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

by

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. kat a and N. cal!fornica and other inteiiidal chitons . 28

2. Two-factor ANOV A of larval settlement rates of N. kat a, 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

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

2

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. .flux a 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 kat a (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. .flux a. Valves

are triangular to rounded triangular in shape. Nutta11ina kat a lives lower in the

intertidal than N. ca!if'ornica and N. .flux a 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.

Stillwater Cove ,_ •co

•

Carmel Bay

Coralli11a vancouveriensis algal mat

•

Cam1el Point

•

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.

7

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

8

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

9

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

settle. Fe1iilized eggs and larvae were examined under a compound microscope

approximately twice daily to monitor development.

10

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

(MANOV A) 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. kat a, 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

45

40-

35 ~

bo ~

..<=

'" 2' ?5 "--.]

<= _.g 20 ..<= u "@ -0 I-

15

10

5

0 I

0

rt 00

0 Cb

2 4

0

0 00

0 0 ° 0

0 0 0

<Ill 0

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0 0 0 0 <!DOCXD 0 0

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00 {]!)

6

em oo OCJ!D®

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8

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10 Fourth Valve Width (mm)

12

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

15

organisms around the manipulations, but the fences may have inhibited or

enhanced movements of some species.

16

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 ANOV As 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 A NOVAs 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 ANOV A, 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

~

Y,

" ""' " --;;; "V

"' § 0 ~

" ::: ·:::;; 0 >--<

11 ~ 60 n 60

f(x) ~ 0.00 16x + 0.238 f(x) 0.0017x + 0.175 0.5 R''2 ~ 0.0012 0.5

R"2 0.039 0.4 - 0.4

0.3 Ao 0.3 o eo o -

~ .. 0 G-0~~0 © oo

0.2 0 0 0 02 0 0

0.1 0.1 -

0 0 0 2 3 4 5 25 30 35 40 45 50 55

n= 50 11 =50 f(x) ~ 0.0092x + 0.237 f(x) 0.0029x + 0.152

0.5 R"2 = 0.049

0.5 W·2 0.075

0.4 ., 0.4

Ill •• ~···§I" -0.3 - .: ~· .,. .. 0.3 -r1:;• I ~ 0.2 --~· ,·~ 0.2 - .1111iai • • ;:: . "' :.

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.

20

21

~

~ 0 L_------------------------------~-------------~~--.= M A M .J .TASOND.TFMAM.TJASO -;;; "0

" " 8 8 -b. N. kala

7

6

5

4

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; mean Jan 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 <mel gonad lacking gametes). A slight increase in mean

gonadal index was recorded in June, but few changes in mean gonad indices were

observed from May through August 1996. Gonad indices declined slightly in

September and October, and a complete spawn occunecl between mid October and

early November 1996 (Mann-Whitney test between October and November;

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

24

U1005 iili(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.

10

9

8

7

X 6 "' -

"0

" -'" 5 "0 rn

" c3 4

3

2

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.

25

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. kat a 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

40 a. N. kata n=209

• males 35

30 D females

?" _)

20

15

10

:>, 5 u §

0 :J

"'" "' '" ;;;;..

30 b. N. cal~fomica n 199

r _,

20

15

10

5

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 and Nuttallina lineata ciliata muscosa lignosa

califomica (I) (2) (3) (3)

Fertilization 0 hrs 0 hrs 0 hrs 0 hrs 0 hrs Hatching 20 hrs 48 hrs 36- 42 hrs :Whrs 19 hrs Larval Eyes 60- 70 hrs 110 hrs N!A 84hrs 72- 8411TS Shell Glands 60 70 hrs 120 hrs 96- 120 hrs 72 hrs 96 hrs Foot/Muscle 110-liShrs 130 hrs N/A N/A N!A Contractions Competent to 150- 160 hrs !50- 160 hrs 144 hrs NIA J 20 hrs Settle (I) Barnes (1972), (2) Thorpe (1962), (3) Watanabe and Cox (1975), and (4) Rumrill and Cameron (1983)

Katharina tunicata

(4)

0 hrs N!A N/A N/A N/A

144 hrs

N 00

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.

29

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

Substrate Mucus Substrate*Mucus

Error

ss

0.65021 0.14872 0.32792

df

5 I 5

0.48582 48

MS

0.13004 0.14872 0.06558

0.01012

F-ratio

12.84826 14.69375 6.47976

P-value

0.00000 ** 0.00037 ** 0.00011 *

0.6

0.5

~

v t:

·;;; 0 OA ~

-< ~·

" "' c: "' ...I

"0 0.3 " -;::: 1U

(J']

t: 0

'E D.2 0 0.. Q ~

"'"

D. I

()

*

Geniculate coralline

algae

*

Crustose coralline

algae

**

Fleshy

algae

• Treatments with mucus

D Treatments without mucus

Sandstone rock

Granite rock

Control

32

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 MANOV A. 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 (mean 1 = 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 = 1 0) 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 = 1 0).

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. kat a 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 (~10w1.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

~

"' < E

8

7

'-' 5 0 0

Area I Area 2

0 N. kala

• N. cali/omica

GJill N. jluxa

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.

35

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.

36

Table 3. ANCOV A 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

Size Class FVW

Error

ss

307.86270 678.20019

656.75889

df

I I

88

MS F-ratio P-value

307.86270 41.25093 0.00000* 678.20019 90.87295 0.00000*

7.46317

37

(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.

38

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 - II N. californica 300 -

CJ N. .f/uxu

200 f-

,--100 -

0 n.

I

200 Area 2

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

>:..

50

0

800 - Area 3

600 -

400 -

200 - -0 1 .......

1 ........ ~

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.

12 f(x) ~ 3.763x + 1.053

R'2 ~ 0.696

10 n =84 0

0 0 0

0 ~ 0

'~ 8-.c :;) "

0 0

" 0 -l 6 § 0

] u a 4 0 f-

0

2-

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.

40

3.5

3

0.5

Area I Area 2

0 N. kala

U Unidentified

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.

41

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 =

42

0.323; Fnnnl = 0.040, Pnnnl = 0.845), fleshy algae (Fini<ial = 0.078, Pinitinl = 0.784; Fnnal

= 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).

Ill Control

I .2 a. Comlline algae

0.8

0.4

~ ~ 1 .2 c. Sessile invertebrates ~

"' ~

0.8-

0.6

OA

0 Decreased Density D Natural Density

I .2 b. Bare rock

0.8

l.2 d. Fleshy macroalgae

April I 997 October 1997 Aprill997 October 1997

43

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. kat a 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

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.

46

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).

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.

47

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.

kat a or N. californica, the asynchronous spawning bet\veen species indicated N.

kat a and N. ca!if'ornica may have been ini1uenced by different species of

phytoplankton present during different oceanographic seasons.

48

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. kat a 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

greater than previously estimated. Densities were approximately 5000/m2,

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. kat a 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

large number of new recruits in each area, however, indicated that larval supply

was not limited.

58

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

spp. in the high intertidal, whereas space and food may have been the limiting

factors in the low intertidal.

60

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

I arge 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. kat a 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

approximately 5000 m·2. 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.

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