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Notice: ©1988 Elsevier Ltd. The final published version of this manuscript is available at http://www.sciencedirect.com/science/journal/00220981 and may be cited as: Young, C. M. (1988). Ascidian cannibalism correlates with larval behavior and adult distribution. Journal of Experimental Marine Biology and Ecology, 117(1), 9-26. doi:10.1016/0022-0981(88)90068-8

J. Exp. Mar. Bioi. £Col., 1988, Vol. 117, pp. 9-26 9 Elsevier

JEM 01042

Ascidian cannibalism correlates with larval behavior and adult distribution

Craig M. Young Department ofLarval Ecology. Harbor Branch Oceanographic Institution, Fort Pierce, Florida. U.S.A.

(Received 24 March 1987; revision received 9 December 1987; accepted 22 December 1987)

Abstract: In the San Juan Islands, Washington, solitary ascidians .that occur in dense monospecific aggregations demonstrate gregarious settlement as larvae, whereas species that occur as isolated individuals do not. All gregarious species reject their own eggs and larvae as food, but nongregarious species consume conspecific eggs and larvae. Moreover, the rejection mechanism is species-specific in some cases. Correla­ tion analysis suggests that species specificity of the rejection response has a basis in siphon diameter, egg density, and larval size, but not in number of oral tentacles, or tentacle branching. One strongly cannibalistic species, Corella inflata Huntsman, avoids consuming its own eggs and newly released tadpoles by a unique brooding mechanism that involves floating eggs, negative geotaxis after hatching, and adult orientation.

Key words: Ascidian; Cannibalism; Distribution; Larva; Settlement behavior

INTRODUCTION

Many sessile marine invertebrates, including filter-feeders such as mussels, oysters, barnacles and ascidians, occur in discrete, dense aggregations. Gregarious larval settle­ ment (defined here as preferential attachment on or near established conspecifics) probably explains aggregation in many species (reviewed by Meadows & Campbell, 1972; Scheltema, 1974; Crisp, 1976) but gregarious settlement is only one of many processes that result in small-scale aggregations among sessile invertebrates. Others include spatial variability in mortality of juveniles or adults (Keough, 1986; Young & Chia, 1984), orientation responses that result in nonrandom distribution oflarvae in the plankton (Grosberg, 1982), larval responses to habitat cues not associated directly with adult con specifics (Scheltema, 1974), and hydrographic processes controlling the supply of larvae (Gaines & Roughgarden, 1985; Butman, 1987). Although postsettle­ ment processes can change the grain size or intensity (sensu Pielou, 1977) of an aggregation by eliminating individuals, small-scale clumps cannot be formed by any mechanism unless recruitment initially occurs (either preferentially or otherwise) near con specifics. Moreover, long-term maintenance of a clump implies recruitment to offset adult mortality.

Contribution 614 of Harbor Branch Oceanographic Institution. Correspondence address: C. M. Young, Department of Larval Ecology, Harbor Branch Oceanographic Institution, 5600 Old Dixie Highway, Fort Pierce, FL 34946, U.S.A.

0022-0981/88/$03.50 © 1988 Elsevier Science Publishers B.Y. (Biomedical Division) 10 C.M. YOUNG

Paradoxically, some of the most formidable predators on planktonic invertebrate larvae are the same benthic filter-feeders that form dense, long-lived aggregations (reviewed by Thorson, 1950; 1966; Young & Chia, 1987). Predation by such filter­ feeders may amount to cannibalism, since conspecific larvae (including kin) are often not distinguished from other food (Cerruti, 1941; Thorson, 1950; Barnes, 1959; Timko, 1979; Young & Gotelli, 1988). In this respect, larval cannibalism can be viewed as a process that counteracts the mechanisms by which aggregations are established and maintained. Intuition dictates that larvae should not recruit into dense aggregations of adult filter-feeders (Thorson, 1950; Woodin, 1976; Cowden etal., 1984; Young & Gotelli, 1988), but such aggregations often include young individuals. Mechanisms by which larvae escape consumption by adults are generally unknown. Have specific mechanisms evolved for minimizing the impact of intraspecific predation on larvae? If so, are the mechanisms present in the adult stage or the larval stage? Larvae of intertidal mussels often settle on filamentous substrata outside adult clumps (Block & Geelen, 1958; Bayne, 1965); juveniles then migrate passively into the clump after they have grown too large to be eaten (Bayne, 1964; Seed, 1969). Eggs of the sand dollar Dendraster excentricus may be protected from adults by their gelatinous coats (Timko, 1979), but this observation begs the question of how larvae (without jelly coats) settle into dense beds of adults (Highsmith, 1982) without being consumed. Although a number of presumed larval defense mechanisms have been documented (reviewed by Young & Chia, 1987), none functions solely in the prevention of cannibalism and very few are thought to be effective against large filter-feeders (Cowden et al., 1984). Larval cannibalism has not previously been documented in the Ascidiacea, a diverse group of benthic filter-feeders. In the present paper, I show that cannibalism occurs in several of the 13 species of solitary ascidians living in subtidal habitats in the San Juan Islands of Washington State and demonstrate that the incidence oflarval cannibalism correlates with small-scale distributional patterns and larval behaviors of the various species. I also discuss the morphological basis of larval rejection, show how brooding reduces cannibalism in one species, and argue that mechanisms preventing cannibalism are appropriate preadaptations for the evolution of gregarious settlement behavior.

MATERIALS AND METHODS

SOURCES OF ADULT AND LARVAL ASCIDIANS Ascidians were collected by dredging or diving in waters surrounding the San Juan Islands, Washington (map in Young, 1985). One species that occurred rarely in the San Juans, Styela montereyensis (Dall), was collected from intertidal pilings in Neah Bay, Washington. Throughout the study, adults of most species were maintained in large running seawater aquaria at Friday Harbor Laboratories, where they filtered actively and survived for long periods of time (over a year in some cases), feeding on plankton ASCIDIAN CANNIBALISM AND LARVAE 11 that entered through the seawater system. These adults were used as predators in experiments, generally without removing them from their tanks or otherwise disturbing them. Larvae were reared in vitro from gametes spawned in the laboratory or dissected from adults (Young, 1982). Following fertilization, cultures were washed repeatedly to remove excess sperm, then maintained in fingerbowls of filtered seawater resting in shallow, running seawater aquaria to control temperature. The water in each culture was changed several times per day until the tadpoles hatched. Larvae were always used within 24 h after hatching.

PREDAnON ON GAMETES AND LARVAE

To assay larval predation in the laboratory, I gently pipetted larvae or eggs, one at a time, into the incurrent flows of filtering adult ascidians. Reactions of the adults were observed. Expulsion of larvae or eggs was easily noted, as it was accompanied by a major contraction of the body wall muscles of the adult. In transparent species (Corella injlata Huntsman, C. willmeriana Herdman), it was possible to observe the fates of larvae through the tunic, using a dissecting microscope. If eggs and larvae of all 12 species were offered to adults of all species, 288 different combinations would have to be attempted with sufficient replication for analysis. Because of time constraints and the unavailability of embryos from all species during appropriate work periods, only a few of the potential heterospecific trials were attempted. Spermatozoa removed by dissection from four ascidian species were diluted in seawater, filtered through 100-.um Nitex to remove large particles, then introduced by pipette into the siphons of adult ascidians. Rejection responses were noted, as explained above.

LARVAL BEHAVIOR

As part of a larger study of comparative larval behavior (Young & Braithwaite, 1980; Young, 1982; Young & Chia, 1985), I investigated substratum preferences of larvae by offering multiple substrata (including adult tunic, rocks, shell fragments, etc.) to groups oflarvae in Petri dishes. Forthis paper, I willpresent only the data comparing settlement on adult tunic and rock, as this comparison gives a simple estimate of gregariousness. Nevertheless, in each experiment, either three or four substratum types were replicated within dishes, arrayed in a Latin square and held in place by agar. During the study, it became apparent that the consistent adult field orientation of Corella injlata (Child, 1927)could reduce the likelihood of cannibalism on an individual's own progeny. I investigated whether the adult orientation originated at settlement or later by allowing tadpoles to settle on a vertical glass plate in a large culture. After settlement, juveniles were cultured vertically on the same plate until their siphons opened. Orientations of the mid-sagittal plane (line between siphons) were then recorded using a dissecting microscope. 12 C.M. YOUNG

DISTRIBUTION, POPULATION STRUCTURE, AND ORIENTATION OF ADULTS Small-scale spatial patterns of adult ascidians were investigated by several tech­ niques. On rocky subtidal cliffs and in boulder fields at Bell Island, Pt. George, Shaw Island and Cantilever Pt., San Juan Island, grids or transects of contiguous quadrats were photographed with a 35-mm camera in an underwater housing. A quadrat attached to the front of the housing delimited a 25 x 25-cm area. Several kinds of distributional data were taken from these data sets (Young & Chia, 1982; Young, 1985, 1986). For the present study, I tabulated the number of ascidians attached to various kinds of substrata, including their own adults. Species that were too uncommon to appear in the photographic quadrats were observed whenever they were encountered. Over a 3-yr period, the substrata occupied by these uncommon ascidians were recorded routinely during dives on rocky slopes, in boulder fields, on rock cliffs, and on floating docks. Substratum use of ascidians living on muddy bottoms was recorded from hundreds of aggregations dredged by the RjVHydah. In Corella injlata, orientations of individuals on vertical surfaces were recorded on an upright slate by drawing arrows to indicate the mid-sagittal planes. After the dive, these drawings were translated to quantitative measurements using a protractor. Size-frequency distributions of species occurring in discrete clumps were taken from aggregations collected at single sites. Individuals were wet weighed or, where this was not possible, measured with calipers. Where sizes are reported as linear measurements, the measurement selected was strongly correlated with wet mass (e.g., Chelyosoma produetum Stimpson, mid-sagittal width-wet mass correlation, n = 76: r = 0.925, P < 0.001).

RESULTS

ASCIDIAN DISTRIBUTION AND LARVAL BEHAVIOR The small-scale distributional patterns of subtidal ascidians in the San Juan Islands (as described in detail in Young & Braithwaite, 1980; Young, 1982; 1985; 1986) are summarized in Table I by a simple measure of aggregation: occurrence on the surface of con specifics. Several general patterns are represented. Some species, such as Aseidia paratropa (Huntsman), Cnemidoearpafinmarkiensis (Kiaer) and Haloeynthia aurantium (Pallas), nearly always occur as isolated individuals. The opposite extreme is represented by Pyura haustor (Stimpson) and Chelyosoma produetum, both of which form discrete single-species aggregations in which virtually all members of a clump are attached to conspecifics. Corella willmeriana and Corella injlata are often found in discrete clumps but the animals are generally attached to a common substratum, not to each other. Eoltenia villosa (Stimpson) sometimes attach to conspecifics, but more often occur as epizooites in multiple-species aggregations (Young, 1986). Styela gibbsii (Stimpson) and Chelyosoma produetum both aggregate in the rocky subtidal, but do so ASCIDIAN CANNIBALISM AND LARVAE 13

TABLE I Occurrence of juvenile ascidians on conspecific adults in rocky subtidal sites, muddy subtidal sites, and floating docks and pilings. Data are summarized from Young (1982), Young (1986), Young & Braithwaite (1980), and unpubl. data. + + +, adults used as substratum more often than would be predicted on basis of available surface areas (tested by goodness of fit statistics; see Young, 1982); +, juveniles sometimes found on conspecific adults but use other substrata more frequently; 0, adults never used as substratum; -, species does not occur in habitat.

Family and species Occurrence on tunic of conspecific

Rocky subtidal Muddy subtidal Docks and pilings

Ascidiidae Ascidia callosa 0 +++ Ascidia paratropa 0 0 0 Corellidae Corella inflata 0 0 0 Corella willmeriana 0 0 0 Chelyosoma productum + +++ +++ Styelidae Styela gibbsii + +++ +++ Styela montereyensis + + Cnemidocarpa finmarkiensis 0 0 Pyuridae Pyura haustor +++ +++ +++ Boltenia villosa + + + Halocynthia igaboja + + Halocynthia aurantium 0 0 to a much greater extent in space-limited habitats such as floating docks (where virtually no primary space is available) and muddy subtidal habitats where other ascidians constitute the major 'hard' surface for settlement. Each of the four families represented in the fauna has at least one species that forms mono specific aggregations and one or more species that do not. Monospecific clumps of Pyura haustor, Styela gibbsii, and Chelyosoma productum generally include many small individuals (Fig. I), and these are generally attached to the larger ones. Thus, clumps are formed by recruitment of larvae onto established individuals, not by simultaneous settlement of larvae on a common underlying substra­ tum. By contrast, size-frequency distributions of Corella injlata, Corella willmeriana, and Styela montereyensis individuals taken from discrete clumps tend to be unimodal. Styela montereyensis sometimes attaches to conspecifics, but clumps of the two Corella species are composed of many individuals of approximately the same size all attached near each other (Fig. 1). Table II gives the number of tadpoles selecting adult tunic and rock in experiments where at least three substratum types were offered. Two species from each family were tested. Styela gibbsii, Pyura haustor, Boltenia villosa, and Corella willmeriana demon­ strated strong preferences for adult tunic over rock. Chelyosoma productum may be added to this list, as it is known to be strongly gregarious from previous work (Young 14 CM. YOUNG

& Braithwaite, 1980). Only two species (Cnemidocarpa jinmarkiensis, and sometimes Corella infiata) preferred rock over tunic. The remaining species appear not to discriminate among substrata. In general, the species that demonstrate gregariousness in the laboratory (Table II) also occur in field aggregations (Table I) in which individuals of many sizes are represented (Fig. 1).

24 24 Corello inf/oto Corello Styelo montereyensis willmeriono 20 Friday Harbor, Wa. 40 20 Neah Bay, Wa. June 7, 1979 Bremerton, Wa, April 25, 197B 16 16 30 January 8, 1981 12 20~ 8 III rll I: 4 JljJfll0~4 65 15- {5 3:5 4.5 5.5 r 2 3 4 "r--/le- 10 12 14 16 LENGTH (em) LENGTH (em) LENGTH (em) 50 Styelo glbbsli 72l Chelyosomo prodoctum 40 Garrison Bay, Wa 60 Fridoy Harbor, Wa, February 24, 1978 1 November 24, 1980 >- I 1 48~ ~ 30~1 I 36J :::J 20 er 24 CD ... 10 U. 12

15 02 0.6' 1.0 -- 14 1.8 2:2 2.6 ANTERIOR MID-SAGITTAL WIDTH (em)

50 Pyuro naustor Garrison Boy, Woo 40 1 March 6, 1978 30

20

10

2

Size Class Fig. 1. Representative size-frequency distributions of ascidians occurring in discrete monospecific aggregations.

Taking the field and laboratory data together, all species in the assemblage can be assigned to one of three behavioral categories, where the classification is based on the tendency to settle gregariously and/or occurrence of discrete field aggregations. 'Strongly gregarious' species include those that demonstrate a laboratory preference for adult tunic and consistently occur in aggregations under field conditions. Three species, Pyura haustor, Styela gibbsii, and Chelyosoma productum are assigned to this category. ASCIDIAN CANNIBALISM AND LARVAE 15

Slightly gregarious species, Styela montereyensis, Boltenia villosa, Ascidia callosa Stimpson, and Halocynthia igaboja Oka, all attach to conspecific tunic in the field under some circumstances, but their larvae are not gregarious in the laboratory. Nongregarious

TABLE II Substratum choices of ascidian tadpoles offered rock and tunic of adult conspecifics in vitro. These data are extracted from larger experiments in which three or four substrata were offered. G statistic tests departure of settlement distribution from expected I : I ratio. All G values have been adjusted by Williams' correction.

Family and species of larva Experiment Numbers of larvae settled G P

Adult Rock

Ascidiidae Ascidia callosa 6 4 0.383 <0.75 Ascidia paratropa 37 34 0.125 <0.75 Corellidae Corella inflata I 125 258 47.100 <0.005 2 7 4 0.793 <0.5 3 17 12 0.852 <0.5 Corella wil/meriana I 75 63 1.041 <0.25 2 95 64 6.064 <0.025 Styelidae Stye/a gibbsii 1 38 9 19.046 <0.005 2 43 19 9.461 <0.005 3 12 12 0.000 1.0 Cnemidoearpa finmarkiensis I 9 20 4.197 <0.05 Pyuridae Pyura haustor I 218 13 219.689 <0.005 2 422 25 426.437 <0.005 3 80 0 105.859 <0.005 4 146 I 191.811 <0.005 Boltenia vil/osa I 12 5 2.885 <0.1

species include Ascidia paratropa, Corella inflata, Corella willmeriana, Cnemidocarpa jinmarkiensis, and Halocynthia aurantium. Corella willmeriana is assigned to this category despite its slight gregariousness in the laboratory because juveniles do not occur on adults in the field, and juveniles attached to adults in the laboratory die shortly after settlement (Young, unpubl. data).

PREDATION ON CONSPECIFIC EGGS AND LARVAE Consumption of eggs and/or larvae by conspecific adults was studied in all 12 species (Table III). Several species rejected their eggs or tadpoles in all or nearly all of the trials. These included all of the strongly gregarious species and one slightlygregarious species, Ascidia callosa. With the exception of Cnemidocarpajinmarkiensis, which rejected 90% of the eggs offered, the 'not gregarious' species all accepted conspecific eggs and larvae 16 CM. YOUNG readily (Table III). The 'slightly gregarious' classification included strongly cannibalistic species, such as Halocynthia igaboja, and two species, Boltenia villosa and Styela montereyensis, that accepted larvae in some trials and rejected them in others.

TABLE III Percentages of conspecific eggs and larvae consumed by adult ascidians in laboratory trials. Behavioral classification is based on data in Tables I and II. Species are classified as "slightly gregarious" if they ever occurred on conspecifics in field, regardless of whether or not they demonstrated labortory preferences for adults. Species that never attached to conspecifics in field were classified as "not gregarious", even if they showed slight gregarious tendencies in laboratory (e.g., Corella willmeriana).

Behavioral classification Eggs eaten Larvae eaten and species % n % n

Strongly gregarious Pyura haustor 0.00 175 0.00 150 Styela gibbsii 20.00 25 0.00 25 Chelyosoma productum 0.00 100 0.00 100 (x ± SD) 6.67 ± 11.55 0.00 ± 0.00 Slightly gregarious Styela montereyensis 20.00 100 Boltenia vil/osa 45.00 220 24.00 25 Ascidia callosa 0.00 25 0.00 25 Halocynthia igaboja 100.00 50 (x ± SD) 48.33 ± 50.08 14.67 ± 12.86 Not gregarious Ascidia paratropa 100.00 50 100.00 50 Corella inflata 100.00 470 99.00 312 Corella wil/meriana 100.00 25 100.00 25 Cnemidocarpa finmarkiensis 10.00 10 Halocynthia aurantium 100.00 50 100.00 50 (X ± SD) 82.00 ± 40.25 99.75 ± 0.50

The differences among mean percentages of con specifics consumed for the three groups was compared by separate one-way ANOVAs on arcsine-tranformed variates for eggs and larvae. There was a highly significant difference among means for larvae eaten (F = 99.35; 2,7 df; P < 0.001) but not for eggs (F = 3.93; 2,8 df; P < 0.1). Two outliers, H. igaboja and C. finmarkiensis appear to have provided the variation that renders egg data nonsignificant. Neither of these species was used in the larval trials. With only these two exceptions, species that occur as epizooites in the field and demonstrate gregarious larval behavior reject their larvae significantly more often than species that occur as isolated individuals in the field and do not select adult tunic during the larval stage. The entire process of consumption or rejection was observed easily in ascidians with large, short incurrent siphons (e.g., Halocynthia igaboja, Styela gibbsii) and in species with transparent tunic (Corella willmeriana, Corella inflatai. The mechanism of rejection in noncannibalistic species consisted of the crossed reflex (Hecht, 1918). A tadpole ASCIDIAN CANNIBALISM AND LARVAE 17 striking the oral tentacle or the inside of the siphon epithelium elicited a response in which the excurrent siphon was closed and body wall muscles were contracted. This action caused the tadpole to be expelled forcefully from the incurrent siphon. Some­ times, several tadpoles or eggs accumulated in the branchial basket before a single large squirt expelled them all. In Corella injlata, tadpoles swimming in the branchial basket sometimes elicited closure of the incurrent siphon by striking the epithelium on the proximal side of the oral tentacles. This closure response prevented escape by the tadpoles. Eventually, C. inflata tadpoles always became entangled in the mucus sheet lining the inside of the branchial basket. They seldom escaped once entangled, despite violent tail thrashing, but instead were rolled in mucus at the dorsal lamina and moved with other food particles into the esophagus. Gut dissections of adult Boltenia villosa, Corella inflata, and Chelyosoma productum confirmed that eggs and larvae were present in the esophagus, stomach, or intestine, and did not reside long in the branchial basket.

SPECIES SPECIFICITY OF EGG AND TADPOLE REJECTION

I expected that species such as Pyura haustor, which reject all of their own tadpoles, would also reject tadpoles of all other species of solitary ascidians, since they would probably all be detected by the same sensory mechanisms. Table IV shows that this was not always the case. Pyura haustor consumed more tadpoles and eggs of other species than of its own species. Indeed, 73.3%of the eggs and 87.5%of the larvae of Corella injlata were consumed. The same sort of species specificitywas observed in Styela gibbsii and Cnemidocarpajinmarkiensis, both of which rejected most conspecifics (Table III), but consumed between 44% and 90% of other eggs and larvae offered. It was also surprising that the cannibalistic ascidian Corella inflata consumed its own tadpoles and eggs more readily than the offspring of other species (Table IV).

MORPHOLOGICAL BASIS OF EGG AND LARVAL REJECTION Why are some species capable of detecting and rejecting conspecific larvae, whereas others are not? I investigated this question by considering morphological characteristics that might facilitate detection of the larvae by adults. These characteristics included egg and larval size, egg density, adult oral tentacle number and form, and adult siphon diameter. Oral tentacle characteristics were taken from Van Name's (1945) monograph in which he summarizes data from the original species descriptions and from Lambert et al. (1981) for the two recently redescribed Corella species. Filiform, unbranched tentacles were present in both cannibalistic (Corella inflata, Corella willmeriana, Ascidia paratropa, Styela montereyensis) and noncannibalistic (Styela gibbsii, Cnemidocarpa jinmarkiensis, Chelyosoma productum, Ascidia callosa) species. Likewise, pinnate and otherwise branched tentacles were found in both types of ascidians (cannibalistic, Halocynthia aurantium, Halocynthia igaboja, Boltenia villosa; noncannibalistic, Pyura haustor). Among solitary ascidians, pinnate, bipinnate, and tripinnate tentacles are TABLE IV Percentages of ascidian eggs and larvae consumed by heterospecific adult ascidians.

Behavioral Eggs Larvae classification and species C. inflata S. gibbsii P. haustor B. villosa C. inflata C. finmark- P. haustor B. villosa iensis

n n % n % n % n % n % n % n % % (') ~ Strongly gregarious -< P. haustor 73.3 60 0.0 25 10.0 40 87.5 40 20.0 10 8.0 25 0 S. gibbsii 83.0 120 76.0 25 60.0 20 50.0 10 44.0 25 c::: Z Slightly gregarious C) H. igaboja 90.0 20 72.0 25 80.0 20 90.0 10 100.0 25 Not gregarious A. paratropa 100.0 20 100.0 50 100.0 20 100.0 10 100.0 25 C. inflata 75.0 20 70.0 20 100.0 20 50.0 10 100.0 50 56.0 25 C. finmarkiensis 55.0 20 84.0 45 90.0 20 60.0 10 H. aurantium 100.0 20 92.0 50 80.0 20 90.0 10 96.0 25 ASCIDIAN CANNIBALISM AND LARVAE 19

found only in the Pyuridae and Molgulidae (Van Name, 1945). More of the pyurids investigated were cannibalistic than noncannibalistic. Therefore, the degree of tentacle branching alone does not explain differences in cannibalistic behavior. The number of oral tentacles is also insufficient to explain interspecific differences in egg or larval consumption (Fig. 2). Both highly cannibalistic species and non-

100 100 ca ca c» ca ca Ho Hi Ap Ci Cw c ] 80 .! 80 .B ~"'" 60 ~ 60 -s: U Bv Q) l o ~ 40 5 40 U U (; ~ 20 .. 20 Bv• Cf o Ph Ac C 5g Cp Ac Ph 20 40 60 80 100 120 2 4 6 10 12 Number of Oral Tentacles Rank Incurrent Siphon Diameter

Fig. 2. Relationships between egg consumption and number of oral tentacles (median and range: left panel), and between consumption of eggs or larvae and incurrent siphon diameter (right panel). Note that siphon diameters are ranked from largest to smallest. Genus and species names are abbreviated (see Table I for species list). cannibalistic species tend to have between 20 and 50 tentacles, although one noncanni­ balistic form, Chelyosoma productum, has an average of 100 tentacles: :::::: 35 more tentacles than the nearest cannibalistic species, Corella willmeriana. A nonparametric rank correlation for percent of eggs eaten vs. number of oral tentacles was not signifi­ cant. A stronger relationship appears to exist between cannibalistic tendency and incurrent siphon diameter. Because of the contractile nature of the siphon, accurate measure­ ments are difficult to make. I therefore ranked absolute siphon diameters from largest to smallest for the analysis (Fig. 2). There was a highly significant correlation between cannibalistic tendency (expressed as rank percent of conspecifics eaten) and rank siphon diameter. Species with smaller siphons tend to be less cannibalistic. Five of the six species with largest absolute siphon diameters consume 100% of their eggs and larvae. It might be expected that larger or denser particles would be more likely to elicit a rejection response than smaller or lighter particles. I tested this hypothesis by correlating consumption rates with egg or larva sizes for two species of adult predators, the highly cannibalistic Corella inflata and the noncannibalistic Pyura haustor (Fig. 3). The corre­ lations were not significant for Corella inflata but were highly significant for both eggs and larvae in Pyura haustor. These latter correlations apparently are significant only because tadpoles and eggs of Corella inflata are consumed much more readily than those of other species. The readily consumed eggs of Corella inflata are larger than eggs of other species not 20 C.M. YOUNG consumed. Eggs of this species also have the unique characteristic of positive buoyancy. This buoyancy is conferred by ammonium ions in the large follicle cells that surround the egg (Lambert & Lambert, 1979). To determine if buoyant folliclecells make the eggs less likely to be rejected by ascidians than normal eggs, I removed the cells from eggs by shaking vigorously in a centrifuge tube (Lambert & Lambert, 1979). Eggs with intact follicle cells have a density of 1.018 g : em - 3, whereas eggs with follicle cells removed by this method have a much greater density of 1.08 g :cm" (Lambert & Lambert, 1979). Experimentally modified eggs (n = 100 per predator species) were all rejected by P. haustor and Styela gibbsii, but consumed by Corella injlata. Thus, egg density or some other attribute associated with the follicle cells is more important than egg size in determining whether eggs will be rejected or accepted by Pyura haustor or Styela gibbsii. Corella injlata fails to detect and reject eggs regardless of their density or size. The tadpoles of Corella inflata are nearly 200 /lm shorter than tadpoles of species that are readily rejected by Pyura haustor. Absolute size may therefore be the basis of species specificity for larvae (Fig. 3).

Pyura naastor 100

• C. inflofO 80 c. ,nflafa "'C (I) • E 60 :J VI c: <3 40 C finmork,.ns;s ~ 20 B. vll/oso • B vil/oso. P. haustor S. gibbsii • I I .Ph~ustor i 100 200 300 900 1000 1100 1200

Corella inflata 100 B. vittosa• C.•inttata C. tnttara• P •nausror

"'C 80 s gibbsii. (I) E •P nausror :J 60 B. vntosa VI c: a a 0 r r U I o.47S! I -o.606!C. finmorki.ns;sI 40 n... n.•. ~ 0 20

iii I 100 200 300 900 1000 1100 1200 Egg Diameter (urn) Tadpole Length (prn)

Fig. 3. Correlations between consumption and egg or larval sizes, using Pyura haustor and Corella inflata as predators. ASCIDIAN CANNIBALISM AND LARVAE 21

REJECTION OF SPERM

Noncannibalistic ascidians generally showed a strong rejection response when detecting sperm clouds, whereas most of the cannibalistic forms accepted the sperm at least half the time (Table V). Because individual sperm are smaller in size than many of the food particles accepted by ascidians, these data suggest that the rejection responses are based at least partly on chemical characteristics of the food, not just size.

TABLE V Rejection of ascidian sperm by adult solitary ascidians. "% eaten" refers to number of trials in which no rejection response was observed following introduction of sperm into incurrent siphon.

Behavioral classification Sperm species % eaten (n) and predator species

Strongly gregarious P. haustor A. paratropa 5 (20) P. mirabilis 0(20) P. haustor 0(20) S. coriacea 0(20) S. gibbsii P. mirabilis 0(20) P. haustor 0(20) S. coriacea 0(20) C. productum A. paratropa o(5) P. haustor 0(20) Slightly gregarious S. montereyensis P. mirabilis 100 (10) B. villosa P. haustor 0(20) A. callosa P. haustor o(20) H. igaboja S. coriacea 50 (10) Not gregarious A. paratropa S. coriacea 100 (10) C. finmarkiensis P. mirabilis 0(10) H. aurantium S. coriacea 90 (10)

BROODING AS MECHANISM THAT REDUCES CANNIBALISM

Although the floating eggs of Corella inflata may render them more vulnerable to consumption, reproductive habits make it unlikely that eggs will be consumed by conspecifics. Child (1927) described a brooding mechanism for Corella in which eggs are retained in the atrial chamber until hatching by virtue of their floating follicle cells. He noted that Corella inflata generally occur on downward-facing or vertical surfaces, and that the atrial siphon tends to be positioned higher than the branchial siphon. This causes the eggs to float up in the expanded atrium, where they are retained until hatching. I confirmed Child's observation of adult orientation by measuring deviations of the mid-sagittal axes from a vertical line for a population of C. inflata on a subtidal rock cliff (Fig. 4). I also measured the orientations of newly metamorphosed juveniles 22 C.M. YOUNG in a laboratory experiment where tadpoles were allowed to settle on a vertical glass plate. The orientation of juveniles was random (Fig. 4). Therefore, the orientations of Corella injlata individuals must change ontogenetically by rotational growth, or else individuals in the population not having the appropriate orientation die before reaching reproductive maturity, leaving only adults with the ability to retain broods.

Juveniles (2 wk after selliement) Reproductive Adults n=20 n=99 Fig. 4. Orientation of the mid-sagittal plane in 2-wk-old and adult Corella inflata on vertical surfaces.

Lambert & Lambert (1979) noted that tadpoles of C. inflata swim upward at hatching, and attributed this to positive phototaxis. I have noted the same behavior. However, the tadpoles do not respond to horizontally directed light (Young, 1982), so the response is probably a negative geotaxis rather than a positive phototaxis. When the adult broods in its typical orientation, eggs and embryos are retained in the atrium, and tadpoles swim upward when leaving the excurrent siphon. By swimming upward, tadpoles also swim away from the incurrent siphon. Such tadpoles are almost never consumed by their parent (Fig. 5). When brooding adults were inverted experi­ mentally and observed, the brood (both eggs and tadpoles) emerged from the atrium (eggs floating, tadpoles swimming) and moved upward into the incurrent stream of the branchial siphon, where they were ingested (Fig. 5). Thus, brooding protects eggs and early tadpoles from consumption by their own parents.

DISCUSSION

Thorson (1950, 1966) and others have argued that predation is the most important source of mortality for the planktonic larvae of marine invertebrates, and that extremely ASCIDIAN CANNIBALISM AND LARVAE 23 high fecundities of marine invertebrates have probably evolved as a compensation for high predation rates in the larval stage. One might expect that invertebrates would compensate for predation not only with high fecundity, but with special defensive adaptations targeted at important predators. Relatively few such defenses have been

Fig. 5. Avoidance of cannibalism by geonegative larvae in Corella inflata positioned in normal orientation (top), and consumption of larvae and floating eggs in individual whose orientation has been reversed (bottom). documented (reviewed by Young & Chia, 1987). Morphological defenses such as spines, setae, and shells seem to be effective against a relatively small assortment of predators (Cowden et al., 1984; Pennington & Chia, 1984), and behavioral mechanisms reduce predation by some predators but not others (Rumrill & Chia, 1984; Pennington et al., 1986). Distasteful chemicals may be useful against large guilds of functionally similar predators such as fishes (Lucas et al., 1979; Young & Bingham, 1987) but their effec­ tiveness against invertebrate predators has not been investigated. The overall assem­ blage of predators a planktonic larva might encounter during its life encompasses virtually all of the major phyla, and includes animals that feed by virtually all known mechanisms. The density and diversity of predators varies at many scales both 24 CM. YOUNG

temporally and spatially. Perhaps It IS not surpnsmg, therefore, that few globally effectivedefenses have evolved among larvae. Fornatural selection to promote defenses, the selective pressure must be more-or-less predictable in form and function as well as in intensity. By virtue of their delay of metamorphosis behavior, most gregarious invertebrate larvae surviving to metamorphosis will encounter adult conspecifics, whereas the suite of predators encountered earlier in larval lifemay be extremely variable. In other words, encounter with a potentially cannibalistic adult may be one of the more predictable events occurring during larval life. Such predictable predation pressure should favor the evolution of specific defenses against ingestion by adults. However, in the cases documented here, protective mechanisms are found not in larval form, behavior, or chemistry, but occur instead as sorting mechanisms of the adults. At the species level, there may be certain energetic and evolutionary advantages to having some cannibals in a population (reviewed by Fox, 1975; Polis, 1981). Most of these advantages occur only when the cannibals consume conspecifics of another genotype; such predation occurs without lowering the fitness of the predator itself. From an evolutionary standpoint, consumption of young by their own parents consti­ tutes wasted reproductive effort and should be eliminated by natural selection. How­ ever, it is impossible to argue on the basis of existing evidence that the mechanisms by which larvae are rejected evolved specifically for that purpose. The adaptationist scenario would invoke group selection. Larvae encountering a particular adult after the dispersal stage are unlikely to have the same genotype as that adult. It is therefore difficult to imagine how an anticannibalism mechanism would confer greater individual fitness to the adult that posesses the mechanism. The rejection mechanism, correlated as it is with siphon diameter, functions not only in rejecting larvae, but also in sorting out food particles of inappropriate size or chemical composition. Moreover, the rejection response functions around the clock and throughout the year, not just during the short periods when larvae are in the water. It is not unreasonable to assume that the sorting mechanisms evolved for feeding efficiency, and that rejection of larvae is simply a fortuitous byproduct. If larval rejection is only a byproduct of sorting efficiency, why would gregarious settlement be strongly correlated with larval rejection? The parsimonious evolutionary explanation is that gregariousness evolved only in those species that already rejected larval-sized particles. This hypothesis is consistent with the tenets of natural selection. Assuming that aggregation confers fitness (advantages of clumping have been docu­ mented: Buss, 1981; Keough, 1984; Grosberg & Quinn, 1986) larvae with gregarious tendencies would have higher probability of survival to reproduction only if the feeding mechanism of the adult permits recruitment nearby. In this context, the rejection of larvae should be viewed as a preadaptation that permits the evolution of gregarious settlement in ascidians. ASCIDIAN CANNIBALISM AND LARVAE 25

ACKNOWLEDGEMENTS

C. B. Jorgensen, J. L. Cameron, L. F. Braithwaite, R. L. Fernald, F. S. Chia, and R. R. Olson provided critical and stimulating discussion. A. O. D. Willows made labora­ tory space available at Friday Harbor. Writing and analysis was supported by NSF Grants OCE-8400406 and OCE-8544845.

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