Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Science in Biology

Committee Membership

Dr. Sean F. Craig, Committee Chair

Dr. Edward C. Metz, Committee Member

Dr. Erik S. Jules, Committee Member

Dr. John O. Reiss, Committee Member

Dr. Michael R. Mesler, Graduate Coordinator

May 2013

ABSTRACT

DISTRIBUTION, GENETIC DIFFERENTIATION, AND REPRODUCTIVE ISOLATION OF DISTINCT MORPHOTYPES OF DIAULULA SANDIEGENSIS, A NUDIBRANCH WITH HIGH DISPERSAL POTENTIAL

Julie A. Kelly

Diaulula sandiegensis (Cooper, 1862), a northern Pacific nudibranch, has considerable phenotypic variation in dorsal spotting pattern throughout its range.

Experiments were conducted to test the possibility that D. sandiegensis is a complex of unrecognized species, even though it has a planktonic larva with a high dispersal potential. Field and laboratory common garden experiments were conducted to investigate whether length of the individual or diet influence spotting pattern. These investigations found individual D. sandiegensis maintained dorsal spotting morphology, indicating that their dorsal spotting pattern was genetically determined.

Field investigations from California to British Columbia and a survey of D. sandiegensis images from the World Wide Web were conducted to describe the variation in spotting pattern of D. sandiegensis and to correlate this variation in dorsal spotting pattern with latitude, depth, and prey. Characteristics from 337 D. sandiegensis were categorized and found to separate individuals into two distinct morphotypic populations, the “many-spotted” (MS) and “few-spotted” (FS) morph, reliably distinguished by presence or absence of mantle-skirt spots, respectively. Dorsal spot number, spot type,

ii and background color can also be used to distinguish between the morphotypes, but slight overlap of these characteristics exists between morphotypes.

Morphotype frequencies of 433 D. sandiegensis showed a strong correlation to differences in latitude and depth. The FS morph was the only form found south of Fort

Bragg (southern region) in intertidal, subtidal, and bay habitats. In addition, the FS morph was the primary morphotype found in the coastal subtidal habitats within the entire study area. The MS morph was the primary morphotype in the coastal intertidal habitat north of Bodega Bay (northern region), comprised of 88 percent MS and 12 percent FS morph.

Field investigations also indicated a dietary difference. Diaulula sandiegensis

(both morphotypes) from coastal intertidal habitats fed on Haliclona sp. A (Hartman,

1975). A fecal analysis indicated that D. sandiegensis collected from the subtidal habitat of Monterey Bay (FS morphotype) fed on Neopetrosia problematica (de Laubenfels

1930). A laboratory common garden experiment indicated a higher growth and survival rate for MS morph from the Crescent City intertidal habitat than FS morph from the

Monterey Bay subtidal habitat, when fed Haliclona sp. A in the laboratory.

A mating study and genetic analysis, using mtDNA sequences of the COI gene, were performed. The results showed that the MS and the FS morphs are reproductively incompatible. MS and FS morphs were reciprocally monophyletic at COI, with sequences differing by a p-distance percentage of between 5.9-7.9 percent.

Examining behavior, habitat partitioning, and genetic variation clearly indicates that D. sandiegensis is recognizable as two species with distinct ranges, habitat, and prey. iii

ACKNOWLEDGEMENTS

This work was supported by the NSF grant DBI-0755466 given to Sean F. Craig.

I especially thank my husband, Mike Kelly, and daughter, Jen Kelly, for their long hours spent in the tidepools. I also want to thank all of the following faculty, staff, and students from Humboldt State University, the Telonicher Marine Laboratory, and the Research

Experience for Undergraduates Program for their guidance, assistance, hard work, and support: S. F. Craig, E. C. Metz, J. O. Reiss, E. S. Jules, G. Eberle, D. Hoskins, A. Baker,

K. Korcheck, R. Koeppel, J. Koeppel, S. Monk, E. Martin, and A. Carter.

I want to thank the faculty and students from San Jose State University for their assistance with the genetic analysis: J. Mackie and N. Taeidi. I want to thank Bret Grasse from the Monterey Bay Aquarium for his help with the collection of nudibranchs from

Monterey Bay. I want to thank D. Behrens, author of Eastern Pacific Nudibranchs, and

Dr. B. Penney from Saint Anselm College for the initial idea for this study.

TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

TABLE OF CONTENTS ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... x

INTRODUCTION ...... 1

METHODS ...... 13

Study Organism - Diaulula sandiegensis ...... Error! Bookmark not defined.

Change in Spotting Pattern over Time ...... 13

Data acquisition - field investigations ...... 17

Data acquisition – laboratory common garden experiment ...... 17

Laboratory set up ...... 19

Data analysis ...... 20

Distinct Morphotypes ...... 21

Data acquisition ...... 21

Data analysis ...... 21

Morphotypes of Diaulula sandiegensis Relative to Latitude and Depth ...... 23

Data acquisition ...... 23

Data analysis ...... 23

Sponge Use Variation...... 23

Data acquisition - field investigation/fecal analysis ...... 24

Data acquisition – laboratory common garden experiment ...... 25

Data analysis ...... 25

Reproductive Compatibility of Morphotypes Using Mating Studies...... 26

Data acquisition – field study ...... 26

Data acquisition – laboratory mating study ...... 26

Laboratory set up ...... 26

Data analysis ...... 27

Phylogenetic Analysis Using COI...... 28

Data acquisition ...... 28

DNA extraction, PCR, and sequencing ...... 28

Data sequence analysis ...... 30

RESULTS ...... 31

Change in Spotting Pattern over Time ...... 31

Laboratory common garden experiment ...... 32

Field investigations ...... 33

Distinct Morphotypes ...... Error! Bookmark not defined.

Spot type ...... 41

Background color ...... 43

Commensal scale worm ...... 43

Radula ...... 43

Morphotypes of Diaulula sandiegensis Relative to Latitude and Depth ...... 46

Sponge Use Variation...... 48

Reproductive Compatibility of Morphotypes Using Mating Studies...... 55

Natural environment ...... 55

Controlled environment ...... 55

Phylogenetic Analysis Using COI...... 59

DISCUSSION ...... 64

REFERENCES ...... 75

vii

LIST OF TABLES

Table Page

1 Spicules found in the skeletons of Haliclona (Haliclona) sp. A, H. panacea, and

N. problematica. These three sponges have only megascleres with no

microscleres present……………………………………………………………...52

2 Results of D. sandiegensis and sponge association analysis from the coastal

intertidal habitat from Monterey to Barkley Sound during the 2009 field season,

showing the numbers of D. sandiegensis found on or near sponge……………...53

3 Results from feces analysis of seven D. sandiegensis collected from the subtidal

habitat of Monterey Bay, showing, type, length, and width of the primary spicule

in feces collected………………………………………………………………....54

4 Mating events among the D. sandiegensis morphotypes at study sites from San

Diego, California, to Bamfield, British Columbia……………………………….60

5 Number of mating events among the D. sandiegensis morphotypes during 319

hours (13.3 days) of time-lapse photography. Six to ten D. sandiegensis of both

morphotypes were placed together, in the same aquarium, giving them a choice of

mating partner. Sixty-four out of sixty-five mating events were between like-

morph pairs. ...…………………………………………………………………...61

6 Number of mating events among the D. sandiegensis morphotypes during 22.3

hours of time-lapse photography. Four dissimilar-morph pairs were placed in

viii

separate aquaria; therefore, they did not have a choice of mating partner. One

mating event occurred……………………………………………………………62

7 Estimates of evolutionary divergence between sequences. Neighbor-joining

method of cytochrome c oxidase I. Shown are the number of base substitutions

per site from between sequences. Standard error estimates are shown above the

diagonal and were obtained by a bootstrap procedure (1000 replicates). The

analysis involved seventeen nucleotide sequences. …………………….……….65

LIST OF FIGURES

Figure Page

1 Variation in spotting pattern and background color of D. sandiegensis. (A) Left is

the MS (or “leopard”) morph. (Intertidal habitat, Crescent City, California.) This

photograph shows evidence of feeding on the sponge Haliclona sp. A. (Hartman,

1975). (B) Right is the FS (or “ringed”) morph. (Subtidal habitat, Monterey Bay,

California). This photograph was taken of an individual being held at the

Monterey Bay Aquarium. This individual is feeding on the sponge, Neopetrosia

problematica (de Laubenfels 1930)……………………………………………...11

2 Distribution of field sites. With habitat and source indicated. D. sandiegensis

were collected for laboratory common garden experiment from Crescent City and

Monterey…………………………………………………………………………16

3 Growth study in a controlled laboratory environment. (A) Growth of a MS morph

for 102 days, from June 14, 2010 to September 23, 2010. Length increased from

0.93 to 3.22 cm. Spot number increased from 54 to 90 spots. Black circles

identify the same spots. The spots have increased in size but the unique spotting

pattern has stayed the same. (B) Growth of FS morph for 34 days, from May 21,

2010 to July 21, 2010. Length increased from 1.77 to 3.89 cm. Spot number

stayed the same with 8 spots (one spot is hidden behind gills). Scale bar equals

one centimeter…………………………………………………………….33

4 Growth Study at Luffenholtz Beach, Trinidad, California. (A) Growth of MS

morph for 69 days, from April 5, 2009 to June 12, 2009. Length increased from

0.72 to 2.76 cm. Spot number increased from 52 to 97. (B) Growth of FS morph

for 44 days, from April 27, 2009 to June 9, 2009. Length increased, from 0.38 to

1.71 cm. Spot number stayed the same with 7 spots. Scale bar equals one

centimeter………………………………………………………………………...34

5 Dorsal spot number as a function of D. sandiegensis length in centimeters. 169

individuals found and photographed during the 2009 field season within the study

area (British Columbia to southern California). There is evidence for a

dependence of dorsal spot number on nudibranch length in the “many-spotted

morph,” dorsal spot number increases with length (Regression: R² = 48.3, N =

106, P < 0.0005). There is no evidence for a dependence of dorsal spot number

on nudibranch length of the “few-spotted morph,” dorsal spot number does not

increase with length (Regression: R² = 1.4, N = 63: P= 0.349).

……………………………………………………………………………………35

6 Variation of D. sandiegensis dorsal spot numbers grouped by presence or absence

of mantle-skirt spots. A histogram of the dorsal spot number distribution of 320

D. sandiegensis showed a bi-modal distribution. Dorsal spot numbers of D.

sandiegensis with mantle-skirt spots are significantly different than dorsal spot

numbers of D. sandiegensis without mantle-skirt spots (T-Test on log-transformed

data, T-Value = -25.80, DF = 138, P-Value < 0.005). Three hundred and twenty

nudibranchs from the 2009 and 2010 field season were used for this analysis. The xi

MS morph curve is not significantly different than a normal curve (Normality

Test, N = 136, P-Value = 0.565)……………………………………………….38

7 Diaulula sandiegensis FS morph with high end spot numbers from (A) Humboldt

County (41 spots) and (B) Pigeon Point, California (38 spots) (Photograph by

Gary McDonald). Both individuals identified as FS morphs due to lack of

mantle-skirt spots. ……………………………………………………………….39

8 Cladogram of D. sandiegensis MS morph and “few-spotted morph.” This

cladogram distinguishes groups by characters. It shows the variation of

characteristics across and within morphotypes. Three hundred and thirty-seven D.

sandiegensis nudibranchs from the 2009 and 2010 field season and from the

World Wide Web were used for this analysis. …………………………………..41

9 Commensal scale worm found in pallial cavity of D. sandiegensis from the

subtidal habitat of Monterey Bay………………………………………………...42

10 SEM micrographs of D. sandiegensis radula. (A) The “many-spotted morph”

showed no sign of denticles at the outer margin. (B) This image shows one row of

teeth on the ribbon of a MS morph. The teeth were smooth and hamate (hook-

shaped) that decreased in size from the center of each lateral side to the outer

margin. (C) The FS morph teeth were also smooth and hamate………………44

11 Geographical and habitat pattern of D. sandiegensis population structure from the

coastal subtidal (C. S.), coastal intertidal (C. I.), and bay (Bay) habitats from

Barkley Sound, British Columbia, to San Diego, California. Pie charts indicate

frequencies of morphotypes. Sample sizes depicted in bold next to sample xii

location. Four hundred and thirty-three D. sandiegensis from the 2009, 2010, and

2011 field season and images from the World Wide Web were used for this

analysis…………………………………………………………………………46

12 Sponge external characteristics, spicules, and skeletal structure used for

identification of sponges. (A) Haliclona sp. A (with the nudibranch D.

sandiegensis) (B) SEM micrograph of Haliclona sp. A. spicules. (C) SEM

micrograph of Haliclona sp. A. skeletal structure. (D) H. panicea (with the

nudibranch Archidoris montereyensis (Cooper, 1862)), (E) SEM micrograph of H.

panicea spicules. (F) SEM micrograph of N. problematica spicule with

mucronate ends. (G) N. problematica (with the nudibranch, D. sandiegensis)

photographed at the Monterey Bay Aquarium. (H) and (I) SEM micrographs of

N. problematica spicules with mucronate ends. ………………………………..50

13 The survival of the MS morph and the FS morph were significantly different

when both morphotypes were kept together in a common garden experiment

(Kaplan-Meier survival analysis, Log-Rank method, P-Value = 0.009). The MS

morph was collected from the intertidal habitat in Crescent City, and the FS

morph was collected from the subtidal habitat in Monterey Bay. Three MS morph

D. sandiegensis survived past the end of the experiment (Eighteen weeks). All FS

morph died by the eightieth day of the experiment ……………………………..55

14 Nudibranch length difference as a function of time between measurements for

both morphotypes grown in a common garden experiment. The MS morph grew

significantly more than the FS morph (ANOVA, P = 0.006). Four FS morph xiii

actually lost length during the experiment. To transform the negative length

values, making them positive for the ANOVA, the maximum length lost, 0.38 cm,

was added to all values. The data was also transformed with the square root for

improving the normality, ( ( + 0.38 ))…………………………………...56

15 Evolutionary relationships �of 푐푚taxa. Phylogenetic푐푚 tree using neighbor-joining

method with mtDNA cytochrome c oxidase I. Fifty percent and higher bootstrap

support values are shown at nodes. …………………………………...64

xiv

INTRODUCTION

Genetically determined phenotypic variation is a fundamental prerequisite for evolution by natural selection (Darwin 1859). With rare exceptions, every species displays phenotypic variation. (For exceptions see Lee & Frost 2002). In response to heterogeneous environments, individuals and populations may display variations in morphology and behavior. This variation can be genetically fixed, environmentally plastic, or determined by both processes (Sotka 2012). Environmentally plastic phenotypes change depending on the environment. In contrast, genetically fixed phenotypes can be specialized for specific environments, or can be the result of genetic drift when populations are separated by a geographical or ecological barrier (Sotka 2012).

Descriptions of phenotypic variation are important for understanding the responses of species to heterogeneous environments. Moreover, correlation of intraspecific phenotypic variation with the distribution and habitat use of a species can illustrate the mechanisms of genetic divergence, reproductive isolation, and species formation (Faucci et al. 2007; Hyde et al. 2008; Knowlton et al. 1993; Sanford& Worth

2010; Stachowicz& Hay 2000).

In the marine environment, the drivers of differentiation are poorly understood

(Palumbi 1994). Most marine species tend to have large population sizes, extensive planktonic dispersal, and large geographic ranges. Consequently, they are predicted to undergo limited genetic differentiation and speciation (Palumbi 1994). However, recent

phylogenetic studies have revealed evidence of genetic differentiation (Burford 2009;

Kelly& Palumbi 2010; Sotka et al. 2004; Yorifuji et al. 2012) and genetically fixed

phenotypic variation (Faucci et al. 2007; Hyde et al. 2008; Stachowicz& Hay 2000;

Stevens 1990) in more species with planktonic larvae than was previously predicted.

Extensive planktonic dispersal can connect distant populations, which in turn can lead to high gene flow and low differentiation between distant populations (Lambert et al.

2003). Planktonic larvae have a high dispersal potential because they can spend weeks or

even months in the water column, there are relatively few physical barriers in the ocean,

ocean currents have an homogenizing effect. Ocean currents have the potential to

transport pelagic larvae hundreds of miles from their birthplace (Bohonak 1999; Kelly&

Palumbi 2010; Shanks 2009). However, tracking microscopic larvae in the ocean is very

difficult. Consequently, actual dispersal distances are often estimated by the length of

time spent as larvae in the plankton (Bohonak 1999; Hellberg et al. 2002).

The difficulty of identifying barriers to larval dispersal and gene flow in the

marine environment may also explain why mechanisms of population differentiation are

poorly understood in the ocean. Distance between populations acts as a physical barrier.

Isolation between populations increases as the distance between them increases, until the

distance between them is greater than the organisms’ dispersal distance. The distance

necessary to become a barrier is difficult to calculate when larval dispersal is difficult to

predict (Lee 2000). Populations may become physically isolated through vicariance

resulting in restriction or complete cessation of dispersal and gene flow. Examples of

vicariance include the separation of the Pacific and Atlantic Oceans by the Isthmus of

Panama (Knowlton et al. 1993), closure of the seaway across north Florida connecting the Gulf of Mexico and the Atlantic (Bert 1986), and the fragmentation of habitats by

glacial advances (Burford 2009; Sotka et al. 2004). The effect historical geological and climatic events may have had on past larval dispersal in marine populations is also very difficult to determine.

When there are no obvious physical barriers, species with planktonic larvae are generally predicted to evolve environmentally plastic phenotypic variation in response to heterogeneous environments (Harvell 1998; Lee 2000; Lively et al. 2000; Palmer 1990;

Warner 1997). In contrast, genetically-fixed phenotypes, or a combination of both plastic and heritable components of phenotypic variation, are frequently attributed to marine species with direct developing larvae that spend little or no time in the plankton. These species tend to have lower dispersal potential, lower connectivity, and lower gene flow across populations (Harris& Jones 1995; Pascoal et al. 2012; Sanford& Worth 2010;

Sotka 2003).

However, it is also possible for reproductive isolation to occur in the absence of physical barriers. Host preference and specialization on the preferred host can lead to assortative mating and population differentiation, such as in the case of the host shift of the apple maggot fly Rhagoletis pomonella in the terrestrial environment (Feder et al.

1988), and the host shift of the aeolid nudibranch in the genus Phestilla (Faucci et al.

2007). Specialization can also occur due to other ecological barriers, such as availability

of food for Nucella canaliculata (Sanford& Worth 2010) and availability of toxic anti-

feedant material for the decarator crab Libinia dubia (Stachowicz& Hay 2000).

Oceanographic processes (Burford 2009; Kelly& Palumbi 2010; Sanford& Worth 2010;

Sotka et al. 2004) and depth (Hyde et al. 2008) can also be a barrier to dispersal and gene flow.

Phenotypic variation has been studied less in marine environments than in terrestrial environments (Sotka 2012). Possible explanations for the lack of studies on

phenotypic variation in the marine environment could be that there is less phenotypic

variation, or that phenotypic variation is more difficult to study in the marine

environment (Sotka 2012). Common-garden experiments and reciprocal-transplants are

frequently used to determine the relative contributions of genetic and environmental

components to phenotypic variation (Sanford& Worth 2010; Sotka 2005, 2012). These

methods identify differences in fitness between dissimilar phenotypes found in differing

habitats when brought into the same environment.

These studies are difficult to do in the marine environment (Personal Observation;

Sotka 2012). Observations and collections of organisms in the intertidal habitat must

wait for low tides and calm water and special equipment, such as SCUBA, are required

for observations and collections of organisms in the subtidal habitat. Planktonic larvae

are difficult to raise in the laboratory for common-garden and reciprocal-transplant

experiments. Planktonic larvae need to remain suspended for days or even months before

they can metamorphose and settle. In addition, some have very specific substrate needs

for settlement.

Many marine species display phenotypic variation in color patterning.

Phenotypic variation in color patterning could be a generalist, specialist, or plastic

strategy in response to environmental heterogeneity. Phenotypic plasticity or genetically fixed variation in color patterning may result from selection pressures, such as predation pressure (Palumbi 1994), thermal stress (Harris& Jones 1995), and mate choice (Levins

1968). Color patterning may provide protection from visual predators in the form of crypsis, disruptive coloration, or warning colors.

Species that rely on coloration for protection against predators often have intraspecific variation in that pattern. Palma and Steneck (2001) found that individuals in phenotypically polymorphic populations that rely on crypsis to avoid detection, and that live in complex environments, have an advantage over individuals in monomorphic populations. A polymorphic population presents many patterns to the predator, preventing the predator from recognizing a discrete search image (Cuthill et al. 2005;

Endler 1978, 1984; Todd et al. 2006). In addition, a bet-hedging strategy of survival, in which many polymorphic offspring ensures that at least some individuals will match the substrate on which they settle, may result in high intraspecific polymorphism (Lloyd

1984; Todd et al. 2009). A change in phenotype over the life of an organism, due to the differential vulnerability of different life stages, may also result in a phenotypically polymorphic population (Levins 1968; Todd et al. 2006).

Finally, Rudman (1991) hypothesized that geographic variation in patterning of chromodorid nudibranchs may result when unrelated nudibranch species mimic

(Müllerian mimicry) each other. Many chromodorid nudibranch species from southern

Australia that rely on aposematic coloration to warn off predators evolved into groups of monomorphic populations (Rudman 1991). The predators learn more easily to recognize

6 and avoid similarly patterned and toxic individuals. When several species of unpalatable nudibranchs share a single pattern of warning color, the avoidance learning by predators more efficiently protects all individuals. As a species comes to resemble the coloration of another species in one region, its coloration may diverge from the coloration of conspecifics in a distant population, leading to geographic variation in phenotype.

Study Organism - Diaulula sandiegensis

The nudibranch Diaulula sandiegensis (Cooper 1862) (Mollusca, Gastropoda,

Opisthobranchia, Nudibranchia, Dorididae) occurs along the north Pacific Coast from

Mexico to Japan (Behrens& Hermosillo 2005) in subtidal and intertidal habitats of the open coast as well as bays. Diaulula sandiegensis exhibits considerable variation in dorsal spotting pattern throughout its range, yet the biological significance of this variation is not understood.

Spot shape on the dorsum of this species can vary from circles to irregular blotches that are solid or oscellated and with or without an outer opaque white ring

(Figure 1). Spot size can vary from one to a few millimeters in diameter. The largest four dorsal spots are usually arranged on the dorsum forming a rectangle. The number of spots on the dorsum can vary from just a few to over three hundred (Behrens& Valdés

2001). Three morphotypes are commonly identified by their dorsal spotting pattern: (1) the “ringed” form with just a few oscellated spots; (2) the “leopard” form with numerous solid spots; and (3) the “speckled” form with a few oscellated brown spots with numerous pale brown specks covering the dorsum (Figure 1).

For the present study, the “ringed” and “leopard” forms of D. sandiegensis were

examined and will be referred to as the few-spotted (FS) morph and the many-spotted

(MS) morph, respectively. The “speckled” form was not included in the present study

because only one “speckled” individual was found during field surveys in San Diego,

California.

The “speckled” morphotype was previously identified as Doris (s.l.)

(MacFarland 1966). Behrens and Valdés (2001) compared the reproductive systems and

radulae of the “speckled” morph to specimens of the other morphs and found no other

morphological differences. They concluded that the “speckled” morph is a synonym of

D. sandiegensis.

The distributions and habitats for each of these morphotypes have not been

described. However, Behrens and Valdés (2001) suggested that there is geographical

variation in the spotting pattern of D. sandiegensis along the Pacific Coast of North

America. The northern populations have many dark solid spots and the southern

populations have just a few spots each with a dark ring around the outside of a light

center (Figure 1). Additionally, Morris et al. (1980) noted that D. sandiegensis is more abundant subtidally at the southern end of its range and intertidally at the northern end of its range. In Bamfield, British Columbia, Brian Penny (personal communication, study in progress) found an association between D. sandiegensis spotting pattern and depth. He

found that the “ringed” form is more common subtidally and the “leopard” form is more

common intertidally.

B A

1 cm 1 cm

1 cm

Figure 1. Variation in spotting pattern and dorsal background color of D. sandiegensis.

(A) The many -spotted (MS) morph. (Intertidal habitat, Crescent City, California.) This photograph shows feeding on the sponge Haliclona sp. A. (Hartman, 1975). (B) The few-spotted (FS) morph. (Subtidal habitat, Monterey Bay, California). This photograph was taken of an individual being held at the Monterey Bay Aquarium, feeding on the sponge, Neopetrosia problematica (de Laubenfels 1930). (C) The “speckled” form from

Orange County, California, intertidal, taken by David W. Behrens.

Diaulula sandiegensis is known to feed exclusively on sponges. However, a distinction has not been made of the sponge prey species fed on by each morphotype.

Elvin (1976) conducted a feeding preference study with D. sandiegensis collected intertidally from Yaquina Point, Oregon. Elvin (1976) found that D. sandiegensis is chemically attracted to Haliclona sp. A. and both are associated in a close grazer-prey relationship. Bloom (1981) found similar amounts of Haliclona sp. A. (Hartman 1975) and Halichondria panicea (Pallas 1766) spicules in the gut contents of D. sandiegensis collected from the intertidal habitats in the San Juan Archipelago. Goddard (1984) found that Haliclona sp. A. is the primary sponge prey of D. sandiegensis from Cape Arago,

Oregon. McDonald and Nybakken (1991) documented instances of D. sandiegensis feeding on thirteen genera of sponges.

However, the grazer-prey relationship between D. sandiegensis and Haliclona sp.

A, found by Elvin (1976) in Oregon, was not found on the shores of San Mateo County,

California (Bertsch et al. 1972). In addition, Neopetrosia problematica (de Laubenfels

1930) is collected from the subtidal habitat in Monterey Bay and regularly fed to those D. sandiegensis kept at the Monterey Bay Aquarium (personal communication by Brent

Grasse, Monterey Bay Aquarium).

Diaulula sandiegensis is a typical nudibranch of the family Dorididae. It has an oval body, the dorsum is highest along its midline and slopes gradually to the margins, and it can grow to twelve centimeters in length (Behrens& Valdés 2001). D. sandiegensis has dorsal caryophyllidia (small spiculate papillae) and six tripinnate branchial leaves and the shape of the radular teeth are hamate (hook-shaped) and smooth

(Behrens& Valdés 2001). D. sandiegensis possesses an annual life cycle (Goddard

1984).

In the coastal intertidal habitat, D. sandiegensis is found on rocky shores. It is common at mid tide levels and less common at high and low tide levels. It is usually located under ledges and on or near the sides of large boulders (Goddard 1984; Ricketts et al. 1985). Diaulula sandiegensis in coastal subtidal habitats are found from the low intertidal to a depth of thirty-five meters (Morris et al. 1980). Diaulula sandiegensis in bay habitats are found on the sides of floating docks and in subtidal rocky areas.

Diaulula sandiegensis is an ideal organism for the study of phenotypic variation, genetic differentiation, and reproductive compatibility. D. sandiegensis is common in the subtidal and intertidal habitats of the open coast and in bays throughout its range.

Diaulula sandiegensis seems to exhibit variation in dorsal spotting pattern with latitude and habitat. In addition, individual populations exhibit differing amounts of variation.

Diaulula sandiegensis is hardy and can survive in laboratory conditions in an open circulating seawater system for up to one year (personal observation). Diaulula sandiegensis readily mate and lay eggs in the lab. The prey sponge Haliclona sp. A is readily available from intertidal habitats and floating docks in Humboldt Bay. These nudibranchs stay close to their prey sponge in the field for an extended period of time, allowing the same individuals to be located repeatedly throughout the year (personal observation).

Since D. sandiegensis has a planktotrophic larva that spends up to four weeks feeding in the water column before settling (Goddard 2004), I predicted that D.

sandiegensis would have a high dispersal potential, which may result in high connectivity, high gene flow, and low genetic differentiation between populations. With high gene flow, D. sandiegensis would generally be predicted to have phenotypic plasticity, with traits that respond to local environmental cues.

The purpose of this study was to investigate variation in the dorsal spotting pattern of D. sandiegensis and to determine whether D. sandiegensis is a complex of unrecognized species. This was accomplished with (1) field surveys as well as surveys for images on the World Wide Web to describe and quantify the latitudinal variation of

D. sandiegensis dorsal spot numbers; (2) a common garden experiment in the laboratory to investigate the effects of growth and diet on the dorsal spotting pattern of two populations of D. sandiegensis (one from a coastal, subtidal population in Monterey Bay and the other from a coastal, intertidal population at Crescent City) to determine if this pattern is genetically fixed or environmentally plastic; (3) field surveys as well as surveys for images on the World Wide Web to investigate the presence of distinct morphotypes distinguished by spotting pattern; (4) light and scanning electron microscopy to examine

D. sandiegensis radulae; (5) field surveys as well as surveys for images on the World

Wide Web to describe and quantify the variation in spot pattern of D. sandiegensis

morphotypes along the Pacific Coast from California to British Columbia; (6) a common garden experiment in the laboratory to investigate the survival and growth of both the D. sandiegensis population from the Monterey Bay subtidal habitat and the D. sandiegensis

population from the Crescent City intertidal habitat, when fed the intertidal sponge

Haliclona sp. A; and (7) a mating study and genetic analysis, using mtDNA sequences of the COI gene to investigate the reproductive compatibility between distinct morphs.

METHODS

Dorsal Spot Number Relative to Latitude

Fully nested hierarchical ANOVA models were used to determine the latitudinal variation of D. sandiegensis dorsal spot numbers.

Data acquisition

Four hundred and twenty-six D. sandiegensis were located and photographed

during field surveys specifically conducted for this project between San Diego,

California, and Bamfield, British Columbia, from 2009 through 2011 (Figure 2). Two

hundred and twenty-five total hours were spent searching coastal intertidal habitats at

thirteen sites. One location was searched in the coastal subtidal habitat of Monterey Bay

with four dives using SCUBA. Diaulula sandiegensis were found at three sites in

Humboldt Bay; two sites subtidally on floating docks and one site intertidally on ballast

rock.

A Nikon Coolpix® P3 995 camera with Ikelite underwater housing was used to

photograph each D. sandiegensis, with a ruler for scale. The area surrounding each D. sandiegensis was photographed to document substrate type and sponge association

(Photographs available to the public on the World Wide Web: keywords are Diaulula and

Kelly). With the help of the scientific community, field sites were selected from locations known to have D. sandiegensis.

Images of D. sandiegensis were obtained from the scientific, SCUBA diving, and tidepooling community, with permission, via photographic records hosted on the World

Wide Web. Photographs from the World Wide Web were included in the analysis only if the photographer’s name, date, and specific location of the photograph were given.

Images from the World Wide Web made up five sites within the study area. Images from the World Wide Web were used only in analyses that did not require the measurement of nudibranch length due to the lack of scale.

Data analysis

Rhinophore to gill length was measured and dorsal spot number counted of each

D. sandiegensis using the computer imaging software program ImageJ (National Institute of Mental Health). The rhinophore to gill length of each nudibranch, measured from the center of the rhinophores to the center of the gills, was used to represent individual size.

This measurement was used instead of total length because it factored out the variation due to mantle-skirt curling. This allowed nudibranchs to be measured in situ.

Photographs showing at least one lateral half of the nudibranch were needed for this analysis, because at times the whole dorsal surface could not be photographed.

Diaulula sandiegensis are sometimes found stuck in cracks and crevices or mating which obscured part of the dorsal surface. A paired t-test showed that the spots on each lateral side of the body were not significantly different (right-left comparison: n = 25, P-Value =

0.245).

The nested ANOVA model consisted of two regions (northern and southern), six

field sites for each region, and twelve nudibranchs for each field site. Of the twenty-one sites within the study area, twelve sites had twelve or more D. sandiegensis. A random sample of twelve D. sandiegensis was chosen from each site for the nested ANOVA at all sites but Bamfield, British Columbia, which had only twelve D. sandiegensis.

Four nested ANOVA models on log-transformed data were used to determine if dorsal spot number varied by latitude among populations within the study area, using the

computer software program Minitab. The first nested ANOVA model was used to

compare dorsal spot number variation between twelve field sites. The second nested

ANOVA model was used to compare D. sandiegensis dorsal spot number variation

between the northern and southern regions. A third and fourth nested ANOVA model

compared D. sandiegensis dorsal spot numbers between field sites specifically within

each of the regions. This nested ANOVA model accounts for the correct degrees of

freedom. In addition, a nested ANOVA partitions the variance into different levels:

between regions, within regions, between field sites, and within field sites.

BC Barkley Sound Puget Sound

Newport OR

Crescent City Trinidad Humboldt Bay N Fort Bragg

Bodega Bay ↑

Pescadero CA Monterey

Habitat Morro Bay Coastal subtidal Channel Islands Coastal intertidal Los Angeles Long Beach Bay San Diego

Pacific Ocean

Figure 2. Distribution of field sites, with habitat and source of photographs indicated. Filled shapes indicate D. sandiegensis photographed by the author or colleagues. Open shapes indicate D. sandiegensis photographs solicited from the

World Wide Web. Diaulula sandiegensis were collected for a laboratory common garden experiment from Crescent City and Monterey, California.

Change in Spotting Pattern over Time

One laboratory and two field studies were conducted to investigate whether

spotting pattern changed over time: (1) 36 Diaulula sandiegensis were followed over

time in the field, (2) 169 D. sandiegensis found in the field were used to investigate

dependence of dorsal spot number on length of nudibranch with a linear regression, and

(3) 28 D. sandiegensis were collected from two separate populations and observed over

time in the laboratory.

Data acquisition - field investigations

From the 426 D. sandiegensis found over the course of this study, thirty-six individuals (thirty-two MS and four FS morphs) were found repeatedly. These 36 D. sandiegensis were followed over the course of two to ten weeks during the years 2009,

2010, and 2011 at three sites in Trinidad, California. Each of these individuals had a unique spotting pattern, which allowed individuals to be identified.

To investigate the relationship between nudibranch size and dorsal spot number,

169 D. sandiegensis found in the field during the 2009 field season were used in a linear regression, using the computer software program Minitab.

Data acquisition – laboratory common garden experiment

A common garden experiment was conducted to investigate changes in D.

sandiegensis dorsal spotting pattern over time. Twenty-eight D. sandiegensis were collected from two populations (the coastal subtidal population of Monterey Bay and the coastal intertidal population of Crescent City) and transported to the Telonicher Marine

Laboratory in Trinidad, California (Figure 2). The D. sandiegensis collected from both

populations were held in a common laboratory environment where all nudibranchs were

fed the same food regime.

Fourteen D. sandiegensis were collected from Shale Reef in Monterey Bay (GPS

coordinates N36 36.531 W121 52.786). This site was accessed by boat. Nudibranchs

were collected using SCUBA at a depth of about fifteen meters on May 18, 2010. These

D. sandiegensis were transported to the Telonicher Marine Laboratory in Trinidad,

California, within twenty-four hours of collection in a large ice chest of ocean water. The

water was aerated with air stones. The water was kept at a temperature between ten and

thirteen degrees Celsius. To chill the water in the ice chest, a fraction of the water was

removed from the ice chest, chilled separately, and then returned to the ice chest containing the nudibranchs. With this method the temperature could be easily controlled within a few degrees.

Each nudibranch was held in a separate container within the ice chest to prevent mating and to separate feces. Each separate container had holes covered with a plastic screen with mesh size large enough to allow for water circulation but small enough to contain feces. Feces were collected from seven of the fourteen separated individuals before they were added to the common garden experiment at the Telonicher Marine

Laboratory, where they were fed Haliclona sp. A.

The second collection site was at Endert’s Beach, Crescent City, California, where fourteen individuals were obtained intertidally on May 29, 2010. All D. sandiegensis collected from Crescent City were found on or within six centimeters of

Haliclona sp. A. The Crescent City D. sandiegensis were transported to the Telonicher

Marine Laboratory in Trinidad, California, within five hours of collection using the same

methods used for Monterey Bay D. sandiegensis.

Laboratory set up

The twenty-eight D. sandiegensis collected from the field included juveniles

(starting at 1mm) as well as adults. These were kept in an aquarium with a closed sea

water system at the Telonicher Marine Laboratory. Water was aerated with air stones

and the temperature was kept at eleven degrees Celsius with a chiller. Eleven degrees

Celsius was suitable for D. sandiegensis collected from both the Monterey Bay subtidal

population and the Crescent City intertidal population. The temperature of Monterey Bay water at the depth of D. sandiegensis collection (about fifteen meters) ranges from nine to

twelve degrees Celsius (U.S. Integrated Ocean Observing System - Central and Northern

California Ocean Observing System). The coastal surface water temperatures of northern

California during the spring and summer months ranges from ten to fifteen degrees

Celsius (National Oceanic and Atmospheric Administration - National Oceanographic

Data Center).

Nudibranchs were held in pairs in smaller containers within the larger tank, either

in similar or dissimilar morph pairs. The nudibranchs were held in pairs in an attempt to

cross the adults from these two populations and raise the offspring in identical conditions

to test whether the resulting spotting pattern corresponded more with parent spotting

pattern or with environment. Unfortunately, the planktotrophic larvae survived in the

laboratory for up to 24 days but died before metamorphosis.

The two populations of D. sandiegensis were fed an ad libitum supply of sponge

collected from the floating docks in Humboldt Bay, California. The sponge consisted

mostly of Haliclona sp. A, an intertidal sponge not common in the Monterey Bay subtidal

habitat (Lee et al. 2007). The diet offered to the nudibranchs also included small

amounts of Halichondria bowerbanki (Burton,1930) and Halichondria panicea (Pallas,

1766). These three sponge species grew side by side on the docks in Humboldt Bay.

When scraped off the sides of docks the small pieces of the other sponges were

inadvertently mixed in with Haliclona sp. A.

Each individual was photographed, using a set scale for measurements, once

every other week over the course of eighteen weeks.

Data analysis

Photographs of each of the thirty-six individuals followed in the field were used

to track changes of individual dorsal spotting pattern. Rhinophore to gill length, dorsal

spot number, and spot sizes were measured, using the computer imaging software

program ImageJ (National Institute of Mental Health), for each nudibranch each time the

individual was found and photographed.

Secondly, 169 D. sandiegensis found during the 2009 field season were used to generate a linear regression. This analysis was used to investigate dependence of dorsal spot number on nudibranch length.

Thirdly, photographs of each of the twenty-eight individuals observed in the common laboratory environment, over the course of eighteen weeks, were used to track changes of individual dorsal spotting pattern. Rhinophore to gill length, dorsal spot number, and spot sizes were measured, using the computer imaging software program

ImageJ.

Distinct Morphotypes

Two studies were used to examine variation in spotting pattern morphotypes. (1)

Images collected from field investigations and the World Wide Web were used to identify characteristics that are distinct for each morphotype. (2) Light and a scanning electron microscopy were used to examine D. sandiegensis radular teeth.

Data acquisition

Three hundred and thirty-seven images of D. sandiegensis collected during the

2009 and 2010 field season (see first section on data acquisition – field investigations) and from the World Wide Web were used for this analysis.

Radular teeth were removed from six nudibranchs, three from the MS and three

from the FS morph.

Data analysis

A histogram was used to examine the spread of dorsal spot number for 320 D.

sandiegensis. A T-test on log-transformed data was used to determine if the total dorsal

spot number differed between D. sandiegensis with mantle-skirt spots and those without.

Chi-Square Tests were used to examine the association between presence or absence of

mantle skirt spots and (1) dorsal spot type, (2) the presence or absence of an opaque

white ring surrounding dark spots, and (3) the background color of the dorsum.

D. sandiegensis characteristics were categorized to identify distinguishing

characteristics that could be used to separate morphotypes. Dorsal spotting pattern

characteristics, of 337 D. sandiegensis, were categorized by (1) presence or absence of

mantle skirt spots; (2) total dorsal spot number (greater or less than 35 spots); (3) dorsal

spot type (solid or ringed); (4) the presence or absence of an opaque white ring surrounding dorsal spots; and (5) the background color of the dorsum (white, light brown, dark brown, or orange). A spot was categorized as a ring if the dark outer ring of the spot was well defined and the color in the center of the ring matched the background color on

the dorsum. A nudibranch was categorized as a “ring bearer” if at least one ringed spot was present. A spot was categorized as a solid spot if the center color was darker than the background color.

Differences in size and shape of nudibranch radular teeth are often used to differentiate species (Hirano 1999). The tooth shape and radular tooth formula were compared for six nudibranchs (three from the MS and three from the FS morph). A D. sandiegensis radula is a ribbon and consists of many rows of teeth. Each row consists of a central tooth (if present), and on both sides of the central tooth, there are one or more lateral teeth, and beyond the lateral teeth, one or more marginal teeth. The arrangement of teeth of the longest row is expressed in a radular tooth formula.

Morphotypes of Diaulula sandiegensis Relative to Latitude and Depth

Morphotype frequencies from different populations were compared across three habitats to describe and quantify the latitudinal and depth variation of D. sandiegensis morphotypes throughout the study area.

Data acquisition

The same photographs of four hundred and thirty-three individual D. sandiegensis

obtained from field investigations and the Word Wide Web were used to determine the

distribution of morphotypes across different habitats from San Diego, California, to

Barkley Sound, British Columbia. Photographs showing only a partial dorsal surface

could be used for this analysis on the condition that the morphotype could be identified.

Data analysis

To determine the distribution of D. sandiegensis morphotypes, frequencies from

twenty-one different populations throughout the study area were compared across three

habitats: (1) coastal subtidal, (2) coastal intertidal, and (3) bay habitats.

Sponge Use Variation

Three studies were conducted to determine whether each morphotype preys on different sponges: (1) a field investigation of sponge association, (2) a fecal analysis of

freshly collected morphotypes, and (3) a laboratory common garden experiment to

24 investigate growth and survival when primarily fed a single, common sponge Haliclona sp. A.

Data acquisition - field investigation/fecal analysis

Indications of feeding in the field include the presence of ragged edges and sections or paths of sponge removed from rock in proximity to nudibranchs (Figure 1).

Sponge species were identified by internal and external structures according to Lee et al.

(2007). To identify sponges, skeletal structure and spicules were examined with light and scanning electron microscopes.

D. sandiegensis is routinely collected for display at the Monterey Bay Aquarium

(Bret Grasse, Monterey Bay Aquarium, personal communication). Neopetrosia problematica is the primary sponge fed to D. sandiegensis at the aquarium and is collected from the subtidal habitat of Monterey Bay. Bret Grasse provided samples of N. problematica, for the sponge analysis.

Observations of D. sandiegensis and sponge association were difficult in subtidal habitats; therefore, a fecal analysis was conducted for the fourteen D. sandiegensis collected from the subtidal of Monterey Bay. Spicules from prey sponge remain intact during digestion and can be found in the feces of dorid nudibranchs (Bloom 1981). After collection from Monterey Bay, all fourteen D. sandiegensis were isolated in separate containers. Feces were recovered from seven D. sandiegensis. Spicules from feces were then isolated and identified.

Data acquisition – laboratory common garden experiment

The common garden experiment was also used to compare growth and survival

between two D. sandiegensis allopatric populations, the 14 FS morph collected from the subtidal habitat of Monterey Bay and fourteen of the 14 MS morph collected from the intertidal habitat of Crescent City. Both morphotypes were fed, ad libitum, on the intertidal sponge Haliclona sp. A, collected from Humboldt Bay.

Data analysis

A Chi-Square Test was used to investigate whether the FS morph used Haliclona

sp. A as a food source, as did the MS morph within the coastal intertidal habitat.

Specifically, to determine if the FS morph would be found on or within six centimeters of

Haliclona sp. A just as often as the MS morph in the coastal intertidal habitat.

Survival and growth were measured as a proxy for D. sandiegensis fitness.

Survival of each morphotype, during the common garden experiment, was compared

using a Kaplan-Meier survival analysis. An ANCOVA was used to compare the growth

of each morphotype, during the common garden experiment. Data were square root

transformed prior to analysis to better meet assumptions of normality. D. sandiegensis

that decreased in length during the study produced negative length values. Negative

length values were transformed by adding the maximum length lost, 0.38 cm, to all D.

sandiegensis length values, making all length values positive for the ANCOVA. The

resulting formula is ( ( ( ) + 0.38 )). The computer software program

Minitab were used for� both퐿푒푛푔푡 analysesℎ 푐푚. 푐푚

Reproductive Compatibility of Morphotypes Using Mating Studies

One field and one laboratory study as well as a genetic analysis, were conducted to determine whether D. sandiegensis morphotypes are reproductively compatible.

Data acquisition – field study

To investigate reproductive compatibility between D. sandiegensis morphotypes in their natural environment, mating events were observed and photographed in the field.

Mating events were also captured on film by the scientific, SCUBA diving, and the tidepooling community and posted onto the World Wide Web. These observed mating events showed whether pairing occurred between similar or dissimilar morphs.

Data acquisition – laboratory mating study

Mating studies were conducted at the Telonicher Marine Laboratory to investigate

D. sandiegensis mating in a controlled environment. D. sandiegensis were collected from the coastal intertidal habitat at Crescent City and Trinidad, California, and from floating docks of the bay habitat in Humboldt Bay in the fall of 2010 and again in the summer of 2011. Ten D. sandiegensis, equal numbers of each morphotype, were collected each year. These D. sandiegensis were kept in aquaria with an open circulating seawater system to be used only for the mating study.

Laboratory set up

For each trial, equal numbers (from six to ten) of each morphotype were placed together in one aquarium. Nudibranch behavior was documented with sequences of

photographs captured with a Nikon Coolpix® 995 camera and an attached Digisnap

2000® intervalometer (Harbortronics). MS morphs and FS morphs were placed together

in the aquarium giving them a choice of mating partner. Six trials were conducted: three

trials in 2010 and three trials in 2011. A different group of D. sandiegensis were used each year. A seventh no-choice trial was conducted with four dissimilar-morph pairs, each in their own aquarium; thus, each D. sandiegensis did not have a choice of mating partner.

Between trials, each nudibranch was held in a separate container with an open circulating seawater system for at least two weeks. These D. sandiegensis were fed an ad libitum supply of sponge. Feeding was suspended during each photographing session in all but the first trial, because feeding behavior made it difficult to differentiate between feeding and mating behavior. The time interval between pictures was 60 seconds.

Length of each session was recorded.

Data analysis

Diaulula sandiegensis mating events and the morphotypes of each partner were identified from the photographs taken during field investigations. A mating event was defined as two individuals that had stopped moving, were positioned right side to right side, and the mantle skirt of both was pushed up exposing the white underside of the mantle. Diaulula sandiegensis are simultaneous hermaphrodites with reciprocal sperm transfer. For the mating events captured with photographs, it was impossible to confirm

sperm transfer, but past observations, by the author, always showed copulation between

nudibranchs when displaying the aforementioned behavior.

Diaulula sandiegensis mating events, morphotype of partners, and premating

behaviors were identified from time-lapse movies created from the sequence of photographs captured in the lab. QuickTime® was used to create the time-lapse movies.

Phylogenetic Analysis Using COI

Data acquisition

Tissue samples were collected from D. sandiegensis populations from Monterey

Bay, Pescadaro, Trinidad, Humboldt Bay, and Crescent City, California. A small clip of

the mantle edge was sufficient and animals were not sacrificed. Tissue samples were

preserved in 95 percent ethanol.

Two D. sandiegensis COI sequences were obtained from an outside source for

this evolutionary analysis. One sequence was obtained from Shields et al. (2009)

GenBank: GQ292030.1. The other sequence was obtained from Dr. Angél Valdés

(California State Polytechnic University, Pomona). A sequence from the dorid

nudibranch Peltodoris nobilis (MacFarland, 1905; GenBank accession: HM162684 658)

was used as an outgroup (Pola& Gosliner 2010).

DNA extraction, PCR, and sequencing

DNA was extracted from tissue samples using Qiagen DNeasy® Tissue Kits

according to the manufacturer’s instructions, and the CTAB extraction protocol as

described by Stewart and Via (1993). For three samples, genetic analysis was carried out at the Humboldt State University Genetics Laboratory. A 674-basepair fragment of mitochondrial DNA COI was amplified, for three samples, with the “universal” primers used for metazoan invertebrates (Primer pair: LCO1490: 5’-

GGTCAACAAATCATAAAGATATTGG-3’ and HC02198: 5’-

TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994). The COI fragment was amplified through PCR (25μl reactions) using the following cycling conditions: melting at 95°C for 90 seconds; 40 cycles of annealing at 50°C for 90 seconds; elongation at 72°C for 45 seconds; and final extension at 72°C for 7 minutes. For eleven samples, genetic analysis was carried out at the Conservation Genetics Laboratory at San

Jose State University. A 712-basepair fragment of mitochondrial cytochrome-c oxidase I

(COI) DNA was amplified, for these eleven samples, and primers were designed considering a consensus of COI sequence of multiple nudibranchs (Forward: OpCOIf –

5’-GTCTTTTTAGGTATGTGATGTGG-3’ and Reverse: OpCOIr1 – 5’-

CAGCAGGATCAAAGAANCTDG-3’) (Shields et al. 2009). The COI fragment was amplified through PCR (25μl reactions) using the following cycling conditions: melting at 95°C for 30 seconds; 35 cycles of annealing at 52°C for 30 seconds; elongation at

72°C for 45 seconds; and final extension at 72°C for seven minutes.

PCR products of the fifteen samples were purified and sequenced bidirectionally on an ABI capillary sequencer by Sequetech, Mountainview, California. Sequences were aligned using ClustalW in MEGA5 (Tamura et al. 2011).

Data sequence analysis

The analysis involved seventeen nucleotide sequences. Evolutionary analyses

were conducted in MEGA5. The evolutionary distances between sequences and standard

error estimates were computed using the Kimura 2-parameter distance model. Sequences were trimmed at the ends to remove sites that were not represented across all samples, obtaining a total of 406 positions in the final dataset.

Relationships among the COI sequences were shown with a Neighbor-Joining tree with node confidence estimated via bootstrapping (1000 replicates) (Felsenstein 1985).

RESULTS

Dorsal Spot Number Relative to Latitude

Dorsal spot numbers were significantly different between twelve field sites in the coastal subtidal and intertidal habitat throughout the study area (Nested ANOVA:

F(11,132) = 64.05, P-Value < 0.0005, R² = 84.22). Dorsal spot numbers differed more

between populations than within populations (Variance components: between sites 1.49, within sites 0.28). Luffenholtz Beach in Trinidad, California was one exception, having more variation within this site than between sites.

Dorsal spot numbers were significantly different between the northern and

southern regions (Nested ANOVA: F(1,10) = 69.66, P-Value < 0.0005, R² = 84.22).

Variation for dorsal spot number was higher between regions than within regions and

within sites (Variance components: between regions 2.40, within regions 0.19, and within sites 0.28). This suggests that dorsal spot numbers differed more between regions than within regions.

In addition, there is considerable variation of D. sandiegensis dorsal spot numbers

between field sites within the southern region. Dorsal spot numbers were significantly

different between sites (Nested ANOVA: F(5,66) = 6.05, P-value < 0.0005, R² = 31.43)

with a higher variation within the southern sites than between sites (Variance

components: between sites 0.096 and within sites 0.229).

In the northern region, dorsal spot numbers were significantly different between

sites as well (Nested ANOVA: F(5,66) = 10.73, P-Value < 0.0005, R² = 44.83), with a

32 higher variation within the northern sites than between sites (Variance components: between sites 0.28 and within sites 0.34). This suggests that dorsal spot numbers also differed more within sites than between sites within the northern region.

Change in Spotting Pattern over Time

Results of the laboratory and field common garden experiments showed that each morphotype maintained its dorsal spotting morphology regardless of experimental conditions. All the FS morphs maintained a fixed number of dorsal spots and all the MS morph individuals from the field and from the laboratory continued to add spots on the edge of the mantle skirt as they grew, with no loss of preexisting spots. The spots of both morphotypes increased in size, but did not change type as the nudibranchs grew. These investigations suggest that D. sandiegensis dorsal spotting pattern is genetically fixed.

However, due to the difficulties of raising D. sandiegensis larvae through metamorphosis, any possible maternal and previous diet effects on dorsal spotting pattern of field- collected D. sandiegensis could not be eliminated.

Laboratory common garden experiment

D. sandiegensis collected from the Crescent City intertidal population had spots that completely covered the dorsum including the mantle skirt and all the way to the edge of the mantle skirt. Spot numbers ranged from 25 to 225. Spot type was exclusively solid. Background color was dark brown. Dorsal spots appeared at the edge of the mantle skirt of all fourteen nudibranchs as they grew. For example, the smallest MS

morph collected over the course of 102 days grew from 0.93 to 3.22 cm and spot number increased from 54 to 90 spots (Figure 3A). Similar results were seen with the other

thirteen MS morphs. This growth pattern seems to maintain the presence of small spots

at the edge of the mantle skirt, while the spots in the center of the dorsum become larger

as the nudibranch grows.

D. sandiegensis collected from the Monterey Bay subtidal habitat population had

spots only on the center area of the dorsum and no spots on the mantle-skirt. Individuals

had either solid or ringed spots or both types. Spot numbers ranged from six to

seventeen. Background color was white. Spot type and spot number for all fourteen

individuals stayed the same with an increase in body length. For example, a FS morph

over the course of 61 days grew from 1.77 to 3.89 cm and spot number stayed the same

with 8 spots (Figure 3B). Similar results were seen with the other thirteen FS morphs.

Field investigations

A field investigation was also conducted to determine if the dorsal spotting

pattern changed over time. Thirty-six D. sandiegensis (four FS morphs and thirty-two

MS morphs) were repeatedly found and photographed, over the course of two to ten weeks, at Luffenholtz Beach, Palmer’s Point, and Houda Point in Trinidad, California.

One individual was found nine times over 72 days.

The results from this study show that each morphotype maintained their initial spot morphology in their natural habitat, also suggesting that there is a genetic basis to the D. sandiegensis dorsal spotting pattern. The results from this study also show that the

Figure 3. Growth study in a controlled laboratory environment. (A)

Growth of a MS morph for 102 days, from June 14, 2010 to September 23,

2010. Length increased from 0.93 to 3.22 cm. Spot number increased from 54 to 90 spots. Black circles identify the same spots. The spots increased in size but the unique spotting pattern stayed the same. (B)

Growth of FS morph for 61 days, from May 21, 2010 to July 21, 2010.

Length increased from 1.77 to 3.89 cm. Spot number stayed the same with

8 spots (one spot is hidden behind gills). Scale bar equals one centimeter.

MS morph increased spot number with an increase in length, whereas the FS morph did

not. For example, the smallest MS morph encountered, over the course of 69 days, grew

from 0.72 to 2.76 cm and increased spot number from 52 to 97 (Figure 4A). Similar

results were seen with the other thirty-two MS morphs. In contrast, the smallest FS

morph encountered grew from 0.38 cm to 1.71 cm over the course of 44 days, but spot

number remained at seven (Figure 4B). Similar results were seen with the other three FS

morphs.

In addition, a field survey of 169 individuals showed that there is evidence for a

dependence of dorsal spot number on nudibranch length in the MS morph. Dorsal spot

number increases with length (Regression: R² = 48.3, N = 106, P < 0.0005) (Figure 5).

There is no evidence for a dependence of dorsal spot number on nudibranch length of the

FS morph. Dorsal spot number does not increase with length (Regression: R² = 1.4, N =

63: P= 0.349) (Figure 5).

Distinct Morphotypes

Three hundred and thirty-seven images of D. sandiegensis collected during the

2009 and 2010 field season and from the World Wide Web were used for this analysis.

The total number of spots that make up the pattern on the dorsum of D. sandiegensis varied from zero to 234. A histogram of dorsal spot numbers of 320 D. sandiegensis showed a bi-modal distribution (Figure 6). However, slight overlap of dorsal spot number existed between morphotypes. The range of overlap is 23 to 41 dorsal spots.

Figure 4. Growth Study at Luffenholtz Beach, Trinidad, California. (A)

Growth of MS morph for 69 days, from April 5, 2009 to June 12, 2009.

Length increased from 0.72 to 2.76 cm. Spot number increased from 52 to

97. (B) Growth of FS morph for 44 days, from April 27, 2009 to June 9,

2009. Length increased, from 0.38 to 1.71 cm. Spot number stayed the same with 7 spots. Scale bar equals one centimeter.

250 Nudibranch Variety Few-Spotted N=63 Many-Spotted N=106 200 s t o p S

a 150 s r o D

f o 100 r e b m u

N 50

0 0 1 2 3 4 5 6 7 8 9 Nudibranch Length (cm)

Figure 5. Dorsal spot number as a function of D. sandiegensis length, in centimeters, of

169 individuals found and photographed during the 2009 field season within the study area (British Columbia to southern California).

70 Mantle-Skirt Spots Present (Length < 1cm)

60 Mantle-Skirt Spots Present (Length ≥ 1 cm)

50 Mantle-Skirt Spots Absent

20 Number of Nudibranchs Nudibranchs of Number 10

0 3 9 30 100 Dorsal Spot Number (Natural Log Scale)

Figure 6. Variation of D. sandiegensis dorsal spot numbers grouped by presence or

absence of mantle-skirt spots. A histogram of the dorsal spot number distribution of 320

D. sandiegensis (located during the 2009 and 2010 field season) showed a bi-modal distribution. The D. sandiegensis with mantle-skirt spots were separated by size in this histogram to show that the MS morphs that fall into the range of overlap are the smallest

MS morphs.

A. B.

Figure 7. Diaulula sandiegensis FS morph with a high number of spots from (A) Humboldt County (41 spots) and (B) Pigeon Point, California

(38 spots) (Photograph by Gary McDonald). Both nudibranchs were identified as FS morphs due to lack of mantle-skirt spots.

One percent of the FS morphs and nine percent of the MS morphs fell within this overlap

of dorsal spot numbers. The individuals that have dorsal spot numbers that fall into this

range of overlap are the smallest MS morphs (fifteen individuals with a length less than

1.2 cm) and the outlier FS morphs (three individuals), such as in Figure 7.

Both morphotypes had spots on the central part of the dorsum. During growth,

these spots increased in size. Arrangement of the largest four spots, in the shape of a

rectangle, is similar across morphotypes. Dorsal spots were distributed over the dorsum,

present all the way to the edge of the mantle skirt, or restricted to the center of the

dorsum with an absence of spots on the mantle-skirt. The MS morph had spots all the

way to the edge of the mantle skirt. New spots appeared at the edge of the MS morph

dorsal surface, keeping the dorsal surface completely covered with spots during growth.

In contrast, the FS morph lacked spots on the mantle skirt and new spots did not appear at

the edge of the mantle skirt on the dorsal surface during growth.

The two peaks in Figure 6 also corresponded to the separation of D. sandiegensis

into two groups defined by presence and absence of mantle skirt spots. Diaulula

sandiegensis with mantle-skirt spots had significantly more spots than those without

mantle-skirt spots, (t-test on log-transformed data, t = -25.80, d.f. = 138, P-Value < 0.005

N=320 nudibranchs). Diaululs sandiegensis without mantle-skirt spots had a mean spot number of eight. Diaulula sandiegensis with mantle-skirt spots had a mean spot number of eighty-two.

The dendogram in Figure 8 distinguishes groups of 337 D. sandiegensis by characters. It shows the variation of characteristics across and within morphotypes.

The results from these field investigations indicated the existence of two distinct

D. sandiegensis morphotypes (the MS and FS morphs), distinguished by presence or absence of mantle-skirt spots, respectively. Other characteristic such as dorsal spot

number, spot type, and dorsal background color generally distinguish the morphotypes, but some overlap of these characteristics exists between morphotypes.

Spot type

Spot type was significantly correlated with presence or absence of mantle-skirt

spots (X² = 185.306, df = 1, P-Value < 0.005). The MS morph had very little variation in spot type: 99 percent of the MS morph population had solid spots and only one individual had a ringed spot. In contrast, the FS morph had much more variation in spot type: 76 percent had ringed spots and 24 percent had solid spots (Figure 8).

The presence or absence of outer rings around dorsal spots was also significantly correlated with presence or absence of mantle-skirt spots (X² = 203.653, DF = 1, P-Value

< 0.005). In this case, the MS morph had more variation in the presence or absence of an outer ring around spots: 83 percent of the MS morph population had an outer light ring around the darker spot and 17 percent did not. In contrast, 99 percent of the population of FS morphs did not have an outer light ring around the darker ringed spot, and only one individual had outer light rings around its ringed spots (Figure 8).

42 BACKGROUND OUTER LIGHT- COLOR RING Dark 59 % Present 84 % Light 38 % SPOT TYPE White 2 % Solid 99 % MANTLE-SKIRT Orange 1 % SPOTS White 86 % Present Light 9 % Absent 16 % Dark 5 %

Ring 1 % Orange (none)

Absent Light (all)

Dark 12 %

Orange 12 % Absent 100 %

Light 22 %

Solid 24 % White 53 %

Light (all) Present 1 % Dark 4 % Absent Orange 7 % Ring 76 % Light 32 % Absent 99 % White 57 %

Figure 8. Dendogram of D. sandiegensis MS and FM morph. This dendogram distinguishes groups of D. sandiegensis by characters. It shows the variation of characteristics across and within morphotypes. Three hundred and thirty-seven D. sandiegensis nudibranchs from the 2009 and 2010 field season and from the World

Wide Web were used for this analysis.

Dorsal background color

Diaulula sandiegensis exhibited considerable variation in dorsal background color. Both morphotypes exhibited a white, light brown, dark brown, or orange dorsal background color (Figure 8). The dorsal background color was significantly correlated with presence or absence of mantle-skirt spots (X² = 111.354, DF = 3, P-Value < 0.005): a large percent of the MS morphs had a dark dorsal color (50 percent dark, 35 percent light brown, and 15 percent white) and a large percent of the FS morphs had a white dorsal color (50 percent white, 40 percent light brown and 5 percent dark).

Commensal scale worm

Ten of the fourteen Monterey Bay FS morph D. sandiegensis collected had a

commensal scale worm present in the pallial cavity (Figure 9). No commensal scale

worms were ever found in association with D. sandiegensis from the northern

populations, however.

Radula

The radular teeth from both morphotypes were hamate (or hook-shaped) and

smooth (Figure 10). The radular formula for the longest row of teeth within the radular

ribbon for both morphotypes ranged from 15.0.15 to 25.0.25 depending on size of

individual. The radular ribbon was bilaterally symmetrical. The teeth were small at the

margins, increased in size at the center of each lateral side, then decreased in size again at

the center. These D. sandiegensis did not have a center tooth. These radular formulas

Figure 9. Commensal scale worm found in pallial cavity of D. sandiegensis collected from the subtidal habitat of Monterey Bay.

A. B. C.

50 µm 100 µm 50 µm

Figure 10. SEM micrographs of D. sandiegensis radula. (A) The MS morph showed no sign of denticles at the outer margin. (B) This image shows one row of teeth on the ribbon of a MS morph. The teeth were smooth and hamate (hook- shaped) that decreased in size from the center of each lateral side to the outer margin. (C) The FS morph teeth were also smooth and hamate.

and teeth descriptions are similar to those found by Behrens & Valdéz (2001) for D.

sandiegensis.

Behrens & Valdéz (2001) found traces of denticles on the marginal teeth of the D.

sandiegensis morphotype commonly known as the “speckled” form and previously identified as Doris (s.l.). There was no trace of denticles on the marginal teeth of the three FS morph and the three MS morph radulae examined for this study, however.

Morphotypes of Diaulula sandiegensis Relative to Latitude and Depth

Morphotype frequencies from different populations were compared across three habitat types to describe and quantify the latitudinal and depth variation of D. sandiegensis morphotypes throughout the study area.

Morphotype frequencies showed a strong correlation to differences in latitude and depth (Figure 11). The FS morph was the exclusive form found south of Fort Bragg

(southern region) in intertidal, subtidal, and bay habitats. In addition, the FS morph was the primary morphotype found in the coastal subtidal habitat throughout the study area.

The MS morph was the primary morphotype found in the coastal intertidal habitat from

Fort Bragg north (northern region). The northern region consisted of eighty-eight percent

MS morphs and twelve percent FS morphs.

Puget Sound, Humboldt Bay, Newport Bay, Alamitos Bay, and Long Beach

Marina are classified as sheltered shores (Ricketts et al. 1985). For this study these field sites were identified as bay habitats consisting of both subtidal and intertidal shores.

C. S. C. I Bay

Barkley Sound (28, 26)

Neah Bay (1) WA Puget Sound (23) N Newport (34)

Crescent City (32) OR Trinidad Beaches Trinidad (4, 188) Palmer’s Point (56)

Humboldt Bay Indian Beach (17) (29) Fort Bragg (7, 7) Luffenholtz (67)

Houda Point (48) Bodega Bay (2) CA San Mateo County (19)

Monterey (68, 13)

Morro Bay (1) Los Angeles (5, 1)

Channel Islands (32) Many-spotted

Long Beach (3) Few-spotted San Diego (16, 1) Pacific Ocean

Figure 11. Geographical and habitat pattern of D. sandiegensis from the coastal subtidal (C.

S.), coastal intertidal (C. I.), and bay (Bay) habitats from Barkley Sound, British Columbia, to

San Diego, California. Pie charts indicate frequencies of morphotypes. Sample sizes depicted in bold next to sample location (subtidal, intertidal, and bay). Four hundred and thirty-three D. sandiegensis from the 2009, 2010, and 2011 field season and images from the

World Wide Web were used for this analysis.

More bay habitat study sites are needed to confirm bay morphotype frequencies.

However, the MS morph seems to be the primary morphotype found within Puget Sound.

The FS morph is the primary morphotype found within Humboldt Bay.

Sponge Use Variation

Field investigations showed that D. sandiegensis from coastal intertidal habitats fed on Haliclona sp. A. The two most common sponges found in the same coastal intertidal habitat as D. sandiegensis were Haliclona sp. A and H. panicea (personal observation). Sponge characteristics used for identification are listed in Figure 12 and

Table 1. Fifty-two percent of D. sandiegensis in the intertidal habitat from Monterey to

Barkley Sound were found on or near sponge. Of the fifty-two percent of D. sandiegensis found on or within six centimeters of sponge, ninety-five percent were found on or near Haliclona sp. A. One was found on Ophlitaspongia pennata (Lambe

1895), two were found on a yellow sponge whose identification was difficult (and may have been H. panicea), and two (FS morphs) were found on a white sponge (Table 2).

Fecal analysis indicated that D. sandiegensis (FS morph) collected from the subtidal habitat of Monterey Bay fed on N. problematica. Feces were recovered from seven D. sandiegensis FS morphs collected from subtidal habitats in Monterey Bay using

SCUBA. The primary spicule found in the feces of these seven nudibranchs was a strongyle with mucronate ends (Table 3). This corresponded to the primary spicule found in the sponge N. problematica (Table 1).

A. D. G.

1 cm 1 cm 1 cm

B. E. H.

100 µm 100 µm 100 µm

C. F. I.

100 µm 5 µm 10 µm

Figure 12. Sponge external characteristics, spicules, and skeletal structure used for identification of sponges. (A) Haliclona sp. A (with the nudibranch D. sandiegensis) (B) SEM micrograph of Haliclona sp. A. spicules. (C) SEM micrograph of Haliclona sp. A. skeletal structure. (D) H. panicea (with the nudibranch Archidoris montereyensis (Cooper, 1862)), (E) SEM micrograph of

H. panicea spicules. (F) SEM micrograph of N. problematica spicule with mucronate ends. (G) N. problematica (with the nudibranch, D. sandiegensis) photographed at the Monterey Bay Aquarium. (H) and (I) SEM micrographs of

N. problematica spicules with mucronate ends.

Table 1. Spicules found in the skeletons of Haliclona (Haliclona) sp. A, H. panacea, and

N. problematica. These three sponges have only megascleres with no microscleres present.

Sponge Megascleres

Haliclona sp. A Oxeas (with oxeate ends): (Hartman, 1975) 55 – 107 µm x 4.5 – 9.1 µm Halichondria panacea Oxeas (with oxeate ends): (Pallas, 1766) 160 – 301 µm x 3.8 – 10.2 µm Neopetrosia problematica (1) Strongyles to strongyloxea, most with (deLaubenfels 1930) mucronate ends: 160 – 175 µm x 9 - 11µm. (2) Oxeas: 130 – 140 µm x 1.5 – 6 µm.

Table 2. Results of sponge association analysis for D. sandiegensis from the coastal intertidal habitat from Monterey to Barkley Sound during the 2009 field season, showing the numbers of D. sandiegensis found on or near sponge.

Coastal Intertidal Habitat

Many-spotted Few-spotted

Total D. sandiegensis 152 26 Not on or near sponge 70 16 On or with six centimeters of 78 9 Haliclona (Haliclona) sp. A On other sponge 4 1

The FS morph seemed to use Haliclona sp. A as a food source in coastal intertidal

habitats. In coastal intertidal sites the FS morph was found on or within six centimeters

of Haliclona sp. A just as often as the MS morph was found on or within six centimeters

of Haliclona sp. A (X² = 2.289, d.f. = 1, P-Value = 0.130).

The laboratory common garden experiment indicated a higher growth and survival rate for D. sandiegensis collected from the Crescent City intertidal habitat than for the D. sandiegensis collected from the Monterey Bay subtidal habitat, when fed

Haliclona sp. A in the laboratory (Kaplan-Meier survival analysis: Log-Rank method; d.f. = 1, X² = 6.92, P-Value = 0.009; Wilcoxon method; d.f. = 1, X² = 4.40, P-Value =

0.036). The fourteen MS morphs collected from the coastal intertidal habitat at Crescent

City lived in the laboratory for an average of 71 days, with three of the individuals sacrificed at the end of the study (eighteen weeks). The fourteen FS morphs collected from the subtidal habitat in Monterey Bay lived for an average of thirty-two days in the lab, and all the FS morphs died by the eightieth day of the experiment (Figure 13).

In addition, the MS morph grew significantly more than the FS morph during the common garden experiment (ANOVA: F(1,19) = 9.62, P-Value = 0.006, R-Sq = 74.70)

(Figure 14), even though the mean initial length of both morphotypes was not significantly different (t-test: t = 2.03, d.f. = 19, P-Value = 0.057). Four of the FS morphs actually lost length during the experiment.

Table 3. Results from feces analysis of seven D. sandiegensis collected from the subtidal habitat of Monterey Bay, showing, type, length, and width of the primary spicule in feces collected.

Nudibranch Primary Spicule Type Length (µm) Width (µm)

1 Strongyles-Ends mucronate 140 – 160 10 - 12

2 Strongyles-Ends mucronate 140 - 160 10 - 12

3 Strongyloxea 140 - 160 10 - 15

4 Strongyles-Ends mucronate 140 - 160 10 - 15

5 Strongyles-Ends mucronate 140 - 160 10 - 15

6 Strongyles-Ends mucronate 140 - 160 10 - 15

7 Strongyles-Ends mucronate 140 - 160 10 - 15

100 Morphotype Few-Spotted 80 Many-Spotted

60 t n e c r e P 40

0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Survival (days)

Figure 13. The survival of the MS morph and the FS morphs were significantly different when both morphotypes were kept together in a common garden experiment (Kaplan-

Meier survival analysis, Log-Rank method, P-Value = 0.009). The MS morph was collected from the intertidal habitat in Crescent City, and the FS morph was collected from the subtidal habitat in Monterey Bay. Three MS morph survived past the end of the experiment (Eighteen weeks). All FS morphs died by the eightieth day of the experiment.

Reproductive Compatibility of Morphotypes Using Mating Studies

Natural environment

Mating studies in the field revealed assortative mating for both the MS and the FS

morphs. Forty-four mating events were captured in photographs. All forty-four occurred

between individuals of the same morph. Twenty-seven of these events were considered

more informative because they occurred at sites where both morphotypes were found;

therefore, when D. sandiegensis had a choice of mate type they still formed like-morph

pairs (Table 4). Minimum size (length) of mating individuals was 1.2 centimeters.

Controlled environment

Mating studies in the laboratory revealed evidence for assortative mating among

morphotypes. Three hundred and nineteen hours of time-lapse photography showed that when given a choice between morphotypes, 64 out of 65 mating events occurred between like-morph pairs (Table 5). Mating events lasted from a minimum of nine minutes to a maximum of 35.5 hours, with an average time of nine hours.

) 2.0 ) 8

3 Morphotype .

0 Few-spotted +

m Many-spotted c ( 1.5 v

(

e c n e r e f

f 1.0 i D

h t g n e L

0.5 h c n a r b i d u 0.0 N 0 20 40 60 80 100 120 140 Time Between First and Last Measurement (Days) Figure 14. Nudibranch length difference as a function of time between measurements for both morphotypes grown in a common garden experiment. The MS morph grew significantly more than the FS morph (ANOVA: R-Sq = 74.70 percent, F= 9.62, P =

0.006). Four FS morphs actually lost length during the experiment. To transform the negative length values, making them positive for the ANOVA, the maximum length lost,

0.38 cm, was added to all values. The data were also transformed with a square root function to conform to assumptions of normality. The resulting formula is

( ( ) + 0.38 ( ).

� 푙푒푛푔푡ℎ 푐푚 푐푚

Table 4. Mating events among the D. sandiegensis morphotypes at study sites from San

Diego, California, to Bamfield, British Columbia.

Mating Events in the Field Location All Sites Only Sites with Both Varieties Number of 664 320

Nudibranchs FS 308 55 MS 356 261 Total Number of 44 27 Mating Events MS with MS 34 26 FS with FS 10 1

MS with FS 0 0

Table 5. Number of mating events among the D. sandiegensis morphotypes during 319

hours (13.3 days) of time-lapse photography. Six to ten D. sandiegensis of both morphotypes were placed together, in the same aquarium, giving them a choice of mating partner. Sixty-four out of sixty-five mating events were between like-morph pairs.

Time-lapse Movie 1 2 3 4 5 6 Total

Oct Nov Dec Jul Jul Aug Date 2010 2010 2010 2011 2011 2011

Time (minutes) 857.5 828.5 3566.5 4259 2543 7114 19168

Nudibranch group 1 1 1 2 2 2

Number of nudibranchs: 10 9 6 8 9 8

MS 6 5 3 4 4 4

FS 4 4 3 4 5 4

Total Mating events: 2 2 3 19 10 29 65

MS with MA 1 0 1 8 5 16 31

FS with FS 1 2 2 11 4 13 33

MS with FS 0 0 0 0 1 0 1

During the second part of the mating study, 22.3 hours of time-lapse photography of dissimilar-morph pairs, separated into isolated aquaria, showed one mating event occurred between one dissimilar-morph pair. This mating event lasted 2.8 hours (Table

6).

Premating behavior was analyzed for physical cues for mate selection. Each nudibranch traveled around the tank touching the other individuals and sometimes circled one another. During the mating study, individuals were three times more likely to touch other like-morph individuals than to touch dissimilar-morph individuals.

Phylogenetic Analysis Using COI

Analysis of the mtDNA COI sequence data from D. sandiegensis produced two distinct clades, with MS and FS morphotypes as separate groups, with high bootstrap support values (Figure 15). The average pairwise divergence between the MS morph and the FS morph clades (±SE) was 6.8 percent (±0.07 percent) with 0.64 percent (±0.04) divergence within the FS morph clade and 0.99 percent (±0.06 percent) divergence within the MS morph clade (Tables 7). Divergence between Peltodoris nobilis (the outgroup) and the MS and FS morph D. sandiegensis was 21.3 percent (0.09 percent) and 19.2 percent (±0.13 percent), respectively.

Table 6. Number of mating events among the D. sandiegensis morphotypes during 22.3 hours of time-lapse photography in no-choice trials. Four dissimilar-morph pairs were placed in separate aquaria; therefore, they did not have a choice of mating partner. Only one mating event occurred.

Date August 2011

Time (minutes) 1340 (22.3 hours)

Number of mating pairs 4 Mating events 1

fewspot - Humboldt Bay Diaulula sandiegensis - Valdez

61 fewspot - Humboldt Bay fewspot - Monterey fewspot - Monterey

fewspot - Humboldt County fewspot - Humboldt Bay

54 fewspot - Humboldt Bay 97 fewspot - Pigeon Point fewspot - Monterey fewspot - Monterey manyspot - Humboldt Bay 100 53 manyspot - Humboldt Bay 55 manyspot - Humboldt Bay 63 manyspot - Crescent City Diaulula sandiegensis GQ292030 - Shields Peltodoris nobilis HM162684 - Pola

0.02 substitutions / site

Figure 15. Evolutionary relationships of taxa. Phylogenetic tree using neighbor-joining method with mtDNA cytochrome c oxidase I. Fifty percent and higher bootstrap support values are shown at nodes.

Table 7. Average genetic distance of mtDNA COI sequences for within morphs and between morphs of D. sandiegensis and outgroup. Standard error estimates are shown in parentheses and were obtained by a bootstrap procedure in Mega5 (1000 replicates).

FS morph (±SE) MS morph (±SE)

FS morph 0.64 % (±0.04 %)

MS morph 6.78 % (±0.07 %) 0.99 % (±0.06 %)

Outgroup 19.2 % (±0.13 %) 21.3 (±0.09 %)

Peltodoris nobilis

The location or morphotype was unknown for the nucleotide sequence obtained

from Valdéz, but from the COI divergence of sequences in Table 4 it seems to group with

the FS morph D. sandiegensis. Location or morphotype is also unknown for the

nucleotide sequence obtained from Shields (2009), but from the COI divergence of

sequences from Table 4 this sequence grouped with the other MS morph D. sandiegensis.

The FS morph with a high number of spots from Figure 7A is found on the FS morph branch of the Neighbor-Joining tree in Figure 15. This individual had 41 spots and mantle-skirt spots absent. We know from the mitochondrial DNA that the mother of this

individual was a FS morph. This might account for the lack of spots on the mantle skirt.

The morphotype of the father is unknown. If the father was a MS morph, and nudibranch

from Figure 7A was a hybrid, this might account for the intermediate number of dorsal

spots. If the father was a FS morph then the high spot number of this nudibranch is at the

high end and over laps the MS morph spot number. Either way, presence of the FS

morph COI gene on the mitochondrial gene tree, accompanied by the lack of mantle skirt

spots, suggests that absence of mantle skirt spots is a stable characteristic of the FS

morph and supports the use of this characteristic to distinguish the two species.

Dorsal spot number was correlated with presence or absence of mantle-skirt spots

in 98.4 percent of D. sandiegensis. Therefore, dorsal spot number as well as presence or

absence of mantle-skirt spots was used to distinguish morphotypes and to determine range, habitat, and prey variation for each morphotype.

DISCUSSION

Clearly, D. sandiegensis is composed of at least two genetically divergent and reproductively isolated morphotypes that have subtle but distinct differences in morphological characteristics. Field investigations, laboratory investigations, and genetic analysis supports the conclusion that two genetically distinct D. sandiegensis morphotypes (the MS and FS morph) exist, and are easily distinguished in the field by presence or absence of mantle-skirt spots. In addition, adult dorsal spot number, spot type, and background color can be used to distinguish morphotypes, but slight overlap of these characteristics exists between morphotypes.

There were many factors that contributed to the difficulty of recognizing and separating D. sandiegensis into distinct morphotypes. First, D. sandiegensis shows significant intraspecific phenotypic variation, and each morphotype also varies in spotting pattern (Figure 8). In addition, characteristics held in common between morphotypes made it difficult to identify the characteristics that distinguish one morphotype from the other. Common characteristics were the presence of dark brown spots and orientation of the four largest dorsal spots. Furthermore, the overlap of characteristics that exists between morphotypes made it initially difficult to identify the characteristics that distinguish one morphotype from the other. Finally, due to the growth pattern of the MS morph (dorsal spot number increased with size of nudibranch), the small MS morph resembled the FS morph, even though the presence or absences of mantle-skirt spots clearly distinguishes the two (Figure 1).

The results of the field investigations, the laboratory common garden experiment, the mating study, and the phylogenetic analysis support the conclusion that the spotting pattern of D. sandiegensis is genetically fixed and not environmentally plastic.

The pattern of genetic differentiation found for D. sandiegensis is unusual.

Population studies along the west coast of North America have typically shown that species with high dispersal potential have low genetic differentiation, such as Cucumaria miniata (Arndt& Smith 1998) and Strongylocentrotus purpuratus (Flowers et al. 2002).

In addition, species with low dispersal potential have strong population differentiation, including the cup coral Balanophyllia elegans (Hellberg 1994).

However, some fish with high dispersal potential have been found with significant genetic differentiation along the west coast of North America (Buonaccorsi et al. 2002;

Burford 2009; Dawson 2001). Burford (2009) found a separation of two lineages of the blue rockfish (Sebastes mystinus) at Cape Mendocino and Hyde et al. (2008) found a separation of two lineages of the vermilion rockfish (Sebastes miniatus) at Point

Conception.

Furthermore, more recent genetic studies of some marine invertebrate species that have high dispersal potential show significant genetic differentiation along the west coast of North America. The barnacle Balanus glandula has a genetic cline between

Cape Mendocino and Pacific Grove, with 2.2 percent (± 0.4 percent) average pairwise divergence within the COI locus between northern and southern populations (Sotka et al.

2004). Thirteen out of forty-one invertebrates with pelagic development that were

examined from Alaska to Santa Barbara showed significant genetic differentiation (Kelly

and Palumbi 2010).

Once a phylogenetic analysis shows genetic differentiation within a species,

further investigations could possibly determine the mechanisms for differentiation. For

example, a phylogenetic analysis suggests the existence of cryptic species of vermilion

rockfish (Sebastes miniatus) (Hyde& Vetter 2007). Hyde et al. (2008) found strong

support for segregation by depth for the two lineages of Sebastes miniatus, and suggested

that the possible mechanism for this differentiation was a truncation of a bathymetric

ontogenetic migration.

In addition, the existence of cryptic species of the aeolid nudibranch in the genus

Phestilla was suggested from a phylogenetic analysis (Faucci et al. 2007). Faucci et al.

(2007) also found strong support for the specialization of cryptic species in the Phestilla

on different host corals, and suggested that the possible mechanism for this differentiation was a host shift.

Examination of ecological and habitat differences of the two distinct D. sandiegensis morphotypes may help to provide clues why they have diverged. D. sandiegensis morphotype frequencies showed a strong correlation between differences in

latitude and depth. The FS morph was the exclusive form found south of Fort Bragg

(Southern region) in intertidal, subtidal, and bay habitats. In addition, the FS morph was the primary morphotype found in the coastal subtidal habitat throughout the study area.

The MS morph made up eighty-eight percent and the FS morph made up twelve percent

of the population found in the coastal intertidal habitat north of (and including) Fort

Bragg (Northern region).

The absence of confirmed records of the MS morph south of Fort Bragg supports a phylogeographic break for this morphotype between Fort Bragg and Bodega Bay that completely restricts dispersal and/or survival of the MS morph. This break may correspond to the biogeographic boundary at Cape Mendocino described by Valentine

(1966). This phylogeographic break, however, does not completely restrict dispersal of the FS morph to the north.

Genetic differentiation has been found to occur at biogeographic boundaries along the west coast of North America. For example, the sibling species Nucella ostrina and N. emarginata have a genetic divergence near Point Conception (Marko 1998), and

Tigriopus californicus show genetic differentiation separating populations at four of the biogeographic boundaries described by Valentine (1966): Point Conception, Monterey

Bay, Cape Mendocino, and Puget Sound, as well as genetic changes at boundaries not previously described (Edmands 2001).

The biogeographic break at Cape Mendocino, suggested by Valentine (1966), results from patterns in oceanographic conditions with regional changes in ocean currents, temperature gradients, changes in geology, and changes in local geographic features. The southern boundary of MS morphs may also be due to some of these changes in oceanographic patterns across the Cape Mendocino biogeographic break. Temperature could be an important factor controlling the range of the MS morph. The biogeographic break at Cape Mendocino separates warmer water, brought into the Southern California

Bight from the south by the Davidson Current, and colder water brought from the north by the California Current (Valentine 1966).

The observed pattern of “many spotted” morph distribution may in part be due to the separation of surface coastal waters originating from Oregon and southern California creating a barrier to larval dispersal. Surface drogue trajectories used to describe currents and movement of surface waters showed that the surface coastal waters originating from

Oregon and southern California rarely mix (Sotka et al. 2004). Surface drogues released off the coast of Oregon, during a four year period, entered the California Current and drifted primarily south, yet none of them returned to the coast south of Point Arena,

California. This suggests that larvae of the D. sandiegensis MS morph that originate north of Fort Bragg enter the California Current and get transported south of Point Arena, but like the surface drifters, may not return onshore to the intertidal habitat. Since the

MS morph is found almost exclusively in the intertidal habitat and larvae need to find a suitable habitat within their life span (about four weeks), it is unlikely that the many MS morph of D. sandiegensis will survive long enough to settle and metamorphose south of

Point Arena, California.

Examination of the spotting pattern of D. sandiegensis may also provide clues to why they have diverged. Phenotypic variation in color patterning could be due to selection pressures, such as predator pressure, thermal stress, and mate choice (Harris&

Jones 1995; Levins 1968). Vermeij (1978) suggests that the intensity of predation increases with both a decrease in latitude, and from high to low shore elevation. Many marine plants and animals show latitudinal variation in prey defenses (Cronin et al. 1997;

Siska et al. 2002; Stachowicz& Hay 2000; Vermeij 1978). If predation pressure is a

mechanism that affects the geographic distribution of D. sandiegensis, then a latitudinal

variation in prey defenses would be expected.

Perhaps populations of D. sandiegensis separated by a difference in latitude or

depth have developed different strategies as defense against different levels of predation

pressure, which created a divergence in dorsal spot pattern.

The loss of the shell in the evolution of nudibranchs has been compensated with a

wide range of defensive mechanisms to protect against predation. Examples of these

defensive mechanisms include considerable divergence in dorsal color and pattern and the sequestration of secondary metabolites gained from prey (Rudman 1991). Dorsal

color pattern can provide protection in the form of background matching, disruptive

coloration, or warning coloration.

Diaulula sandiegensis MS morphs seem to be cryptic on Haliclona sp. A

(personal observations). The dorsal spots of D. sandiegensis resemble the volcano-like

oscula (or excurrent pores that protrude from the body) of its sponge prey. The dark

spots resemble the dark holes, the light rings resemble the light raised rims of the oscula,

and the distance between spots resembles the distance between oscula (personal observation, paper in prep).

Many nudibranchs resemble their host prey. Cook (1962) noticed that the nudibranch Rostanga pulchra (MacFarland 1905) almost perfectly matches the color and texture of its sponge prey, Ophlitaspongia pennata (Lambe 1895). Nudibranchs of the

genus Corambe can closely resemble the pattern of the bryozoan colony of the genus

Membranipora on which they feed (Gosliner 1990). Marin et al. (1997) found the shape

and color of the nudibranch Paradoris indecora (Bergh 1881) is similar to the sponge

Ircinia variabilis (Schmidt 1862).

In addition, the mantle-skirt spots of the D. sandiegensis MS morph possibly

create a disruptive coloration that obscures the edge of the nudibranch. Disruptive

coloration is thought to optimize the effects of background matching (Cuthill et al. 2005).

The ringed spots of the FS morph lack the dark center and the light outer ring, and thus do not resemble Haliclona sp. A to the same degree as the spots on the MS morph.

The FS morph may have developed cryptic coloration on a subtidal sponge other than the intertidal sponge Haliclona sp. A or developed an aposematic coloration to warn predators of the presence of secondary metabolites gained from its sponge prey.

Dorid nudibranchs, including D. sandiegensis, can remove secondary metabolites

(anti-feedant chemicals) from their prey sponge, and sequester these unchanged, in their own tissues for re-use as an anti-feeding defense of their own (Marin et al. 1997; Rudman

1991). Rudman (1991) suggested that even with anti-feeding chemicals, fish predation plays a major role in influencing selection within the Chromodorid family of nudibranchs.

Secondary metabolites within the skin extracts taken from D. sandiegensis in a northern population are different than those from a southern population (Kubanek&

Andersen 1999; Walker& Faulkner 1981; Williams et al. 1986). However, morphotypes were not distinguished in these studies. Further research is needed to determine if this difference in metabolites is due to a difference in morphotype, latitude, or sponge prey.

It is doubtful that the latitudinal variation in spotting pattern of D. sandiegensis is

due to aposomatic Müllerian mimicry of sympatric groups of similarly colored, but

unrelated, species as described by Rudman (1991) for nudibranchs in southern Australia.

Australia has many more dorid species with much more extensive variation in spotting

pattern than the North American coast dorids. Rudman (1991) found fifty-eight species

in the family Chromodorididae in southern Australia. However, there are only six

species within the suborder Doridina, with a range from Mexico to Alaska, which have a

dark dorsal spotting pattern.

Adaptive differentiation of color pattern in other animals may result from mate

choice (Levins 1968). However, it is unlikely D. sandiegensis spotting pattern is related

to mate choice because the eyes of D. sandiegensis appear to detect only changes in

general light intensity (Barnes 1987).

Divergence in color pattern related to heat absorption could affect species in

intertidal habitats, though this effect has been studied more in terrestrial habitats than the

marine environment (Levins 1968; Michie et al. 2010). Thermal stress significantly

affects the distribution of color morphs in the marine snail Nucella lapillus (Harris&

Jones 1995). Dark individuals may absorb heat more readily than light ones, which could be an advantage in colder environments. In intertidal habitats, D. sandiegensis MS morph had a darker dorsal surface with many dark spots. A large percent of the population had a dark dorsal color (50 percent dark, 35 percent light brown, and 15 percent white) which may absorb heat during low tides when the cold air temperatures of the north could be stressful. In contrast, the FS morph more often had a light dorsal color

(55 percent white, 40 percent light brown and 5 percent dark) and may stay cooler during low tides when the warmer air temperatures of the south could be stressful.

The results of this study suggest that perhaps the sponge use of these two morphotypes is a mechanism underlying differentiation. Fifty-two percent of the MS morphs were found on or near Haliclona sp. A in the coastal intertidal habitat. The results of this study, as well as similar results of feeding studies by Elvin (1976) and

Goddard (1984) support the conclusion that the MS morph feeds almost exclusively on

Haliclona sp. A in the intertidal. In addition, a fecal analysis indicated that D. sandiegensis collected from subtidal sites in Monterey Bay feed on N. problematica. In this study, the D. sandiegensis MS morph survived significantly longer and grew significantly during a common garden experiment where both morphotypes were kept under the same conditions and fed the intertidal sponge, Haliclona sp. A. This evidence seems to suggest that even though the FS morphs can eat Haliclona sp. A in the laboratory, they do not thrive on it.

Further research with a multiple-choice feeding assay is needed to distinguish between local adaptation and phenotypic plasticity of sponge prey use for these morphotypes. A multiple-choice feeding assay should compare growth and survival of each morphotype fed with Haliclona sp. A as well as growth and survival of each morphotype fed with N. problematica. The multiple-choice assay could not be performed in northern California due to the inability to collect N. problematica locally and the difficulty of keeping sponge healthy in a closed circulating seawater system. This type of assay will need to be performed at another location, possibly in central California, where

73 both sponges, Haliclona sp. A and N. problematica, are easily accessible and can be maintained in an open circulating seawater system for successful survival of this sponge.

Alternatively, growth and survival may have differed due to other factors. The extended transportation from Monterey Bay to Trinidad may have affected the health of the FS morph collected from Monterey Bay. A change in temperature (from field temperature to laboratory temperature) may have affected the health of either morphotype, even though both populations were collected from water with approximately the same temperature range. However, the survival of both morphotypes was similar for the first thirty days and then diverged (Figure 17), casting doubt on both of these possibilities.

Understanding the dependence of D. sandiegensis on its sponge prey has implications for evolution, specialization, speciation, and the dynamics of populations.

The connection between D. sandiegensis and its sponge prey may be particularly strong because the sponge not only provides nutrition, but is likely to be a source of defensive chemicals and/or spicules used for protection against predators. Each morphotypes’ host sponge may provide protection in the form of background matching. These morphotypes’ host sponges may also be a source of secondary metabolites or spicules gained from the sponge that are sequestered in the dorsal tissues for self-defense. Further research identifying D. sandiegensis secondary metabolites gained from the sponge prey and the sponge feeding preference of each morphotype may help to understand the contribution of diversifying selection in the separation of these morphotypes into genetically and reproductively isolated species.

The substantial genetic divergence of the morphotypes of D. sandiegensis suggests that future investigations of the genetic structure of each morphotype will help to identify the mechanisms of divergence (Burford 2009). Further information on demographic and evolutionary history will help to determine if these two possible species diverged in sympatry or allopatry, how both may have expanded and contracted in the past, and if one of the species may have been isolated from the other and persisted in a northern refugium during a past ice age similar to other marine species suggested to show northern persistence, such as the acorn barnacle (Sotka et al. 2004) or the marine gastropod Nucella lamellosa (Marko 2004).

In conclusion, differences in morphology, range and depth of occurrence, and reproductive isolation suggest these two morphotypes are separate species. It is likely that such strong genetic differences were generated when D. sandiegensis subpopulations became historically separated. The subpopulations may have diverged by genetic drift or local selection, or a combination of both processes. Diversifying selection for these species could appear in the form of host shifts, dependence on different habitats, divergence of the chemical cues for mate recognition, or divergence in anti-feeding chemicals or spicules obtained from their prey.

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