Invertebrate Biology J22(4): 293-298. 0 2003 American Microscopical Society, Inc.

Internal brooding of clonal propagules by a sea anemone, Anthopleura sp.

Naoko Isomura," Keiko Hamada, and Moritaka Nishihirah

Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan

Abstract. We investigated the seasonal prevalence of reproductive activities and of the devel- opment of brooded propagules in an intertidal sea anemone, Anthopleura sp., on the rocky shore of Mutsu Bay, in northern Japan. A monthly examination of anemones, by dissection and histological techniques, revealed no sign of gonad development, but did reveal that they produce and internally brood propagules throughout the year. Release of propagules was ob- served in the field. This anemone population appears to be entirely asexual and agametic, and may persist solely through clonal propagation.

Additional key words: Cnidaria, Anthozoa, Actiniaria, asexual reproduction

Anthopleura sp. lives in the intertidal zone of Mutsu Bay, in northern Japan and broods internally (Atoda 1954). Modes of clonal replication, such as fission, pedal laceration, or budding have not been reported for this species. However, because anemones of the same color morph form aggregations and are aggres- sive towards different color morphs, these aggrega- tions may be clones (Hamada 1999). Moreover, be- cause no gonads have been found in these anemones (Hamada 1999), it is possible that the brooded prop- agules of this anemone represent clonal replicates.

Young at various developmental stages have been observed in the coelenteron in most species of Actinia (Carter & Miles 1989). Several hypotheses have been proposed to explain the origin of these young (Chia & Rostron 1970; Gashout & Ormond 1979; Orr et al. 1982), especially in Actinia equina FARQUHAR 1898, which is known to be an internal brooder.

Because sexual reproduction of Anthopleura sp. has not been investigated in detail, it is possible that the young brooded in the coelenteron are derived from several processes, including sexual reproduction, as in A. equina (Chia & Rostron 1970; Yanagi et al. 1996). The aims of this study were (1) to document any sea- sonality of brooding in Anthopleum s ~ . , (2) to deter- mine whether the brooded offspring are of sexual or asexual origin, and (3) to describe their development.

Author for correspondence. Present address: Graduate School of Humanities and Sciences, Ochanomizu Univer- sity, Tokyo 1 12-86 10, Japan. E-mail: iso@cc.ocha.ac.jp

Present address: Meio University, Nago, Okinawa 905- 8585, Japan.

Methods

Study site and collections

Sea anemones were collected on the rocky shore of Mutsu Bay near the Marine Biological Station, Tohoku University (40'54' N, 140'51' E). The topography of the intertidal area is rugged and irregular, with many tide pools of various sizes. All color morphs of Antho- pleura sp. are distributed in the area, and other \ea anemones are also found there, including A. juscoviridis CARLGREN 1949, A. pacijica UCH~VA 1938, A. uchidui ENGLAND 1992, Metridium senile LINNAE~JS 1767, and Cnidopus juponicus VERRJLL 1869 (Hamada 1999).

We collected and examined 20 anemones monthly from January to December 1998, and 15 individuals monthly from July 2000 to June 200 I . A preliminary study clarified that the anemones begin to brood when they are larger than 8 mm. All the individuals studied were from 3 aggregations of a single color morph, 10 in apart. The anemones were collected using a spatula and kept individually in plastic bags filled with natural seawater.

Laboratory maintenance and measurements

In the laboratory, anemones were kept separately in plastic containers ( I 1 .5 cm diameter, 9.5 cm high) that were filled with unfiltered natural seawater and left undisturbed. When the anemones had attached to the bottom of the container and expanded, we recorded the number of tentacles. We measured the longest and shortest diameters of the pedal disk after individuals retracted in response to a gentle touch. If a propagule was released, we collected the entire brood.

The size of the anemones was quantified using the

204 Isomura, Hamada, & Nishihira

geometric mean of the longest and shortest diameters of the pedal disk. In the 1998 samples, pedal disk di- ameter ranged 2.6-27.4 mm: in the 2000-2001 sam- ples, sizes ranged 2.5-40.1 mm. For propagules, we measured the diameters of the pedal disk for fixed propagules. Since the oral/aboral length changes with fixation, all propagule measurements were converted to estimated lengths of live propagules using the for- mula y ? 1.2467~ + 0.4233 (R2 = 0.7193, n = 43, p<.O01), where y is the value for a live propagule and x that of a tixed propagule.

Fixation, dissection, and histology

After being measured, the anemones were left in the plastic container undisturbed until they expanded, when they were anesthetiLed with menthol and fixed in neutral 10% formalin for 24 h. Finally, they were transferred to 50% ethanol for 24 h and then to 70% ethanol. We dissected the anemones to determine brooding condition after they had been stored for at least 24 h in 70% ethanol.

Longitudinal dissection of the preserved individuals allowed us to collect with tweem-s the entire brood found in a gastric cavity. For all propagules, we re- corded the number of tentacles, if any, and measured the height of the body and the longest and shortest diameters of the pedal disk, to the nearest 0.01 mm under a stereoscopic microscope (Olympus VMZ) with an ocular micrometer.

Each month from July 2000 to June 2001, we made histological preparations of 10 large individuals which had been dissected. Serial sections 10-pm thick were double-stained with hematoxylin-eosin and mounted using Eukitt. The sections of mesenteries were examined under a light microscope (Olympus ER).

Field observations of the behavior of small individuals after release

We made dctailed observations of the behavior of 7 small individuals, presumably from brooders, in tide pools at low tide, for 2 days in October and 2 days in November 1998. Unlike larger anemones, small anem- ones do not have small sand grains or pieces of shell fragments on their body wall. in addition, small anem- ones appear paler than larger ones because they con- tain fewer zooxanthellae, and the coloration used to distinguish color morphs is not fully differentiated. We used these morphological characteristics to distinguish newly released anemones. Because no other anemone species were present in the tide pools used for these observations, we assume that the small anemone$ we observed were released by brooding individuals of

Results

Propagule development

In 1998, we looked for gonads and buds only by dissection, and in 2000-2001 we looked for them by dissection and histology.

in 1998, we examined 240 large individuals of An- thopleura sp. in dissections. In 2 individuals we ob- served, on the basal parts of mesenteries, spherical protuberances about 200 p m in diameter, which ap- peared to be developing buds (Fig. 1A). Of the prop- agulcs found free in the gastric cavity, the smallest wa\ ovoid (400 X 300 pm). In early developmental stages, the propagules lacked mesenteries (Fig. 1 H), whereas larger propagules (500 X 400 pm) had mesenteries and a mouth (€ig. IC). They swam by ciliary move- ments and altered their shape with muscular contrac- tions. Tentacles were differentiated in the larger prop- agules that had well-developed mesenteries (Fig. I I)) . I’ropagules at all developmental stages had moxanthellae.

From July 2000 to June 2001, we dissected 120 anemones (I0 anemones each month, sim >6 mm). We observed no signs of gonads (ovaries and testes). In our histological study, we found no gonads either.

Size of brooders and timing of brooding

In 1998 and in 2000-200 I , the smallest brooder had a pedal disk of 5 mm. When anemones were grouped into 4 size classes, i.e., <5 mm, 25-9 mm, 99-13 mm, and > 13 mm, the proportion of brooders differed between size classes, and was significantly smaller in the smallest 1 or 2 e classes than in the larger siLe classes (ANOVA and Scheffk’s multiple comparison. p<.05, Fig. 2). At no time were the developmental stages of the propagules synchronized among different brooders or even within a single brooder. Typically, a brooder simultaneously continued various stages, rang- ing from those without tentacles to those with as many as 20 tentacles.

The number of propagules brooded in the gastric cavity, counted during monthly dissections, showed no seasonal trend, nor were there consistent seasonal pat- terns in the size of propagules or number of their tentacles (Fig. 3).

Propagule size and number of tentacles per propagule

The sizes of propagules found in the gastric cavity of brooders were quite variable; the s k e was 2.07 i 0.71 mm (average ? 1 SD, n=836, range: 0.40-5.63 mm) in 1998, and 2.15 2 0.78 (n=124, 0.20 -4.00

Anthopleura sp. mm) in 2000-2001, and 80-90% of propagules mea-

Brooding of clonal propagules by Anthopleura sp. 295

sured 1-3 mm. The number of tentacles per brooded propagule ranged from 0 to 26, but most of the prop- agules had either 0 or 12 tentacles (Fig. 4). None of the propagules had 1 to 6 tentacles, and few had more than 12. The number of tentacles was correlated pos- itively with size (Fig. 5, 1998: r2=0.573, pC.05, 2000- 2001: r2 = 0.493, p<.05).

Field observations of propagule release and fate

Our field observations were made at low tide around noon. We observed the release of 3 propagules by 3 different brooders on 1 day. These brooders had pedal disks that were >8 mm, and the number of propagules released was 1, 3, and 3. In the 2 brooders that released 3 propagules, the small anemones emerged one after the other within 10 min. All propagules were expelled from the mouth of the brooder. The single offspring from the first brooder stayed on the mouth of the

Fig. 1. Early developmental stages of propagules of Anthopleura sp. in the gastric cavity of a brooder. a. Propagules (arrows) at the base of mesenteries (m). Pedal disk (pd). b. Propagule just after detachment from the mesentery; its own mes- enteries have not yet developed. c. Propagule with a mouth (mo) and mesenteries; tentacles not yet devel- oped. d. Propagule with 12 rudi- mentary tentacles (t); developed mesenteries are visible. Scale bars, 100 km.

brooder for a few minutes after release, before it was carried away by a wave.

The 3 propagules from the second brooder similarly remained near the mouth of the brooder for about 1 min and then fell down beside the brooder. Two were carried away by a wave within 30 min. One of these 2 was found clinging to the wall of the tide pool by its tentacles and, over a period of 5 min, it quickly moved along a crack in the wall to the bottom of the pool, then moved a further 5 cm until it reached the edge of an aggregation of anemones that included its brooder, where it stopped moving. However, it was not found there the following day. The third propagule re- leased by the second brooder moved down the body of its brooder and settled in a gap between its brooder and adjacent anemones, but it too was not found there the following day.

All 3 propagules from the third brooder, as soon as they were released, fell into a gap between the brooder

Isomura, Hamada, & Nishihira

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100

80 n * L 60 cn

2 40

20

a, c a c

b

C

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Fig. 3.

1

A 6 c D Fig. 2. Proportions of brooders in 4 size classes of sea anemones. A, <5 mm; B, 25-9 mm; C, 29-13 mm; D, >13 mm. Bars indicate 5 1 SD. Anemones examined in 1998: A, n=15; B, n=42; C, n=75; D, n=48. Anemones examined in 2000-2001: A, n=12; B, n=68; C, n=86; D, n= 14. Values with different letters are significantly different (Scheffk's multiple comparison, p<.05).

anemone and some mussels (S. virgatus WIEGMANN) where they settled, using the pedal disk, and were found in the same spot the next day.

Discussion

We found that many individuals of Anthopleura sp. were brooding propagules in their gastric cavities. Be- cause no gonads were observed during a full year of dissections and histological examination, we think it

J F M A M J J A S O N D J A S 0 N D J F MA M J

Month Monthly records of the number and size of propa-

gules and the number of tentacles. Bars indicate 2 1 SD.

likely that these propagules are produced asexually, and that the study population is entirely asexual. The broods consisted mainly of propagules with 0 or 12 tentacles, though a few had from 6 to 24 tentacles. It has been reported that Anthopleura sp. broods develop rapidly to the 12-tentacle stage (Atoda 1954), although we could not determine the rate of development. Brooding anemones were found throughout the year, and no seasonal patterns were detected.

Some form of clonal proliferation occurs in all ma- jor groups of sea anemones, but these anemones also reproduce sexually; these 2 modes are not alternatives, and the rarity with which anemones have abandoned sexual reproduction and relied exclusively on cloning may be related to the fact that such a strategy would be an evolutionary dead end (Shick 1991, p. 250). Shick (1991) cited, in this context, locally introduced populations of Haliplanella lineata that are unisexual and monoclonal; they sometimes flourish transiently, but most became extinct in the long run. However, these animals had not fully abandoned sex, as they

Brooding of clonal propagules by Anthopleura sp. 297

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were still producing gametes (Shick 1976). In all spe- cies of Anthopleura studied, sexual reproduction yields planula larvae, which settle and grow into polyps, and some species clone by fission (Geller & Walton 2000). However, we found no sign of gametes in local pop- ulations of Anthopleura sp., nor did we observe fission or pedal laceration, and it seems likely that the brood- ed propagules found throughout the year are produced asexually by some unknown mechanism.

In clonal plants, the relative importance of sexual vs. clonal recruitment may vary between populations because clonal replication allows populations to persist in habitats or regions where, for various reasons, sex- ual reproduction cannot occur (Preston & Croft 1997; Eckert 2002). Moreover, it is likely that clonal plants probably experience reduced dispersal compared to offspring produced through sexual reproduction, be- cause clonal offspring are also usually much larger than seeds and lack prolonged dormancy or specialized dispersal mechanisms (Ashton & Mitchell 1989; Star- finger & Stocklin 1996). In Mutsu Bay, there remains the possibility of recruitment into the local anemone population of sexually produced young that originate from elsewhere and are (for unknown reasons) unable to reproduce sexually in the local environment. We cannot offer any explanation for the apparent absence of gametes and of sexual reproduction in our study population of Anthopleura sp. However, from our field

I I

0 1 2 3 4 5 6

Size of propagules (mm)

Fig. 5. of tentacles in Anthopleura sp.

Relationship between propagule size and number

observations of propagules and of the aggregations of individuals of Anthopleura sp., we suggest that clonal replication of brooded propagules might by itself allow populations of Anthopleura sp. to persist in the habitat of Mutsu Bay.

So far, our studies are only suggestive; it is possible that our dissections and histological analyses somehow failed to detect gametes that were indeed regularly pre- sent. In order to identify the ecological and/or genetic factors underlying the local population characteristics of Anthopleura sp., genetic analyses are essential to confirm the genetic identity of brooding individuals and their offspring. If the propagules are indeed pro- duced by cloning, our next goal would be to elucidate the mechanism by which they are produced, presum- ably a kind of somatic embryogenesis. These anemo- nes may represent a case rare or even unique for Ac- tiniaria, of an entirely agametic population maintained by vegetative cloning alone, but further studies are needed to test this exciting possibility.

298 Isomura, Hamada, & Nishihira

Acknowledgments. We would like to thank S, Tamura and M. Washio for their support in collecting sea anemones and their help with the fieldwork. We would also like to thank M. Ishimura, S. Yaguchi, and N. Wakayama for their assis- tance in the histological studies, and S. Takeda for his valu- able advice and suggestions. This work was partially sup- ported by a grant-in-aid from the Research Institute of the Marine Invertebrates Foundation, and a grant-in-aid for cre- ative basic research (DIVER) and scientific research (C) from the Ministry of Education, Science, Sports, and Cul- ture, of Japan. This study is a contribution to knowledge from the Asamushi Marine Biological Station.

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