Na Spp. Along the Central Coast of California

ABSTRi\CT

REPRODUCTION AND GROWTH OF THE CHITON NUTTALL!NA SPP. ALONG THE CENTRAL COAST OF CALIFORNIA

Analyses ofreproductive cycles, larval settlement, recmitment, and growth rates of Nuttallina spp. within a coralline algal mat were conducted in Stillwater Cove, Califomia. Nulfallina kata exhibited discrete spawning events in spring and autumn, whereas N. cal!fomica was reproductive throughout the study period. Within the algal mat, increases in mean density of Nuttallina spp. were related to slight decreases in mean size, indicating Nuttallina spp. may have been at or near its saturation level in the algal mat. Significant recruitment occurred in the algal mat, and the majority of Nuttallina spp. in the algal mat were estimated at less than 2 years old. Larval settlement of N. kata occUlTed in the presence of C. vancouveriensis with and without adult mucus, and in the presence of P. neof'arlowii with adult mucus. Grazing activities of Nutta!lina spp. within the algal mat did not signiticantly limit the growth of fleshy macroalgae.

Michelle Diane White December 1998 REPRODUCTION AND GROWTH OF THE CHITON

NUTTALLJNA SPP. ALONG THE CENTRAL COAST OF CALIFORNIA

by Michelle Diane White

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Marine Science in the School ofNatural Sciences Califomia State University, Fresno December 1998 Copyright© 1998

Michelle Diane White ACKNOWLEDGMENTS

1 would like to thank my committee members Dr. James Nybakken, Dr. Michael Foster, and Dr. Stephen Ervin for their guidance, patience, encouragement, wisdom, and friendship. I have enjoyed working with them and look fmward to continued friendship in the years to come. There are many people at Moss Landing Mmine Laboratories that I would like to acknowledge, but a special thank you goes out to Gail Jolmston, Aldo DeRose, and Joan Parker. It is the combination of students, faculty, and staff that creates a wonderful and unique educational environment. There were many students that helped with my research, including Caren Braby, Jean DeMarignac, Jason Flores, Lori Gant, Satina Giammanco, Michele Jacobi, Korie Johnson, B1ynie Kaplan, Eli Landrau, Mark Pranger, and Kyra Schlining. I was able to convince these students that chitons were cool and that

seeing the sunrise can be a ve1y rewarding experience. 1 also need to thank Stephanie Flora for her lmowledge and patience in answering my endless questions. I owe a huge debt of gratitude to the gang at Granite Canyon, including Brian Anderson, John Hunt, Susy Jacobson, Patty Nicely, B1yn Phillips, Witold Piekarski, Max Puckett, and Mickey Singer. Not only did they let me use their lab facilities, they paid me to hang out with them for a couple of years. 1 consider myselffmiunate to have worked with such a wonderful group of people. Financial support was provided in pa1i by the Dr. Earl and Ethyl Myers Oceanographic and Marine Biology Tmst, the Conchologists of America, and the Graduate Studies Depmiment at California State University, Fresno. vi

I owe a special thank you to my parents, Everett and Delores White. Throughout my life they have supported me in every endeavor, no matter how long it may have taken me to accomplish my goals. I would not be where I am today had it not been for their physical, emotional, and financial support. Finally, a very special thank you goes out to Tomoharu Eguchi. More than anyone else, he provided daily support and motivation to get the work done and to do it well. He has not only made me a better scientist, but continues to encourage me to grow as a person. His love and support have been inspirational and greatly appreciated. TABLE OF CONTENTS Page

LIST OF TABLES. Vlll

LIST OF FIGURES 1x

INTRODUCTION I MATERIALS AND METHODS 6 Reproductive Cycles. 8 Larval Development and Larval Settlement 9 Density, Size, and Species Composition within the Coralline Algae Mat II Reduction in Density, Algal Assemblages, and Growth Rate 14 RESULTS 19 Reproductive Cycles . 19 Larval Development and Larval Settlement 26 Density, Size, and Species Composition within the Coralline Algae Mat 33 Reduction in Density, Algal Assemblages, and Growth Rate 42 DISCUSSION 44 Reproductive Cycles . 44 Larval Development and Larval Settlement 49 Density, Size, and Species Composition within the Coralline Algae Mat 54 Reduction in Density, Algal Assemblages, and Growth Rate 58 CONCLUSIONS 62 REFERENCES 64 LIST OF TABLES

Table Page I. Larval development stages of N. kata and N. cal!fornica and other inteiiidal chitons . 28

2. Two-factor ANOV A of larval settlement rates of N. kata, measured as prop01iion of settled larvae among different substrate types and in the presence or absence of adult mucus 31 3. ANCOVA analysis of the relationships between fomih valve width ( FVW) and total chiton length for large and small size classes of N. ca!ifornica . 37 LIST OF FIGURES

Figure Page l. Study area in Stillwater Cove in Cannel Bay, California 7 2. Fomih valve width versus total chiton length for N. cal(fornica I 5 3. Arcsine transfonned gonad indices versus chiton mass (g) and chiton length for all chitons collected prior to natural spawning events 20 4. Mean(+/- SE) monthly gonadal indices of N. cal!fornica and N. kata 21 5. Mean(+/- SE) monthly gonadal indices of N. kata and N. californica. 25 6. Size frequency distributions of male and female chi tons 27 7. Mean proportion of settled larvae ofN. kata among substrate type and in the presence or absence of adult mucus 32 8. Mean density (square root+/- SE) of N. kata, N. califomica, and N. .fluxa from three areas within the C. vancouveriensis algal mat 35 9. Mean size (mm Fomih Valve Widtl1 +/- SE) of N. kata, N. californica, and N. .fluxa from three areas within the C. vancouveriensis algal mat 36 I 0. Estimated age class distributions of N. kata, N. californica, andN. .fluxa in three areas within the C. vancouveriensis algal mat . 39 II. Relationship of foutih valve width versus total chiton length for juvenile N. kata 40

12. Mean density (square root+/- SE) of newly recruited N kat a and unidentified chitons estimated less than 2 months old from three areas within the C. vancouveriensis algal mat 41

13. Mean percent cover (arcsine+/- SE) of coralline algae (a), bare rock (b), sessile invetiebrates (c), and fleshy macroalgae (d) among experimental plots at the initiation (April l 997) and conclusion (October 1997) ofthe experiment 43 INTRODUCTION

Distribution and survival of intertidal organisms are affected by many interacting processes, including physical factors such as desiccation and heat stress, and biological factors such as intra- and interspecific competition, larval settlement, and recruitment (Connell 1961, Wolcott 1973, Underwood and Jemakoff 1981 ). Larval settlement can influence the structure of the adult intertidal community (Roughgarden et al. 1985, 1988, Gaines and Roughgarden 1985), whereas inter- and intraspecific competition may negatively influence growth, survivorship, and distribution of individuals after recruitment has occuned (Haven 1972, Underwood 1978, Peterson and Andre 1980). Species migrations also may be induced by interspecific competition (Branch 1975a, 1975b, Underwood 1978, Chow 1989). Studies on reproduction, recruitment, growth, and competition provide important infonnation on the interaction of biological factors influencing community structure in the intertidal. Although many studies have examined factors influencing gastropod assemblages (Underwood 1979, Branch 1981, Underwood and Jernakoff 1981), few have focused on chitons. Approximately II 0 to 125 species of chi tons inhabit the west coast of North America, yet few of these have been studied extensively (Ricketts and Calvin 1968, Strathmann and Eernisse 1987). The majority of information on chi tons pe1iains to reproductive cycles and the timing of spawning events (Pearse 1979, Strathmann and Eernisse 1987). Chitons graze macroalgal spores and gennlings and can significantly reduce the abundance of foliose algae in some areas (Dethier and Duggins 1984, Black et al. 1988, Scheib ling 1994). Grazing activity of chi tons prevents overgrowth of 2 foliose algae and allows the persistence of chi tons and other grazers, such as limpets, which would otherwise be excluded by algae (Dethier and Duggins 1984 and 1985, Bany 1988, Scheibling 1994). Chitons within the genus Nutta//ina are common in the mid to high intertidal along the central coast of Califomia (Ricketts and Calvin 1968, Andrus and Legard 1975). Taxonomists and ecologists have considered Nuttallina to consist of one, possibly two, species along the Pacific Coast ofNmih America based on morphological similarities. Using electrophoretic teclmiques, however, Piper ( 1984) showed the existence of tlu-ee distinct Nuttal /ina species; N calif"omica, N flux a, and N kata. Differences between these three species are apparent in gill morphology, coloration, valve shape, geographical range, abundance, and intertidal distribution (Piper 1984). Nutta//ina cal!f"omica (Reeve 1847) is primarily a cold water species that ranges from the Straits of Juan de Fuca, Washington, to Baja Califomia, Mexico (Burghardt and Burghardt 1969, Smith 1977). This species is typically found noiih of Point Conception, although it is found in cold-water upwelling areas along the Pacific Coast of Baja California, Mexico (Piper 1984). Gills of N ca!ifornica are abanal and holobranchial, and extend from beneath valve vii to beneath valve ii (Piper 1984). The girdle is often dark brown with shoJi brown spines. Incomplete white stripes may extend from the valves to the girdle edge. Uneroded valves tend to be rounded triangular to very triangular in shape. Individuals typically inhabit the high to mid inteiiidal at densities less tl1an 200 m·2 and are often associated

with mussels and barnacles (Piper 1984). Larger N cal!f"omica are found higher in the inteiiidal than are smaller chi tons (Itumie 1981 ).

Nutta/linafluxa (Carpenter 1864) is considered a warm-water species and occurs primarily in high intertidal areas south of Point Conception (Piper 1984), 3 although it is found from Monterey, Califomia, to the Gulf of Califomia, Mexico

(Burghardt and Burghardt I 969). Gills of N. .fluxa also are abanal and holobranchial, yet they extend from beneath the suture of valves vi and vii to beneath valve ii or iii. The girdle typically consists of alternating stripes of light brown and white extending from the valves to the girdle edge. White spines are more abundant than brown spines and are longer than spines on N. ca!if'ornica, giving the girdle a "fuzzy" appearance. Valves of N..fluxa are wider than valves of

N. ca!ifornica and N. kata, and are rectangular in shape. Nutta11inafluxa occurs in the mid to high intetiidal and does not exceed densities of 200 m·2 (Piper I 984).

Nutta11ina kata (Piper I 984) is a recently described warm water species and ranges from Monterey, Califomia to Baja California, Mexico, although it is most abundant south of Point Conception (Piper 1984 ). Gills of N. kata are abanal and merobranchial and extend from beneath valve vii to beneath the suture of valves iii and iv. The girdle typically consists of alternating stripes of white and light brown extending from the valves to the girdle edge. Long white and light brown spines are numerous and give the girdle a "fuzzy" appearance, similar toN. .fluxa. Valves are triangular to rounded triangular in shape. Nutta11ina kata lives lower in the

intertidal than N. ca!if'ornica and N. .fluxa and individuals typically occupy small depressions in the substratum among geniculate coralline algae mats (Vesco I 980,

cited as N..fluxa, Piper 1984).

Although individuals of the three Nutta11ina species are found on various types of substrata, the greatest densities are found in association with the

geniculate coralline alga Cora11ina vancouveriensis on soft, porous sandstone substrata. Quantitative determination of species composition within the algal mat

has not been determined, yet N. kata appears to be the most abundant Nutta11ina species within the algal mat (Piper 1984). 4

Vertical distributions of size classes along the shore have been repmied previously for Nutta!!ina spp. Small chitons typically occur lower in the inteiiidal than large chi tons (Kues 1969, Louda 1972, Vesco 1980, Ituarte 1981, Piper 1984). Similar distribution patterns have been observed for other species of inteiiidal chitons, including Chiton tuberculatus (Glynn 1970), Sypharochito11 pe!lise1pentis (Boyle 1970), and Mopalia muscosa (Fitzgerald 1975). These size gradients along the shore may result from differences between species, differential growth rates, differential mortality, migration, and larval recruitment (Venneij 1972, Denley and Underwood 1979, Underwood 1979, Branch 1981). Piper (1984 ), however, detennined the vertical distribution of size classes for Nuttall ina spp. did not result from species migrations. Because N. kata is newly described, information is not available regarding reproductive cycles, larval settlement, abundance, and growth rates. Nuttallina kata is not recognized in current literature because prior studies on Nutta!!ina spp. were conducted before this species was described. Previous authors shtdying N. califomica and N. flux a may have mistaken N. kata for one or both of these species. Therefore, it would have been impossible to detennine the reason for the vertical distribution of size classes if differences along the shore resulted from differences between species in larval settlement, growth rates, or mmiality. Some infonnation on the ecology of N. kata may be inferred from previous studies based on differences in geographical range, location within the inteiiidal, habitat, and associated organisms between the three Nutta!lina species (Piper 1984). lnfonnation is limited, however, on reproduction, growth, larval settlement, and recruitment of Nutta!lina spp. Therefore, this study was designed to determine differences between the Nuttallina species in reproductive cycles, larval settlement, recruitment, and growth rates of individuals within a C. 5 vancouveriensis algal mat along the central coast of Califomia. The objectives of this study were to: I) detennine the annual reproductive cycles of the Nuttallina species, 2) detennine if spawning occurs synchronously between the Nuttallina species, 3) determine if spawning occurs synchronously among males and females of each species, 4) determine the species composition, mean density, and mean size of Nuttallina within the C. vancouveriensis algal mat, 5) detennine if recruitment occurs within the C. vwzcouveriensis algal mat, 6) detennine if larvae of Nuttall ina spp. are induced to settle by the presence of C. vancouveriensis, or in the presence of mucus from adults, 7) determine if Nuttct!lina spp. significantly decreases macroalgal growth in the C. vancouveriensis algal mat, and 8) detennine if the growth rate of Nuttallina spp. is density-dependent in the C. vancouveriensis algal mat. MATERIALS AND METHODS

The study was done in the intertidal region of Stillwater Cove, approximately 5 km south of Monterey, California, in Cannel Bay (36° 34'N, 121° 56'W; Figure I). The site consisted of a friable sandstone bench where three Nutted/ina species coexisted. Nutted/ina californica and N flux a occulTed most frequently in the mid- to high-intertidal region, dominated by the algae Endocladia muricata, Mastocwpus papil/atus, Cladophora columbiana, and Chaetomorpha /inum, and patches of the mussel, Myti/us cal!fomianus. The limpet Lottia gigantea and barnacles, Balanus glandula, Po//icipes po/ymerus, and Tetraclita rubescens also were common. The greatest densities of the three species of Nuttall ina occmTed within a conspicuous mat oftl1e coralline alga Corallina vancouveriensis in the mid- to low-intertidal (pers. obs.). The lowest intertidal zone was dominated by the urchin Strongylocentrotus plllpuratus and encrusting coralline algae. All field manipulations were done in the C. vancouveriensis algal mat (referred to hereafter as the algal mat). Because field experiments were conducted simultaneously, the algal mat was divided into three areas to minimize trampling of experimental plots (Figure 1). The rock was extremely friable and porous sandstone, and individual Nutted/ina within the algal mat formed deep pits or depressions within the substrate. Pits were approximately the size of the chitons occupying them, and in some cases were more than 2 em deep. The great number of pits in the algal mat made the substrate extremely heterogeneous. Similar assemblages of Nutted/ina spp. and C. vancouveriensis have not been documented 011 hard substrata. 7

Stillwater Cove ,_ •co •

Carmel Bay •

Cam1el Point

•

Coralli11a vancouveriensis algal mat

Figure l. Study area in Stillwater Cove in Carmel Bay, California. Areas 1, 2, and 3 refer to areas where different field experiments were condueted. 8

Reproductive Cycles To detennine reproductive activity, 10 adult specimens of N. californica

(> 30 mm) and N. kata (> 25 mm) were collected monthly from Stillwater Cove from March 1996 to October 1997. Reproductive activity of N. flux a was not examined because densities were not great enough to collect I 0 individuals consistently every month. Jn the lab, total length was measured after placing chi tons in petri dishes containing ambient seawater until they were relaxed and fully extended. Total length of each chiton was measured to the nearest 0.2 mm using Vernier calipers. Chi tons were blotted dry with a paper towel to remove excess water, and the wet weight of each was obtained by weighing to the nearest milligram using a Mettler balance. Each chiton was dissected and the gonadal tissue was separated from the remainder of tl1e body tissue. Wet weight of each gonad was detennined to the nearest milligram. Each gonad then was visually inspected for the presence of gametes.

To detennine the proportion of gonad material to body weight, gonad indices were calculated for each individual using the following fonnula:

Gonad index = (wet weight of gonad/wet weight of total animal)* I 00.

Mean gonad index then was calculated for each month.

To detennine if a spawning event occurred, monthly mean gonad indices were compared to the previous month. A significant decrease in mean gonad index indicated a spawning event had occuned. All data were transformed using the arcsine transfonnation (Zar 1984). A one-tailed t-test was used when data

were normally distributed and variances were equal, and a one-tailed Mmm Whitney U test was used when assumptions of normality and homoscedasticity

were violated. Spawning events were categorized as complete or pmtial spawns.

Complete spawns resulted in gonads devoid of gametes, whereas partial spawns 9 resulted in a significant decrease, but not a depletion. To ensure data were not biased by sexually immature chitons, transformed gonad indices were regressed against total chiton length and chiton mass. The sex of each specimen was detennined by the color of the gonad: gonadal tissue from males of both species ranged from bright orange to red, whereas tissue from female gonads ranged from dark green to brown. Sex ratios for N. californica and N. kata were detennined using a X2 goodness of fit test with an expected ratio of 1:1. Heterogeneity among monthly samples was tested for each species by subtracting the pooled X 2 values from the sum of all monthly values. Data were pooled if they were homogenous (Zar !984).

Larval Development and Larval Settlement

To describe larval development stages and to conduct larval settlement experiments, adult specimens of N. californica and N. kata were collected prior to natural spawning events. Natural spawning events were determined from monthly analyses of gonads as described above. Specimens of each species were maintained in separate static aquaria. If animals were ripe, spawning typically occurred the evening after collection. Upon feiiilization, eggs were removed from the aquaria and placed into a 90 ~tm mesh screen tube. Fertilization was determined by the presence of a space between the egg hull and the cell membrane. Eggs were rinsed gently with I ~tm filtered seawater to remove excess sperm and debris. Fertilized eggs were maintained in static screen tnbes at approximately 14"C. Once eggs had hatched, swimming larvae were placed in 90

~tm screen tubes with slow-flowing seawater. Larvae were maintained in flow

through screen tubes until development was complete and they were competent to 10 settle. Fe1iilized eggs and larvae were examined under a compound microscope approximately twice daily to monitor development. To detennine if substrate type and the presence of adult mucus affected the settlement of Nuttallina larvae, fully developed trochophore larvae were used in a laboratory settlement experiment. These larvae were recognized by the presence of larval eyes, shell glands, differentiated foot, and an elongated post-trochal region (Barnes 1972, Strathmann and Eemisse 1987). Trochophore larvae were competent to settle when they became negatively phototactic and alternatively swam and crawled along the bottom of the screen tube (Strathmann and Eemisse

1987). Selective settlement of fully developed trochophore larvae of N kat a on five substrata was tested using five treatments and a control. Substrata included the geniculate coralline alga Cora11ina vancouveriensis, the encrusting coralline alga Pseudolithophyllwn neoj"arlowii, the fleshy red alga Endocladia muricata, and sandstone and granite rock devoid of any biota. The sandstone and granite substrata were biological controls, and a treatment containing only filtered seawater was used as a substrate control. All substrata were collected from the study site at Stillwater Cove, except for the bare granite rocks, which were collected from Carmel Point (Figure I, p. 7). To test effects of adult mucus on larval settlement, all substrata were separated into two sets; one set contained mucus from adult specimens of N kata and the second set did not contain adult mucus. Mucus was obtained in all applicable treatments by placing two adult specimens of N kata on the substrate in each vial 24 hours prior to initiation of the experiment. Each treatment was replicated five times. Allmacroinvertebrates were removed from each 11 experimental substrate, and all substrata were leached in seawater 24 hours prior to use. To conduct the larval settlement experiment, fully developed larvae were combined into one liter of 1 ~tm filtered seawater and the density oflarvae was detennined. Equal aliquots of larval solution were inoculated into each test container. Four additional containers were inoculated and immediately preserved with 5% buffered fonnalin to detennine the mean number oflarvae added to each test container. The experiment was conducted in static 30 ml shell vials for 7 days. All test containers were maintained at ambient seawater temperature (approximately 14"C). Test containers were observed daily using an invet1ed microscope, and the presence or absence of settled larvae was noted. At the conclusion of the experiment, all vials were preserved with 5% buffered formalin. The number of settled larvae then was counted in each container. The proportion of settled larvae for each container was estimated by dividing the number of settled larvae in the container by the mean number of larvae put into each container. A two-way analysis of variance was used to test differences in mean numbers of settled larvae among substrate types and between the presence or absence of adult mucus.

Destructive sampling methods were used to precisely estimate the density, size, and species composition of Nuttallina spp. within the algal mat. The algal mat was the only habitat sampled using destructive sampling methods within the intertidal at Stillwater Cove. Because two field experiments were done simultaneously within the algal mat, the study site was separated into disturbed ,and undisturbed areas to reduce possible effects of trampling. Disturbed areas 12

(areas I and 2; Figure I, p. 7) ineluded habitat encompassing field experiments and were subject to consistent trampling. The undisturbed area (area 3; Figure 1) was habitat in which no experimental manipulations were being conducted. Because areas I and 2 were smaller than area 3, more samples were collected in area 3 than in the other two areas. All sampling was conducted between August 17 and August 20, 1997, using I 0 em x I 0 em randomly located quadrats. A total of 45 samples were collected: I 0 samples fi·om area I, 10 samples fi·om area 2, ru1d 25 samples from area 3. San1ples were located in all three areas using a transect tape placed parallel to shore tlu-ough the C. vancouveriensis habitat. A second meter tape was placed perpendicular to the shore at random points on the transect. Random numbers were chosen along the second tape in either a positive (toward the shore) or negative (toward the water) direction. The upper lett comer of the quadrat was placed at the randomly chosen point. In areas I and 2, any quadrats that overlapped an expelimentalmanipulation were discarded and additional random numbers were chosen. To collect srunples, all substrate within each quadrat was removed to the level ofthe deepest pit by chipping away pieces with a hrunmer and small chisel. Substrate pieces from a sample were placed in a plastic bag and taken to the lab, where visible chitons were removed and placed into labeled vials. Substrate pieces from a single sample then were placed in a 90 Jlm screen tube and soaked in a warm seawater bath of30% ethanol for approximately 2-3 minutes. Tllis removed the smallest chitons from the substrate by relaxing the muscles in their foot. Substrate pieces then were rinsed with seawater and removed from the screen tube. Small chi tons and debris remaining in the screen tube were rinsed into a labeled vial and preserved in 5% buffered fom1alin for identification. 13

All specimens of Nuttall ina were counted, measured, and identified to species when possible. Because chitons contracted when preserved, estimates of total length were impossible to obtain on most specimens. Fourth valve width is linearly related to total chiton length in many species of chi tons (Wells and Sellers 1987, Otway 1994), including Nuttallina spp. (Piper 1984). Therefore, measurement of fourth valve width (FVW), measured to the nearest 0.2 nnn using Vernier calipers, was used as an indication of size. Some small chi tons, however, did not contract and their total length also was measured. Mean density and size of N. kata, N. californica, and N. flux a were determined for each of the three areas. A multivariate analysis of variance (MANOVA) was clone to test for significant differences in mean density and size of the three Nuttallina species in areas I, 2, and 3. Mean density data were transformed using the square-root transformation to satisfy the assumption of homoscedasticity (Zar 1984 ). Because a greater number of samples were collected in area 3 than in areas I and 2, ten samples were chosen randomly from the area 3 samples and were used in the analysis. Because the relationships between FVW and total chiton length were different among N. kata, N. californica, and N. flux a (Piper 1984), FVW measurements were converted to estimates of age in years. The relationships between FVW and age were detennined by Piper (1984) for N. kata and N..fluxa. Although Piper (1984) did not detennine the relationship between FVW and age for N. californica, Itumie ( 1981) described the relationship between age and total chiton length. To estimate the age of N. californica, it was necessary to determine the relationship between FVW and total chiton length. Measurements of total length, however, were obtained only from large and small individuals. Because measurements were not available for all size classes, a linear regression between 14

FVW and total chiton length for all size classes could not be done (Figure 2). Therefore, an analysis of covariance (ANCOV A) was done to determine if relationships between FVW and total length were the same for large and small size classes. If the relationships were not different, the relationship for the middle size classes could have been estimated. Mean number of new recruits was determined for each area. Although many individuals smaller than 0.5 mm FVW could not be identified to genus or species, all identifiable recruits, with the exception of one individual, were N. kata. To obtain approximate ages of juvenile N. kata, total length was estimated from a linear regression on FVW and chiton length for N. kala between 0.7 and 2.0 mm FVW. Total length estimates were then compared with growth rates of other chi tons to estimate age. One-way ANOV As were conducted to detennine differences in mean number of unidentified recruits and N. kata recruits among areas I, 2, and 3.

Reduction in Density, Alytl Assemblages, , and Grmvth ate A field experiment was conducted fi'om June 5 to October 30, 1997 to detennine the effect of decreased chiton density on the algal mat assemblage and growth rates of Nuttalli11a spp. within the algal mat. Densities of Nuttallina spp. were manipulated within fenced plots. Fences were constructed of 0.2 em thick clear polypropylene Naltex@ mesh (1.0 mm opening), and were approximately 35 x 35 em and 5 em high. They were attached to the substrate using stainless steel screws and washers fastened into plastic anchors. Z-spar@ marine epoxy was used to seal the base of the fence to the substrate. A 5 em boundaty sun·otmded each 25 x 25 em experimental plot and was used to reduce effects of the fence on the animals within the plot. No attempt was made to control movements of other 15

0 45 0 0

0 I 0 2 4 6 8 10 12 Fourth Valve Width (mm)

Figure 2. Fomih valve width versus total chiton length for N. califonzica. Each circle represents measurements from one individual, n = 91. 16

organisms around the manipulations, but the fences may have inhibited or enhanced movements of some species. Growth rates of chi tons and change in algal cover were estimated for three treatments and a control. The first treatment consisted of fenced plots at one-third the visual!y estimated density of Nuttailma spp. within the algal mat. This density approximately conesponded to estimates of Nuttallina spp. within the mussel and bamacle zone. The second treatment was exactly the same as the first except new macroalgal growth was trimmed manually to mimic levels at natural Nuttallina spp. densities. The third treatment consisted offenced plots at natural densities. A natural control contained chitons at natural densities in unfenced plots. All experimental plots were visually inspected twice monthly. Chiton densities were adjusted by adding or removing chitons, and algal growth was trimmed as needed. To determine the impact of Nuttallina spp. on the community species composition within the algal mat, percent cover of algae and sessile inveriebrates was dete1111ined at the initiation and conclusion of the experiment. Four categories were created tbr percent cover analysis: coralline algae, fleshy algae, sessile invertebrates, and bare rock. The point quadrat method described by Foster eta!.

(1991) was used to detennine percent cover in each quadrat. A 35 x 35 x 1.2 em Plexiglas plate was perforated by I 00 holes in a grid. The grid covered the entire area of each experimental plot, 25 x 25 em. Adjustable legs were used to hold the plate parallel to the substrate. A pointed stainless steel rod was slid through thirty random holes in the grid and each organism in contact with the point was recorded. Multiple layers were recorded by carefully moving aside the uppermost layer and observing the next contacted layer. In this way, multiple organisms could be recorded at a single point and the total percent cover could be greater than I 00 percent Multiple contacts of the same organism were not recorded. Percent cover 17 for each organism recorded in a plot was calculated as 3.33 * the total number of occunences. Percent cover data were transfonned using the arcsine transformation (Zar 1984), and a series of one factor ANOVAs were used to compare mean percent cover of coralline algae, fleshy algae, sessile invertebrates, and bare rock among treatments at the initiation and conclusion of the experiment. Analyses of percent cover did not include the decreased density plots that had been manually trimmed. This treatment was used only as an algal growth control to examine effects of decreased chiton density on chiton growth rates.

To detem1inc the effect of density on growth rate of Nutted/ina spp., a total of 579 chi tons were tagged between May 8 and June 4, 1997, using the method described by Piper (1984: four pound test monofilament (-0.2 mm diameter) and 16 gauge hypodennic needles). Tags were constructed of0.13 mm thick stainless steel cut into 5 x 5 mm squares with unique number and letter combinations stamped into each for identification. A hypodermic needle was inserted into the girdle on the left side of each chiton in the area of the fourth valve. Monofilament was fed through the needle and, once secured, the needle was removed fi·om the girdle. A stainless steel tag was then attached to each chiton using the monofilament. A drop of Loctitc@ instant adhesive was used to secure the knot. Chitons were tagged on two separate occasions in each experimental plot. Twenty chitons initially were tagged in each plot. All plots were revisited 2 to 3 days after initial tagging and additional animals tagged to maintain the total at 20 per plot. In some cases, no additional tagging was necessary. One plot, however, required 9 additional chi tons to be tagged 3 days after the initial tagging. Total length and tburth valve width were measured for each tagged chiton. All chitons were chosen haphazardly from each plot. 18

Total length and foutth valve width of each recaptured chiton were measured again after 6 months. Growth rates were calculated for both total length and fourth valve width using the following equation:

R = ln(L/L0)/t1 where R is the instantaneous rate of growth per unit length, L, and L0 are chiton lengths or fomth valve widths at times t and 0. One factor ANOVAs were used to compare mean size of tagged individuals among treatments at the initiation and conclusion ofthe experiment. Homoscedasticity among cells was tested using Cochran's test prior to conducting ANOVA, and transfonnations were conducted when heteroscedasticity was found. Power analyses were conducted when differences between means were not signil1cant. When the statistical power was below 0.8, the number of samples necessary to achieve a power of0.8 was calculated (referred to as n'). RESULTS

Reproductive Cycles Gonad indices were used to estimate reproductive condition of adult chitons. To ensure all collected chitons were reproductively mature, gonad indices for all individuals collected prior to natural spawning events were plotted against chiton mass and total length. There was no significant linear relationship between gonadal index and mass, or between gonadal index and total length for either N cal(fornica or N kata (Figure 3). These results indicated all animals used in analyses were reproductively mature and variation in gonadal indices did not result from differences in chiton size. If reproductively immature animals had been present, mean gonadal indices would have decreased and a linear relationship between gonadal index and mass or length would have been detected. Reproductive cycle of N californiccl was variable with no apparent annual cycle fl-mn March 1996 to October 1997 (Figure 4). Two spawning events were evident in 1996 as indicated by significant decreases in monthly gonad indices. The first occuned in March 1996 (t-test between March and April; meanM"' = 0.234, SEMur = 0.015; meanApr = 0.163, SEAr• = 0.0 13; t = -3.528, p = 0.006). By the beginning of April, gonads of60% of chi tons were empty (GI's less than 3 and gonad lacking gametes). Although there was a slight increase in mean gonad indices during April, 50% of chi tons at the beginning of May had empty gonads. A complete spawning event had occurred by the begi1ming of June, however, as 80% of chi tons in mid-June were devoid of gonad material (t-test between May and June; meanMuy = 0.191, SEMay = 0.0 14; meanJun = 0.151, SEJun = 0.008; t =-

2.417, P = 0.039). Gonadal growth occwTed in summer and early autumn during June, July, August, and September 1996. A partial spawn occurred in early 20

11 ~ 60 n 60 f(x) ~ 0.00 16x + 0.238 f(x) 0.0017x + 0.175 0.5 0.5 R''2 ~ 0.0012 R"2 0.039 0.4 - 0.4 o o 0.3 - 0.3 eo 0~~0Ao © oo G- 0.2 0 0 0 02 ~.. 0 0 0

~ 0.1 0.1 - Y, ""'" 0 0 -;;;-" 0 2 3 4 5 25 30 35 40 45 50 55 "V §"' 0 ~ ::: ·:::;;" 0 n= 50 11 =50 -<>- f(x) ~ 0.0092x + 0.237 f(x) 0.0029x + 0.152 0.5 0.5 R"2 = 0.049 W·2 0.075 0.4 ., 0.4 0.3 - 0.3 .: ~· .,. Ill •• ~···§I" -r1:;•.. I ~ - 0.2 - .1111iai • • 0.2 --~·;:: ."' :. ,·~ 0.1 0.1 0 0 0 2 3 4 5 6 7 25 30 35 40 45 50 55 Mass (g) Length (mm)

Figure 3. Arcsine transfonned gonad indices versus chiton mass (g) and chiton length (mm) for all chitons collected p1ior to natural spawning events. Each symbol represents one individual. Open circles represent N. kata; closed circles represent N calij'omica. 21

~ ~ 0 L_------~------~~-- .= M A M .J .TASOND.TFMAM.TJASO -;;; "0 " 8" 8 - b. N. kala 7

3 ~ 2 -

0 L_------~ M AM J J AS 0 N D .J F M AM J .J AS 0

1996 1997

Figure 4. Mean(+/- SE) monthly gonadal indices of N cal!fornica (a) andN kata (b). Each asterisk(*) represents a significant decrease in mean monthly gonad index. Closed symbols represent females, open symbols represent males, n = 10. 22

October 1996 (t-test between September and October; meanscp = 0.225, SEscp =

0.013; mean0 ,, = 0.177, SE0 ,, = 0.018; t = -3.152, P = 0.012). Although 40% of chi tons at the end of October had relatively small gonads compared to other sampling months, gametes were present in the gonads of these individuals. Other chi tons contained large gonads full of gametes. Rapid gonadal growth occurred from late October 1996 through early January 1997. The decrease in mean gonadal index during December 1996 was not significant (t-test between

November and December; meanNnv = 0.272, SENnv = 0.021; mean0 ,, = 0.241, SE0 " = 0.016; t=-1.057, P = 0.318; Power= 0.27, n' =52). Eightypercentofchitons had large gonads full of gametes; only two individuals had gonad indices less than 3. The reproductive cycle of N ca!ifornica was variable dming 1997 (Figure 4). The greatest decrease in mean monthly gonadal index during the study period occulTed between December 1996 and January 1997 (t-test between January and

February; meanJan 0.309= ' SEJan = 0.015; meailr-cb = 0.194, SEFeb = 0.0 13; t =- 5.106, P = 0.001 ). This spawning event, however, was not a complete spawn, as most gonads of chitons in early FebruaJ·y 1997 contained gametes. Rapid gonadal growth occulTed in February 1997. Although a decrease in mean gonadal index occulTed during March and early Aprill997, the decrease was not significant (t test between MaJ·ch and April; mean~hn· = 0.255, SEMar = 0.0 16; meanApr = 0.208,

SEArc = 0.015; t = -1.944, P = 0.084; Power= 0.62, n' = 16). Gonadal growth was evident from mid April to early May 1997. A spawning event begaJl in mid May

(t-test between May and June; meanMay = 0.265, SEMay = 0.012; meaJlJun = 0.207,

SEJun = 0.019; t = -3.199, P = 0.011) and continued through June 1997 (t-test between June and July; meaJlJun = 0.207, SEJun = 0.019; meanJul = 0.165, SEJul = 0.016; t = -2.346, P = 0.043). By the beginning of July, half of the animals had 23

spawned completely. A short period of gonadal growth was recorded during July, followed by another spawning event during August and early September 1997 (t test between August and September; mean Aug= 0.219, SEAug = 0.01 0; meanscp = 0.122, SEscr = 0.016; t = 6.274, P = 0.0002). By early September, 80% ofchitons had empty gonads. A slight increase in gonadal indices was observed from late September to mid-October, 1997. Spawning between males and females of N. califomica was synchronous for the entire study period (Figure 4). Mean gonadal index of females was greater than males in March, August, and September 1996, whereas mean gonadal index of males was greater than females in May, November, and December 1996, and January, February, March, and October 1997. Dming all other months, mean gonadal indices of males and females were similar. Although differences in mean gonadal indices between males and females may have occmTecl in most months, spawning periods were evident by a steep decline in gonad indices of both sexes. The reproductive cycle of N. kat a was less variable than N. califomica between March 1996 and October 1997. Two spawning events were documented for N. kata during 1996 (Figure 4). Gonadal growth occurred from mid March to early April. A partial spawning event was recorded during April 1996 (t-test between April and May; mean Apr= 0.241, SEApr = 0.009; meanMay = 0.192, SEMay = 0.009; t = 3.491, P = 0.007), and only 10% of chitons in early May had empty gonads (GI's less than 2.5

l11eano,, = 0.162, SE0 , 1 = 0.014; meanNov = 0.122, SENov = 0.006; U = 80, 24

U1005iili(JOii lUi 73 ). At this point, all gonads of chitons were devoid of gametes. Rapid gonadal growth was evident from early November through December 1996. In contrast to 1996, only one spawning event was recorded for N. kata during I 997 (Figure 4). Following a rapid increase in mean monthly gonadal indices in November and December of 1996, and a slight decline in Jannary 1997

(t-test between January and February; mean1nn = 0.217, SE1,10 = 0.023; meanFch =

0.204, SEFcb = 0.0 17; t = 0.424, P = 0.682; Power 0.11, n' = 300), gonadal growth ceased in March and April 1997 and peaked in early May. A spawning event did not occur in spring 1997, and chi tons retained their gametes through July. Spawning began between mid August and mid September 1997 (t-test between August and September; mean"""= 0.224, SEAug = 0.012; meanscp = 0.150, ·

SE5,r 0.011; t = 6.037, P = 0.0002), and by mid October all chitons had spawned completely (t-test between September and October; meanscr = 0.150, SE5,P =

0.011; mean0 ct = 0.091, SE0 " = 0.010; t = 4.323, P = 0.002). Although complete spawning events of N. kata oecmTed in October in both I 996 and 1997, a partial spring spawn occulTed during 1996, but did not occm· during spring of 1997. Spawning was synchronous between males and females of N kata from March 1996 through October 1997 (Figme 4). Males typically had greater mean monthly gonadal indices than females. Spawning events, however, were evident by significant decreases in gonad indices by both males and females.

Spawning events of N. califomica and N. kala were not synchronous between species from March 1996 through July 1997 (Figure 5). Nullallina ca/ifomica typically spawned prior toN kala. During spring 1996, N. californica spawned in March and May, whereas N. kala spawned in April. Only N. ca/{fomica exhibited a major spawning event in January 1997. During autumn !997, N. cal{fornica spawned in September, whereas N. kata spawned in October. 25

X 6 - "0"' " - 5 "0 '"rn " c3 4 3

I -

0 L______

MAMJ J A SOND J FMAM J JA SO

1996 1997

Figure 5. Mean(+/- SE) monthly gonadal indices of N kata and N cal!fornica. Closed circles represent N. kata, open circles represent N. californica, n = I 0. 26

The only time during the study period that spawning appeared to occur simultaneously between these two species was during August and early September 1997. A total of 199 specimens of N. californica and 209 specimens of N. kata were analyzed to detennine sex ratios. Because male to female sex ratios did not differ among months for N. ca!ijornica (Heterogeneity= 16.3276, F = 28.869) and

N. kata (Heterogeneity I 0.4096, F = 31.41 ), monthly samples were pooled for both species. Although males were more abundant than females for both species, results indicated N. kata confonned to a 1:1 male to female sex ratio (X2 = 2.9904, F = 3.841, P = 0.2333), whereas N. cal[fomica had a male to female sex ratio of 1.4:1, significantly different from I (X2 = 5.4724, F = 3.841, P 0.0419).

Mean length of N. cal(fomica was 38.9 111111 (SE = 0.3559, n = 199), whereas mean length of N. kata was 36.7 mm (SE = 0.3168, n = 209). Mean length of 1V. ca/(fomica was significantly greater than that of N. kata (one-tailed t test, t = -8.918, P < 0.000 I). The largest N. ca/ifornica collected was 54 mm in length, whereas the largest N. kata was 50 111111 in length. Males of N. califomica were significantly greater in length than females (one-tailed t-test, llmales = 116, n1cmab = 83, t = 6.978, P < 0.000 I). Mean length of N. kata males was significantly greater than mean length of N. kata females (one-tailed t-test, nmnles =

117, nlemalcs = 92, t 5.329, P < 0.000 I; Figure 6).

Larval Development and Larval Settlement Larval development of 1V. cal[/ornica and N. kata was similar to that of previously studied chitons (Strathmann and Eernisse 1987; Table 1). All development times were recorded as hours after fertilization. Observations were made on chi tons that developed at 14"C. 27

a. N. kata n=209 40 • males 35 30 D females

?"_)

20 15 10

:>, 5 u § :J 0 "'" ;;;;.."''" 30 b. N. cal~fomica n 199 r_,

0 27-31 31-35 35-39 39-43 43-47 47-51 51-55

Length (mm)

Figure 6. Size frequency distributions of male and female chitons. Table 1. Larval development stages of N. kata and N. ca/ifomica and other local intertidal chi tons.

STAGE Nutta/lina kala Toni cella lvfopa/ia Mopalia lvfopalia Katharina and Nuttallina lineata ciliata muscosa lignosa tunicata califomica (I) (2) (3) (3) (4) Fertilization 0 hrs 0 hrs 0 hrs 0 hrs 0 hrs 0 hrs Hatching 20 hrs 48 hrs 36- 42 hrs :Whrs 19 hrs N!A Larval Eyes 60- 70 hrs 110 hrs N!A 84hrs 72- 8411TS N/A Shell Glands 60 70 hrs 120 hrs 96- 120 hrs 72 hrs 96 hrs N/A Foot/Muscle 110-liShrs 130 hrs N/A N/A N!A N/A Contractions Competent to 150- 160 hrs !50- 160 hrs 144 hrs NIA J 20 hrs 144 hrs Settle (I) Barnes (1972), (2) Thorpe (1962), (3) Watanabe and Cox (1975), and (4) Rumrill and Cameron (1983)

N 00 29

Hatching: Hatching occutTed approximately 20 hours after fet1ilization.

Oocytes were approximately 300 ~un in diameter, and eggs with hulls were 460 rm1 in diameter. Upon hatching, the egg hull cracked and the beating of the prototroch, in addition to rapid body flexions, enabled the larvae to break free of the egg hull. Larvae hatched as free swimming trochophores approximately 330 11m in length. Larvae were bulbous in shape and were swimming actively in helical patterns in the water column after hatching. Forty-eight hours: External appearance of the larvae did not change dramatically. Larvae continued swimming in the water column near the surface. Sixty to 70 hours: The anterior, or pre-trochal, region of each larva was more bulbous in shape than that of newly hatched larvae. The posterior, or post trochal, region appeared more dorso-ventrally flattened, giving the larvae a torpedo shape. The ridge at the prototroch was more defined and development of the blastopore had occurred. Larval eyes were present as small, lightly pigmented spots on either side of the larvae just posterior to the prototroch. Initial development of the shell glands was also visible as small ridges along the dorsal surface. Small cilia were visible over the entire surface of the larvae. Eighty-five hours: Larval eyes were fully developed at this time. Larvae began to appear more dorso-ventally flattened as the dorsal and ventral sides became distinct. Larvae continued swimming near the surface of the water. Shell glands were slightly more distinct. One hundred ten to 115 hours: The foot began to differentiate from the rest of the body as a small, raised circular area on the post-trochal ventral surface. General muscle contractions were evident througout the body, and under tl1e microscope, larvae could be seen changing their body shape from oval to elongate. 30

Larvae continued to become more dorso-ventrally flattened and the development of the shell glands was complete. Larvae continued to swim in the water column. One hundred thirty to 135 hours: The foot appeared to be well developed, and little additional change in external morphology or behavior was noted. Although most larvae remained swimming in the water column, larvae observed under the microscope attempted to crawl on the slide using the foot with the pre trochal region slightly elevated. One hundred fifty-five hours: The foot and shell glands were fully developed. Larvae were spending most of their time near the bottom of the screen tnbe, alternately swimming and crawling along the bottom. All larvae observed under the microscope were crawling along the surface of the slide. Larvae were competent to settle at this point and were approximately 460 11111 in length. Only larvae of N. kat a were used in the laboratory larval settlement experiment as a large proportion of N. califomica larvae did not develop nonnally, resulting in insufficient sample size for the larval settlement experiment. The mean nwnber oflarvae added to each test container was 48.5 (SE = 1.58, n = 4). The test was concluded after 7 days when it was apparent that some settled juveniles died and stm1ed decompose. Results of the two-factor ANOVA indicated significant differences in the mean proportion of settled larvae among treatments (Table 2). Because a significant interaction was detected between the two main effects, i.e .. substrate type and the presence of adult mucus (P = 0.0001, F = 6.4798; Table 2), Ryan's Q post -hoc multiple comparison test was perfon11ed on arcsine tranfonned data (Day and Quinn 1989). The greatest mean prop011ion of settled larvae occurred in the cmstose coralline algae treatment with adult mucus (mean= 0.535, SE = 0.046, n ""5; Figure 7). Geniculate coralline algae treatments with mucus (mean= 0.321, 31

Table 2. Two-factor AN OVA of larval settlement rates of N kata, measured as propmiion of settled larvae among different substrate types and in tl1e presence or absence of adult mucus. SS =sum of squares, MS =mean square, * = significantly different, ** see text for post hoc comparisons.

Source ss df MS F-ratio P-value

Substrate 0.65021 5 0.13004 12.84826 0.00000 ** Mucus 0.14872 I 0.14872 14.69375 0.00037 ** Substrate*Mucus 0.32792 5 0.06558 6.47976 0.00011 *

Error 0.48582 48 0.01012 32

0.6 * • Treatments with mucus

D Treatments without mucus 0.5

~v t: ·;;; 0 ~ * -< OA ~· ""'c: ...I"' "0 0.3 -;:::" 1U (J'] t: 0 'E 0 D.2 0.. Q ~ "'"

D. I

() Geniculate Crustose Fleshy Sandstone Granite Control coralline coralline algae rock rock algae algae **

Figure 7. Mean proportion of settled larvae(+/- SE) ofN. kata among substrate type and in the presence or absence of adult mucus. Each asterisk (*) represents a treatment with significantly greater larval settlement than the other treatments. Each double asteric (**} represents a set of treatments where a signficant interaction was detected, n = 5. 33

SE = 0.051, n = 5) and without mucus (mean= 0.329, SE = 0.081, n = 5) contained the next greatest mean proportion of settled larvae. Mean proportion of settled larvae in these two treatments were not significantly different from each other, but were different from all other treatments. The control with mucus had the next greatest mean proportion of settled larvae (mean= 0.178, SE = 0.029, n = 5) Although few larvae settled in the control treatment with mucus, settlement was significantly greater in this treatment than in the control treatment without mucus, the encrusting coralline algae treatment without mucus, and the fleshy algae, sandstone, and granite treatments, with and without mucus (Figure 7, p. 32).

Density, Size, and Species Conwosition withm the Corallme Algae 1at

No con·elation was found between density and size (r = 0.096). Therefore, both variables were used in the MANOVA. Results of the MANOV A indicated density of Nuttallina spp. was significantly different among areas (P = 0.03237, F = 3.57).

No difference in mean density was detected between area I (mean1 = 7.519, SE =

0.250, n 10) and area 3 (mean3 7.344, SE = 0.201, n 10; P 1.000), nor between area 2 (mean2 = 6.021, SE = 0.224, n = 10) and area 3 (P = 0.073 ). Significant differences in mean density of Nuttallina spp., however, were detected between areas l and 2 (P = 0.0243). Area 1 contained the greatest mean density of Nuttaffina spp., whereas area 2 contained the lowest mean density. Significant differences in mean density also were detected between the three Nuttallina species (P < 0.0001, F = 414.0395). A priori tests indicated there was a significant difference in mean density between N. kata, and the average density of N. califomica and N. flux a (P < 0.000 I, F = 172.6718). Mean density of

N. californica (mean= 1.425, SE = 0.152, n = 30) was significantly greater than mean density of N . ./lltxa (mean= 1.232, SE = 0.125, n = 30; P < 0.0001, 34

F = 601.4056). Mean density oflV. kata (mean= 6.614, SE = 0.182, n = 10) was greater than that of N. californica and N. .fluxa in areas all areas, and mean density of N. californica was greater than N. fluxa in all areas (Figure 8). No difference was detected in mean size of Nuttallina spp. among areas (P

= 0.126, F = 2.126; Power 0.2, n' =52). Although differences were not statistically significant, mean size of Nuttallina spp. was greater in area 2 (mean2 =

4.637, SE 0.382, n = 10) than in areas 1 (mean1 = 3.447, SE = 0.478, n = 10) or 3

(mean3 3.89, SE 0.466, n = 10). Significant differences in mean size were detected between the three

Nuttallina species (P = 0.001, F = 7.995}. Mean size ofN. kata was significantly different n·om the average of N. califomica and N. fluxa (P = 0.002, F = I 0. 755). Although mean sizeofN. kata (mean= 3.928, SE=O.I03, n= 30) was less than

N. fluxa (mean= 4.888, SE = 0.423, n = 30) in all areas, mean size of N. kata was greater than N. cal!fomica (mean= 3.144, SE = 0.323, n = 30) in all areas (Figure

9). Mean size of N. fluxa was significantly greater than N. califomica (P = 0.033, F 4.659). Results of the ANCOV A indicated the relationship of FVW to total length of large and small chi tons of N. calijornica did not represent all size classes accmately (Table 3). The slopes of the two linear equations, however, were not significantly different (~ 10 w 1 .g 5 = 2.635, t = 0.848). Although the estimated regression did not accurately represent the relationship between FVW and total length, additional data were not available to provide estimates of total length from FVW. Therefore, the single linear equation was used to approximate total length of N. califomica from FVW for all size classes. Estimates of yearly age classes ofN. kata, N. califomica, and N . .fluxa indicated the majority of chi tons from the algal mat were less than 3 years old 35

0 N. kala

• N. cali/omica

GJill N. jluxa

~ < "'E '-' 5 0 0

Area I Area 2 Area3

Figure 8. Mean density (square root+/- SE) of N kata, N califiJrnica, and N jluxa from three areas within the C. vancouveriensis algal mat. n = I 0. 36

D N. kala

• N. califomica

N.flw:a

Area 1 Area2 Area 3

Figure 9. Mean size (mm Fourth Valve Width+/- SE) of N. kata, N californica, and N .flux a fi·om three areas within the C. vancouveriensis algal mat. n = I 0. 37

Table 3. ANCOVA analysis of the relationships between fomih valve width (FVW) and total chiton length for large and small size classes of N. calijomica. SS =sum of square, MS =mean square, * significantly different

Source ss df MS F-ratio P-value

Size Class 307.86270 I 307.86270 41.25093 0.00000* FVW 678.20019 I 678.20019 90.87295 0.00000*

Error 656.75889 88 7.46317 38

(Figure I 0). All chi tons measured were less than the maximum total length for each species. The results indicated chi tons within the algal mat were relatively small in size and young in age. There was a significant linear relationship between FVW and chiton length for N. kata between 0.7 and 2.0 mm FVW (r2 = 0.696; Figure I I). Although actual ages of Nutta!lina were not known, estimates were detennined based on previous studies of Nuttal!ina (Ttuarte 198 I, Piper I 984) and other chiton species (Strathmann and Eemisse 1987). Many species of chitons grow 4 mm in the first month after settlement (Pearse 1979). A FVW measuring 0.78 111111 corresponded to an estimated total length of 4 mm (Figure II). Therefore, new recruits were defined as individuals less than I mm FVW and were estimated to be 1-2 months old. Although some individuals were identified at 0.5 mm FVW, most individuals up to 0.5 111111 FVW were unidenifiable. Significant differences were detected in mean number of N. kata recruits among areas. The one factor AN OVA indicated significant differences inN. kata recruits among areas (P < 0.000 I; F 16.603 }. Recmitment of N. kata in area I

(mean1 = 2.045, SE OJ I 9, n = I 0) was greater than area 2 (mean2 0.1, SE =

0.1, n = 10; P < 0.0001) and area 3 (mean3 = 0.941, SE = 0.239, n = 25; P = 0.017), and greater in area 3 tl1an area 2 (P 0.00 I; Figure 12). The mean number of unidentifiable recruits also was significantly di tferent among areas (P = 0.009; F = 5.250). Recruitment of unidentifiable chitons was significantly less in area 2 (mean2 = 2.051, SE = 0.147, n = I 0) than in area 3

(mean3 = 2.848, SE = 0.470, n = 25; P = 0.007). Data were insufficient to detect a difference in recruitment of unidentifiable chitons between areas I (mean 1 = 2.909,

SE = 0.355, n I 0) and 2 ( P = 0.10 13) and between areas I and 3 (P = 1.000; Figure 12). 39

400 r Areal D N. kala - 300 - II N. californica CJ N. .f/uxu 200 f- ,-- 100 - n. 0 I

200 Area 2

!50 >-. u c

c:;-~ 100 2 >:.. 50

800 - Area 3

600 -

400 -

200 - -

1...... 1 ...... ~ 0 I 0-l l-2 2-3 3-4 Estimated Age (years)

Figure 10. Estimated age class distributions of IV. kata, N. cal(fomica, and N. jlu.'m among three areas within the C. vancouveriensis algal mat. 40

12 f(x) ~ 3.763x + 1.053 R'2 ~ 0.696 n =84 0 10 0 0 0 0 ~ 0 '~ 8- .c :;) 0 0 " 0 -l" 6 § 0 ] u

a0 4 f- 0

0 I 0.6 O.R l.2 1.4 1.6 l.S 2 Fourth Valve Width (nun)

Figure II. Relationship of fomth valve width versus total chiton length for juvenile N. kata. Each circle represents measurements from one individual. 41

0 N. kala U Unidentified

3.5

0.5

Area I Area 2 Area 3

Figure 12. Mean density (square root+/- SE) of newly recruited N kata and unidentified chitons estimated less than 2 months old from three areas within the C. vancouveriensis algal mat. 11 10 for areas I and 2, 11 25 for area 3. 42 Reduction in Densit;t. Al~al Assemblages, atiffGfowth{atc The benthic assemblage was not affected significantly hy the density of Nuttaflina spp. Results of the one factor ANOVAs indicated there were no significant differences in percent cover of coralline algae (F; 11 ;,1, 1 = 1.056, Pinitial =

0.323; Fnnnl = 0.040, Pnnnl = 0.845), fleshy algae (Fini

= 1.197, Pnnal = 0.294), sessile invertebrates (Finitinl = 0.612, Pinitinl = 0.809; Fnnal =

0.545, Pnnal = 0.819) or bare rock (F; 11 ;,1, 1 = 0.002, 1'; 111 ,1•1 = 0.966; Fnnal = 0.181, Pr.nal

= 0.677; Figure 13) between treatments at the initiation or conclusion of the experiment. A total of 13 chitons were recaptured at the conclusion of the experiment between October 30 and November 23, 1997. Because very few tagged chitons were recaptured, effects of reduced chiton densities on growth rates could not be tested. Growth rate data for total length indicated 6 specimens grew, whereas 7 specimens shrank (mean= 0.8~un/day, SE = 0.1 ). Growth rate data for FVW indicated 1 specimen shrank and 12 specimens grew (mean 0.2~m/day, SE 0.1). 43

Ill Control 0 Decreased Density D Natural Density

I .2 a. Comlline algae I .2 b. Bare rock

0.8 0.8

0.4

~ ~ 1.2 c. Sessile invertebrates l.2 d. Fleshy macroalgae ~ ~"'

0.8-

0.6 OA

April I 997 October 1997 Aprill997 October 1997

Figure 13. Mean percent cover (arcsine+/- SE) of coralline algae (a}, bare rock (b), sessile invettebrates (c), and fleshy macroalgae (d) among experimental plots at the initiation (April 1997) and conclusion (October 1997) of the experiment. Control treatments were unfenced plots, decreased density treatments were fenced plots at reduced densities, and natural density treatments were fenced plots at natural densities. DISCUSSION

Reproductive Cycles Although N. kala, N. calijornica, and N. flux a coexisted at the study site at Stillwater Cove, Califomia, N. kata and N. califomica comprised greater than 96% of all chi tons. Too few specimens meant that the reproductive cycle of N. .fluxa, therefore, could not be determined. Seasonal trends in gonadal growth were apparent for N. kata and N. califomica. Maximum gonadal growth occurred during autumn and winter for both species. This pattem is common for intertidal invertebrates in temperate and polar areas and may be stimulated by declining sea surface temperatures (Himmehnan 1980). Although productivity of the environment may not be optimal during this period, nutrients stored in the digestive gland may be used for nutritive demands of gonadal growth during periods of decreased productivity (Tucker and Giese 1962, Pearse 1979). Evidence of inverse relationships in size of gonads and digestive glands have been repm1ed for C!jptochiton stelleri (Tucker and Giese 1962) and Katharina tzmicata (Giese and Pearse J 974). Large quantities of nutrients may concentrate in gonad material during periods of gonadal growth at the expense of other body systems. Nuttallina kata had discrete spawning events in spring and autumn, whereas N. californica was reproductive throughout the study period. Although a bi-annual reproductive cycle was recorded for N. kata during 1996, only a single spawning event was recorded in late summer-autumn during 1997. Previous data on the reproductive cycle of N. kata were not available. Prior research on N. califomica by ltuaJ1e ( 1981) from March 1978 to June 1979 in central Califomia indicated N. cal((ornica was reproductive tluoughout the year. These data concur with 45 spawning events of N. califomica recorded during the present study. Although individual chitons may spawn repeatedly during a season, most species of chi tons examined along the Pacific coast ofNo1th America have well-defined annual reproductive cycles (Pearse 1979). Spawning events typically occur in late winter and early spring when sea sUJfacc temperatures decrease (Pearse 1979, Strathmann and Eernisse 1987). Although species such as Mopalia muscosa spawn throughout the year, it is uncommon for most species of chi tons to be reproductive throughout the year (Pearse I 979). Both N. kata and N. californica exhibited complete and pa1tial spawns at different times of the year. Pattial spawning events have been reported for many species of chi tons, including Katlwrina tunicata (Himmelman 1978) and

Acantlwpleura granulata (Glynn 1970). Pattial spawns result from incomplete development of gametes at the time of the spawn; only those gametes that have completed gametogenesis are released (Nimitz and Giese 1964). Additional gonad growth may occur after partial spawns, as the remaining gametes continue to develop. In contrast, a complete spawn depletes the gonad of all viable gametes. Nuttct!lina kata did not exhibit gonadal growth after a partial spawn occurred in spring 1996. Gonads remained relatively small until the next spawning event occuued in October 1996. Pearse ( 1978) noted, however, that even gonads ranked as small contained great numbers of gametes. Viable gametes may have been retained in the gonads of N kata, and further development, though not necessarily an increase in volume, may have occurred after the partial spawn in spring 1996. Pattial spawning events of N. califomica always were followed by rapid gonadal growth and were more frequent than partial spawning events of N. kat a. Because N. ca/ffomica was reproductive throughout the year, grunetes may have been produced at various times of the year. Consequently, at any one time 46 there may have been differing stages of developing gametes within the gonad. Histological infonnation is necessary to determine the developmental stages of gametes within the gonad. Spawning events hetween N. kat a and N. californica were asynchronous except during August 1997. Although spenn of chitons are chemotactic, they are not species specific (Pearse 1979). Spenn from three chitons, Mopa!ia muscosa, Katharina tunicata, and Ton ice!!a lineata, fe1tilized eggs of any of the three species (Himmelman 1976). Complete larval development, however, did not occur in those individuals where cross fe1tilization occurred. Because chiton spe11n are not species specific, asynchronous spawning between species prevents cross-fertilization and reduces gamete wastage. Spawning events were synchronous within the species. Although mean gonadal indices of males and females were not equal each month, spawning events were evident by concurrent decreases in gonadal indices between males and females. Synchronization of spawning within species increases the rate of fcitilization and reduces gamete wastage (Himmelman 1978). Strong synchronization of spawning within species also indicates possible influence of an extemal factor. Although seasonal trends in spawning events were apparent for N. kata and N. calijomica, the timing of spawning events differed among years. During 1996, N. kata spawned during April and October. Duling 1997, however, N. kata did not begin spawning until late July to August. Nutiallina calijomica spawned during March, May, and September, 1996, and during Januruy, May, June, and August, 1997. Variability in spawning among years at the same locality is common in chi tons that exhibit discrete spawning periods tln·oughout the year (Giese et a!. 1959, Boolootian 1964, Himmelmru1 1975, 1978, Pearse 1978, Otway 1994). 47

Many of these chitons also spawned in response to changes in environmental factors (Himmelman 1975, 1980). If spawning events are int1uenced by envirorunentat factors, differences in the timing of spawning events among years may result fi·om yearly changes in environmental conditions. Spawning events in many species of chitons are correlated with environmental factors. Although precise cues have not been identified in most cases, spawning may be influenced by phytoplankton blooms (Himmelman 1975, 1978, Stan· et a!. 1990, 1991 ), tidal cycles (Yoshioka l989a), tidal cycle and photoperiod (Nagabhushanam and Deshpande 1982, Yoshioka 1989a), and lunar cycles (Strathmam1 and Eemisse 1987, Yoshioka 1989b). Envirom11ental cues often do not directly affect larvae, yet they may signal that conditions within the water column or the intertidal are favorable for survival of planktonic larvae or settled juveniles. The synchronization of spawning within the species of Nuttallina indicated extemal factors may have affected their spawning events. The asynchronous spawning between the species indicated that the cue may have been species specific. Enviromnenta1 factors coinciding with oceanographic seasons may have stimulated spawning events of N. kara and N califomica. Three oceanographic seasons have been defined for the Monterey Bay area: the Davidson current (November through Februa1y), the upwelling period (February through September), and the oceanic period (September through October; Bolin and Abbott 1964 ). The begiillling of each season is defined by significant changes in the taxonomic complexity of phytoplankton (Bolin and Abbot 1964 ). As the seasons progress, the phytoplankton becomes more homogenous. Individual species of phytoplankton stimulate spawning in some invertebrates (Himmelman 1976, 1978, Stan-eta!. 1990, 1991 ). If phytoplankton was a stimulus to spawn for either N. 48 kata or N. californica, the asynchronous spawning bet\veen species indicated N. kata and N. ca!if'ornica may have been ini1uenced by different species of phytoplankton present during different oceanographic seasons. Yearly changes in reproductive periodicity of N. kata and N. californica indicated that reproductive activities of these species are affected by factors that vary among years, rather than predictive factors that are constant fi·om year to year. Although most species of chitons live in intertidal or shallow subtidal areas and have photoreceptors (aesthetes) in their shell valves, reproductive cycles of most chi tons are not related to constant environmental factors, such as photoperiod, lunar cycle, and tidal cycle (Pearse 1979). The seasonal variability in reproductive cycles indicated that constant factors had little effect on reproductive cycles of Nuttal!ina spp. Males were more abundant than females for both N. ca!ifomica and N. kata. Although unequal sex ratios in gonochoristic molluscs generally indicate a greater number of females dmn males (Pearse 1978), several hypotheses have been proposed to explain the greater abundance of males than females in many species. For animals with external fe1tilization, an increase in d1e abundance of males might alleviate problems of spenn dilution in d1e water column (Glynn 1970). This hypothesis was not probable for Nutta!!ina because males and females occmTed within close proximity of each other. Another hypothesis suggests that if an increase in females is observed in small size classes, females may exhibit earlier sexual maturity d1an males and perhaps have a high rate of mortality in large size classes (Pearse 1979). This hypothesis did not apply toN. calif'ornica and N. kata from the CUITCnt study. An increase in the number of females in small size classes of N. ca!(f'omica and N. kata was not observed. Finally, because several members of the family Chitonidae have biased sex ratios in favor of males, Otway ( 1994) 49 suggested that the greater abundance of males than females may be restricted to pmiicular chiton families. Nuttallina belongs to the fmnily Lepidochitonidae. Although it is possible that a biased sex ratio in favor of males pertains to specific chi tons families, it is clear that further research is necessary to detem1ine the exact cause of unequal sex ratios among N cal!fornica and N. kala. Mean total length ofN. califomica was greater tha11 N kata. Measurements of chiton total length were biased towards large adult animals, yet the bias was consistent between species. These results confinned previous repmis of differences in total length between the two species (Piper 1984). The largest specimen ofN. kata collected was 50 mm in length. Piper (1984) reported the maximum size of N. kata was approximately 40 mm in length from southern Califomia. Variability in morphological characters has been reported within Nuttall ina species (Piper 1984). Monterey Bay marks the northemlimit of N. kata and, thus, slight differences in morphology may be expected between individuals in Monterey Bay and those from the type locality in La Jolla, California.

Larval Development and Larval Settlement Larval development patterns of chitons are quite uniform among species (Pearse 1979, Strathmann and Eernisse 1987). Variation in developmental rates, however, has been reported within the smne species depending on envirorunental conditions, including temperature (Pearse 1979). The timing oflarval development in N kata and N. calijomica was similar to many species of chitons studied thus far. Eggs hatched approximately 20 hours after fertilization, and were competent to settle within approximately 5 days. Immediately after hatching, larvae were swimming in the water column near the surface. As development so progressed and the larvae became competent to settle, they began to spend more time near the bottom of the container, perhaps searching for appropriate substrate. Settlement did not appear to occur indiscriminately, as larvae that were not presented a suitable substrate died after approximately 2-3 weeks. Some species have been reported to delay settlement until the appropriate substrate is encountered and, in the absence of preferred substrate, settle indiscriminately rather than perish (Pearse 1979). It was previously thought that delayed metamorphosis in the absence of a settlement inducer resulted in indiscriminate settlement as larval systems began to deteriorate while the larva was in the plankton. It has recently been suggested, however, that stringency (degree of dependence) and specificity (biological and chemical resolution) do not deteriorate with aging of larvae in the plankton, and delayed metamorphosis may enhance dispersal and substrate specificity (Morse 1990). The number of settled larvae in tl1e laboratory settlement experiment may have been reduced significantly by the presence of ciliates in test containers. A great number of ciliates began to appear in the test containers within a few days after the initiation of the experiment. Although no attempt was made to quantify them, there appeared to be more ciliates in the algae and mucus treatments than in bare rock and control treatments. In many cases, the ciliates appeared to attack the larvae and many larvae died be tore settlement. Therefore, the proportion of settled larvae in these treatments may have heen artificially reduced.

A significant number of N. kata larvae settled on C. vancouveriensis with or without mucus of adult chi tons during the larval settlement experiment Several explanations for this result were possible. Although repmts oflarval settlement in response to geniculate coralline algae are limited, crustose coralline algae is thought to induce settlement in many marine molluscs, including the chiton 51

Tonicella lineata (Bames and Gonor 1973) and the abalone Ha/iotis rufescens (Morse 1985, 1990). In both cases, the alga inducing settlement was the main food source for adults. Many gastropods reportedly settle in response to soluble materials present in, or emanating from, food items (Crisp 1984). Significant amounts of the geniculate coralline alga C. vancouveriensis have been found in the guts of adult Nuttallina spp. from central Califomia (Andrus and Legard 1975). Coralline algae, including C. vancouveriensis, provide little caloric value (Littler and Littler 1984). The ingestion of C. vancouveriensis may have been the result of incidental scraping as Nuttallina spp. grazed other algae and diatoms associated with the algal mat. Because the presence or absence of mucus did not affect settlement rates on C. vancouveriensis, the cue to settle in the coralline algae mat may have been nutritional in nature. The settlement cue in the C. vancouveriensis treatments may have originated from epiphytes associated with the alga. Although N. kata juveniles may not feed on C. wmcouverie11sis, they may feed on epiphytic phytoplankton, bacterial films, or diatoms associated with the algal mat. The isolation and complete structural character of natural settlement inducers have not been identified for most species. It has been detennined, however, tl1e abalone Haliotis 111fescens was induced to settle on crustose coralline algae by a small oligopeptide (Morse 1990). It was originally repmted that the oligopeptide was algal in origin, yet Morse and Morse (1984) reported the settlement inducer could be removed from crustose coralline algae by lightly brushing the surface. An inducer emanating from crustose coralline algae most likely would not be easily removed from the surface. Because organisms such as b1yozoans, spirorbids, scyphozoans, and bivalves are known to settle in response to bacterial organic films (Garland et a!. 1985, Keough and Raimondi 1995), and unique bacterial populations occur on 52 the surface of cmstose corallines (Johnson eta!. 199 J a, 199 l b), it was hypothesized that the settlement inducer of H ruf'escens was bacterial in origin (Johnson et al. 1991 a). Although bacterial biomass is limited in marine environments and may be of minor nutritional significance, bactelia may perform essential metabolic transformations within the gut. and thus, promote larval settlement (Garland et al. 1985). Mucus from adult conspecifics induces settlement in many marine molluscs, including the abalone Haliotis spp. (Slattery 1992, Seki and Taniguchi 1996) and the gastropod Concho/epas concho/epas (Rodriguez eta!. 1995). The presence of adult mucus caused pre-competent larvae to stop swimming and drop to the substrate (Rodriguez eta!. 1995). Settling near conspecifics would promote reaggregation after dispersal and propagation of the species. Larval settlement of N kata on the cmstose coralline alga Pseudo/ithophyl/um neofarlowii occurred only in the presence of adult mucus and was significantly greater than treatments containing C. vancouveriensis. For lru-vae of N. kata, however, mucus alone was not enough to induce settlement. Biologically inert substrates with adult mucus, such as sandstone and granite, did not induce settlement. Although significantly more larvae settled in the control treatment with mucus than in the control without mucus, the difference was not biologically significru1t. Only 3% of lru-vae settled in the control with mucus. An interaction occurred between the cmstose coralline alga P. neofarlowii and the presence of adult mucus, resulting in a high proportion of settled larvae in this treatment. Laboratory experiments indicated the settlement cue of P. neof'arlowii and adult mucus of Nuttallina was stronger than the C. vancouveriensis settlement cue. The difference in intensity of settlement cues may have resulted from the position P. neojatlo1Nii occupies in the inte1tidal. Pseudolithophyllum neofarlowii is a 53 high-intertidal encrusting red alga found in pits associated with large adult N. kata. The mid-intertidaJ zone occupied by C vancouveriensis is immersed for longer periods of time and, thus, planktonic larvae have a longer exposnre time to this habitat. Because the presence of a settlement cue in the C. vancouveriensis algal mat may cause many larvae of N. kat a to drop out of the water colum11, few larvae may reach the high-intertidal area. Distribution of larvae within the water colmrm has been shown to decrease dramatically after exposure to a suitable substrate, causing variability in the distribution of settlement rates over small distances (Connell 1985, Gaines et al. 1985). fn addition, tl1e short immersion time of the P. neofarlowii habitat may limit the amount oftime larvae are exposed to the surface. The cue may need to be stronger in this habitat because few larvae are exposed to the substrate for a short peliod of time. The interaction between P. neofarlowii and adult mucus of Nuttallina in response to larval settlement may be explained by competitive processes in the C. vancouveriensis zone. Because of extremely high densities of Nutted/ina spp. in the algal mat, intraspecific competition may limit larval settlement Settlement rates of many invertebrates were reportedly affected by the density of conspecifics (Gaines et al. 1985). The bamacle Semibalanus balanoides recruits to areas in the low intertidal at densities exceeding the adult holding capacity by an order of magnitude, creating intense competition for space and causing high mortality rates in the low intertidal (Bertness 1989). ln contrast, intra- and interspecific competitive processes are less important in physically stressful habitats, such as the high intertidal, than physically benign habitats, such as the low-intertidal (Bertness I 989). A great number of larvae may settle in the high-intertidal, yet few may survive because of the harsh physical conditions (Denley and Underwood 1979). The presence of conspecifics, however, increases survival by reducing 54 desiccation and heat stress (Bertness 1989). Settlement of N. kata larvae may be limited in the physically benign algal mat because of great densities and intraspecific competition. Larvae unable to settle in algal mat may seek refuge in the high intertidal near adults. The presence of conspecifics may increase survival by reducing physical stress, while ti1e alga or associated epifauna may fulfill nutritional requirements. Because laboratory experiments cannot mimic exact conditions in ilie field, there may be other factors influencing larval settlement of N. kata. Larval settlement and metamorphosis are not random events in some species, but highly detennined by chemosensory recognition of lllOI]Jhogenic and regulatory molecules in the environment (Morse I 990). Although identifying the precise molecular nature of settlement inducers is impmiant, interactions of multiple factors also may influence larval settlement. Only a few combinations of factors were tested in this experiment and many more may be important in detennining larval settlement and distribution of adult N. kata in the field.

Density of Nuttallina within the C. vancouveriensis algal mat was much

2 greater than previously estimated. Densities were approximately 5000/m , whereas previous estimates of density in ti1e algal mat were 1500 to 2000/m2 (Piper 1984). The large discrepancy between estimates resulted fi·om differences in sampling method. In cont·ast to ti1c dcstntctive sampling method employed in the cmTent sh1dy, previous estimates relied on visual counts. Because the substrate within this habitat was extremely heterogeneous, many chitons in deep pits could not be seen from ilie surface and would have been missed in visual counts. Another source of enor in visual estimates resulted from the difficulty in seeing 55

chi tons less than 5 mm in length. Although some small chi tons were visible in the field, the majority were highly cryptic. Estimates of the density of Nuttallina were somewhat misleading, however, clue to the heterogeneous nature of the substrate. The presence of pits within the sandstone substratum created a three-dimensional habitat for Nuttallina and greatly increased surface area. Although no attempt was made to quantify actual surface area within the l 0 em x I 0 em plots, previous studies indicated a significant increase in surface area due to pit fom1ation (Louda 1972). Data from the cuJTent study provided useful infonnation on the actual densities of Nultalfina and species composition within the C. vancouveriensis algal mat. Differential growth rates may have contributed to differential distribution of size classes along the shore. Size gradients of Nutta/lina spp. along the shore in southern California have been explained by slow growth rates of N. kala in the C. vancouveriensis algal mat (Piper 1984). During the cmTent study, the majority of chi tons within the algal mat were significantly smaller than the maximum size for each species. Although mean size of N kat a was less than N. flux a, mean size of N. kata was greater than N. californica. These results indicated the differential size distributions along the shore applied to all tlu·ee Nuttallina species from the intertidal region of Stillwater Cove. Differences in size of Nuttallina spp. along the shore may be due to different growth rates associated with specific habitat<> rather than differences in growth rates among species. Estimates of mean size of Nuttalfina spp. within the algal mat were somewhat misleading based on measurements ofFVW. Because valves ofN. fluxa were wider per body length than valves of N. kata and N. cal!fornica, individuals of N fluxa appeared to be larger than individuals of N kala and N. caltfornica of similar size. Distribution of the three Nuttall ina species among 56 yearly age classes in the algal mat was relatively similar based on conversions of FVW measurements to approximate age in years. Although individuals of N. jluxa appeared to be large in size, no specimen of N. fluxa was estimated over 3 years in age. Differences in age were most likely not significant between N. californica and N flw.:a. Statistical analyses were not conducted on yearly age classes, however, because the estimates of age could not be continued. A linear regression of data from large and small size classes was used to estimate total length of N. califomica from FVW measurements even though significant differences were detected between the two equations. The slopes of the equations were similar, and the discrepancy between the two equations may have resulted from differences in growth rates between juveniles and adults. Juveniles of Nutwllina spp. have faster growth rates than adults and growth rates appear to be asymtotic (Piper 1984). Because data from all size classes were not available, however, estimates of age for N cal!fomica may be inaccurate. Nuttallina spp. within the algal mat can be categorized as "K strategists," organisms that tend to live in stable habitats at or near their saturation level (Wilson and Bosse1i 1971). These organisms have a competitive ability to occupy an environment for long periods and utilize the energy produced within the envirorunent (Wilson and Bossert 1971 ). Because Nuttallina spp. may have been at or near their saturation level in the algal mat, slight changes in mean density may have had an impact on mean size. Of the tlu·ee areas studied at Stillwater

Cove, the area with the lowest mean density of Nutted/ina spp. also contained the largest mean size. If resources available in the algal supported a specific biomass of chitons, density of Nuttallina spp. may have been negatively related to size. Approximately 90% of chitons within the algal mat were N. kat a. Although species composition of Nuttallina within the algal mat previously had not been 57

detem1ined, it was estimated that N kata was the most abundant chiton in the algal mat in southem California (Piper 1984). Nuttallina ca/ifornica and N jluxa were found in the algal mat at very low densities. In contrast toN. kata, N. califomica and N. flux a may not have recruited to the algal mat. One new recruit of N. calif'omica was found among all samples collected, whereas recruits of N. flux a were absent fi·om the algal mat. Because sampling occulTed at one point in time, the recruitment period for these species may have been missed. The low number of adults, however, indicated individuals of N. califomica and N. fluxa may not recmit to this habitat. Although many new recmits were present at the time the substrate was sampled, recmitmcnt was not equal among areas. Settlement and recmitment of benthic marine organisms often varies considerably in space and time (Connell 1985, McShane 1991 ). Differences in recmitment among areas may have been due to biological and physical factors, such as local cunents or eddies adjacent to the substratum, abundance of larvae in water column above the substratum, and predation (Stratlm1ann and Branscomb 1979, Connell 1985). Substrate texture and availability of microhabitats also may influence larval settlement and survival (Emson and Faller-Fritsch 1976, Raffaelli and Hughes 1978, Raimondi 1988, James m1d Undervvood 1994). Post-settlement mmiality also may have influenced the number of recruits among areas. Mmiality during the early juvenile period is often high for mm1y mm·ine invertebrates and may be impmiant in detem1ining distribution and abundance of species (Gosselin and Qian 1996, David eta!. 1997). Because density-dependent moJiality may have a significant impact at increased densities (Gosselin and Qian 1996), intraspecific competition of Nuttallina in tl1e C. vancouveriensis habitat may have limited the growth and survival of recruits. The 58 large number of new recruits in each area, however, indicated that larval supply was not limited. The size of adult Nuttallina also may have negatively influence post settlement survival oflarvae (Louda 1972). Although mean size of Nuttallina was only slightly greater in area 2 than in areas I and 3, the difference may have had a significant negative impact on survival of recruits. Grazing activities of mobile invertebrates are detrimental to survival ofrecmits of some species of invertebrates (Zamorano et al. 1995). Although the exact reason for lower post settlement survival in area 2 could not be determined in the present study, movements oflarge Nuttallina spp. and limited resources were likely causes of mortality.

Reduction in Density, Altl Assemblages, and Growth {ate Reduced densities of Nuttallina spp. in the mid to low intertidal of Stillwater Cove did not have a significant impact on the algal assemblage. Decreases in Nutta/lina spp. density, however, resulted in a slight increase in abundance of fleshy macroalgac. Increased growth of C. vancouveriensis due to branching also was observed in plots with reduced densities of Nuttallina spp. If the experiment had been conducted for a longer period of time significant increases in macroalgal growth may have been observed in treatments with reduced densities of Nuttal!ina spp. The importance of Nuttallina spp. in determining community structure previously has been reported from southem Califomia (Bany 1988). Intense grazing by Nuttall ina spp. prevented settlement and growth of spores and gem1lings of macroalgae in the C. vancouverieusis algal mat. Reduced densities of Nuttallina spp. enabled fleshy macroalgae to colonize the algal mat (Bany 1988). 59

Over a long time period, macroalgal growth completely excluded Nuttallina spp. fi·om the mid to low intertidal by occupying the substratum and eliminating space for attachment (Bany 1988). The abundance of intertidal limpets also was reduced dramatically due to a lack of substrate on which to adhere (Barry 1988). Limpets and other species of chitons significantly impact algal distribution and abundance by grazing microbial films, and spores and gennlings of macroalgae (Branch 1981, Dethier and Duggins 1984, 1985, Black et al. 1988). The upper limit ofmacroalgae often is limited by grazing of intertidal invertebrates (Underwood and Jemakoff !981 ). Although the absence of grazers may allow some algal species to colonize areas in the intertidal above their usual limits, most do not grow to maturity due to physiological stresses of the habitat (Underwood and Jemakoff 198 I). Because coralline algae are often resistant to grazing (Littler and Littler 1984), it is often one of the few types ofmacroalgae located in areas with increased densities of chi tons and gastropods (Scheib ling 1994). Great densities of Nuttallina spp. in the mid to low intertidal may be necessmy to maintain community stmcture of the C. vancouveriensis algal mat Although previous studies have alluded that growth of Nuttallina spp. may be limited within the algal mat due to great densities (Louda 1972, Piper 1984 ), the advantages of great densities may outweigh the disadvantages of a reduction in growth rate and a loss of habitat. Increases in mean size of Nuttall ina spp. may have been sacrificed for increases in mean density. In contrast, great densities in the high intertidal may not be necessary to maintain the habitat Algal growth may be limited in the high intertidal due to harsh physieal conditions (Unde1wood and Jemakoff 1981 ). Food resources available in the high inteJ1idal, however, may not be abundant enough to support great densities of Nuttctllina spp. Food resources and stresses of the physical habitat may have been limiting factors for Nuttallina 60 spp. in the high intertidal, whereas space and food may have been the limiting factors in the low intertidal. An unexpectedly low number of individually tagged chitons were retrieved during the mark and recapture experiment. Although Piper ( 1984) reported nearly 50% recovery of tagged Nwtallina spp. in southem Califomia, less than 1% of tagged chi tons were recaptured in the current study. One reason for the difference in recovery rates between these studies may have been the size of chi tons that were tagged. Chitons Jess than I 0 mm total length were tagged to include a wide range of sizes. The smallest chiton recaptured in the cunent study was 17.7 mm in total length, and only two recaptured chi tons were less than 20 mm in total length. Piper (1984) only tagged chitons that were greater than25 mm in total length. Because tags were attached through the girdle, and small chitons had a significantly smaller girdle area than large chi tons, tl1e chance of a tag pulling out of the girdle due to strong wave action or stuface drag was greater in small tlmn Iarge c hitons. Secondly, reactions of other organisms to the tags may have increased tag loss and predation rates on tagged Nuttalli11a spp. Altl10ugh tags were made as inconspicuous as possible, significant diatom growth began to occur on the monofilament within days after chitons were tagged (pers. obs.). Small crabs (Pachygrapsus crassipes) were observed witl1 detached tags in their pinchers on a few occasions. Although crabs were not observed pulling tags out of chiton girdles, it was possible that tags were removed as the crabs hied to feed on small ctustaceans located in the diatom growth. Predators also may have been attracted to the tags and increased predation rates on tagged Nuttallina spp. Many authors have reported problems tagging chi tons (Boolootian 1964, Brousseau 1979, 61

Rhoads and Lutz 1980). Although the method of Piper ( 1984) may be suitable for large chi tons, the method needs to be revised for small chitons. Previous reports of growth rates of tagged Nuttallina spp. from sou them Califomia (Piper 1984) were greater than growth rates obtained from the cunent srudy. Inconsistent growth rate data from individually tagged chi tons may have resulted fi·om the short period of time during which the study was conducted. Data collection in the cmrent study extended only 6 months due to unfavorable weather conditions during winter and early spring. Piper ( 1984) collected growth rate data for approximately 2 years and recaptured nearly 50% of the chi tons that were tagged. E1rors in measurements also may have resulted in inconsistent growth rates. Because a great number of chitons were tagged in a short pe1iod of time, many people were involved in the tagging process, resulting in inconsistent measurements. The season of data collection also may have impacted grov,'th rates in the cunent study. Summer has been detennined as a period of minimal growth for Nuttallina in southem Califomia (Piper 1984). Tagging was initiated during spring 1997 and the experiment originally was to be conducted for an entire year. Because of winter stonn activity dm·ing 1997, however, the expe1iment was concluded after 6 months to minimize the loss of data. An additional 6 months of data collection may have reduced valiability in growth rates of tagged chi tons. Inconsistent growth rates most likely resulted from the short sampling period and the season of data collection rather than a negative effect of the tags. CONCLUSIONS

Nutta!liua kata exhibited discrete spawning events in spring and autmnn during the study period, whereas N calif'ornica was reproductive throughout the study period. Maximal gonadal growth for both species occurred during autumn and winter. The timing of spawning events for both N. kata and N californica differed among years. Spawning events were synchronous within the species, but most often asynchronous between species. The synchronization of spawning within species indicated external factors may have influenced spawning, and the asynchronous spawning between species indicated the cue may have been species specific. Larval development of N. kata and N. cal!t'ornica was similar to many species of chi tons studied thus far. Eggs hatched approximately 20 hours after fertilization and larvae were competent to settle within 5 days. Larvae of N. kata did not settle indiscriminately; settlement occtmed in the presence of C. vancouveriensis with and without adult mucus, and in the presence of P. neofarlowii with adult mucus. Density of Nuttallina spp. in the C. vancouveriensis algal mat was

2 approximately 5000 m· . Nuttallina kata was the most abundant chiton in the alga mat, comprising nearly 90% of all Nuttallina. Slight increases in mean density of Nuttallina spp. within the algal mat were related to decreases in mean size, indicating Nuttallina spp. may have been at or near its satmation level in the algal mat. Significant recruitment of Nuttallina spp. occurred in the algal mat. The majority of Nuttall ina spp. in the algal mat were estimated at less than 2 years old. Grazing activities of Nuttallina spp. within the algal mat did not 63 significantly limit the growth of t1eshy macroalgae. A slight increase in the abundance of macroalgae was apparent with a reduction in the density of Nuttallina spp. Although growth of Nuttall ina spp. may have been limited in the algal mat, the great densities may have been necessary to maintain the habitat. A settlement cue for larvae ensures the propagation of the species in the C. vancouveriensis algal mat. REFERENCES

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