Shannon et al.\ Evidence for sexual reproduction in Petalonia fascia 511 Botanica Marina Vol. 31, pp. 511-513, 1988 Evidence for Sexual Reproduction in Petalonia fascia (O. F. Müller) 1 Kuntze R. K. Shannon*, G. E. Crow* and A. C. Mathieson**** * Department of Botany and Plant Pathology and ** Jackson Estnarine Laboratory, University of New Hampshire, Durham, New Hampshire 03824, U.S.A. (Accepted 23 July 1988) Abstract Fertile fronds of Petalonia fascia were collected from January through April 1985 at four sites in New Hampshire, U. S. A. in order to investigate their ability to reproduce sexually. Swarmers from separate fronds were allowed to mix on microscope slides and were observed over a period of 18 to 24 hours. Clumping of swarmers was common. Settled swarmers possessing two chloroplasts and two eyespots were observed on slides from eight of the 24 collections. The number of such cells on any slide was less than 1 % of the number of cells on the slide. Introduction Recently the life histories of the scytosiphonaceous brown algae Petalonia and Scyiosiphon have been intensively studied (Wynne and Loiseaux 1976) and shown to involve a heteromorphic alternation between an upright frond in the form of a blade or tube, and a small crustose or cushion-like thallus (often described as 'ralfsia-like'). Whether the upright and crustose stages represent gametophyte and sporophyte stages, respectively, has largely been a matter of speculation, as the alternation between the two stages is not obligatory. Some investigators have reported that swarmers from the upright stages of Petalonia and Scytosiphon function as gametes, fusing to form zygotes (Tatewaki 1966, Nakarxiaura and Tatewaki 1975, Clayton 1980), while others have reported that fusion was not observed (Wynne 1969, Edelstein et al 1970, Rhodes and Connell 1973, Roeleveld et al 1974, Fletcher 1981). Most reports describing the occurrence of sexual reproduction in the Scytosiphonaceae are for Scy1 Scientific Contribution Number 1495 from the New Hampshire Agricultural Experment Station and Contribution Number 212 from the Jackson Estuarine Laboratory tosiphon lomentaria (Lyngb.) Link (Tatewaki 1966, Clayton 1980), although the phenomenon has also been reported for Petalonia zosterifolia (Reinke) Kuntze (Nakamura and Tatewaki 1975). Sexual reproduction has never been reported in the upright stage of Petalonia fascia (O. F. Müller) Kuntze. The present report describes observations on swarmers from the upright stage of P. fascia, as part of a larger study on P. fascia and S. lomentaria (Shannon et al. 1988). Methods Fertile fronds of Petalonia fascia were collected from January through April 1985 in tidepools and in mixed sand and cobble areas at the following sites in New Hampshire: Fort Constitution, New Castle; Great Island Common, New Castle; Plaice Cove, Hampton; and Beckman's Point, Seabrook. In the laboratory, fronds that were dark brown (indicating mature plurilocular reproductive organs) or mottled with tattered apical areas (indicating previously discharged swarmers) were selected as potential sources of swarmers. Individual fronds were washed Botanica Marina / Vol. 31 / 1988 / Fasc. 6 to you by | University of Queensland - UQ Library Brought Copyright © 1988 Walter de Gruyter · Berlin · New York Authenticated Download Date | 7/14/15 1:40 PM Shannon et ai: Evidence for sexual reproduction in Petalonia fascia 512 in filtered seawater and then placed in separate crystallizing dishes. Swarmer release was stimulated by incubating the fronds overnight in covered crystallizing dishes without water at 2-7 °C in the dark, then flooding them with 150 ml of filtered seawater (Clayton 1984). Drops of seawater containing swarmers were removed with a Pasteur pipette and swarmers from two fronds were allowed to mix on a microscope slide. Slides were observed under a compound microscope at ÷ 400 immediately after inoculation to check for swarmer motility and signs of clumping. Slides were also observed one or two times during the next several hours until most or all warmers had lost their motility. They were surveyed the next day (at ÷ 1000) for evidence of fusion — e. g. two plastids or eyespots. Slides with clumps of swarmers were carefully surveyed for the presence of zygotes. Between observations, slides were kept in covered petri dishes in an incubator at 2—7 °C and light: dark regimes of 10:14 (January and February collections), 12:12 (March collections), and 14:10 (April collections). Twenty-four collections were processed as described above between 7 January and 26 April 1985 (Table I). Fronds that released swarmers are deposited as voucher specimens in the Hodgdon Herbarium, University of New Hampshire (NHA). Table I. Summary of collections with swarmers yielding zygotes (cells with two chloroplasts and two eyespots). Date Site No. fronds releasing swarmers No. slides prepared No. zygotes 24 JAN 7FEB 11 FEB 12FEB 28 FEB 13 MAR 18 MAR 28 MAR New Castle New Castle New Castle New Castle Seabrook Seabrook New Castle New Castle 6 3 5 4 9 11 5 6 12 4 7 5 7 8 7 13 4 1 8 10 3 1 3 8 Results Swarmer release occurred from within a few minutes up to an hour after flooding. The swarmers were typical for the Scytosiphonales, being pyriform, laterally biflagellate, and containing an eyespot, a single chloroplast with a conspicuous pyrenoid, and fucosan vesicles (Lee 1980, Bold and Wynne 1985). Swarmers were generally very active when first released. They gradually became more sluggish, and eventually lost their motility entirely. As they settled and attached to the slide they became spherical and withdrew their flagella. Settling occurred anywhere from a few minutes to 24 hours after release. All swarmers were very similar in size, measuring approximately 5 ìðé in diameter when settled. Swarmers on approximately 78% of the slides showed some degree of clumping. A careful survey of slides with clumped and settled swarmers revealed cells with two chloroplasts and two eyespots (Table I). The number of such cells on any one slide from these eight collections was very small, being fewer than 1% of the cells in every case. Discussion We are unaware of any other reports of apparent fusion of swarmers from the foliose stage of Petalonia fascia. Although Kunieda and Arasaki (1947) reported sexual reproduction in P. fascia, they claimed that gametes were produced on microscopic garnetophytes and that the fronds discharged asexual zoospores. The results of Kunieda and Arasaki (1947) have never been confirmed. It is interesting to note that of the many researchers who have recently investigated the life histories of Petalonia and Scytosiphon very few have found sexual reproduction (Tatewaki 1966, Nakamura and Tatewaki 1975, Clayton 1980). Our criteria for identifying zygotes (two chloroplasts and two eyespots) are the same as those used by Clayton (1980). There is some presumption in basing a conclusion of sexual fusion solely on these criteria. M ller (1975) reports that apparent 'motile zygotes', supposedly representing fusion of swarmers from uiiilocular sporangia of Ectocarpus siliculosus (Dillwyn) Lyngbye, are really 'twins', released from the sporangia before cell division is complete. It is not surprising that he was suspicious of reports of swarmers fusing from unilocular sporangia, as they normally produce spores rather than gametes. In the case of Petalonia and Scytosiphon the apparent fusion of swarmers from plurilocular organs is more likely, as plurilocular organs are normally the sites of gamete production. Also, unlike accounts of Ectocarpus siliculosus, none of the apparent zygotes observed in the present study were motile, suggesting that fusion may have occurred after the swarmers had settled. Although clumping of swarmers was common, this event does not necessarily indicate a mating reaction. Clayton (1980) described clusters that formed immediately as male gametes were attracted to and encircled female gametes. Clusters eventually dispersed with one male gamete retaining flagellar contact with the female. No clumps in the present study were Brought to you by | University of Queensland - UQ Library Authenticated Botanica Marina / Vol. 31 / 1988 / Fasc. 6 Download Date | 7/14/15 1:40 PM Shannon et .: Evidence for sexual reproduction in Peialonia fascia 513 observed to disperse. Kristiansen and Pedersen (1979) and Roberts and Ring (1972) describe clumping of Scytosiphon lomentaria swarmers when they settled. In their reports, clumps formed as motile swarmers used already settled swarmers as substratum. Some of the clumping observed in the present study appeared to be of this type. Neither Kristiansen and Pedersen (1979) nor Roberts and Ring (1972) report fusion of swarmers. While our study does not present conclusive evidence for sexual reproduction, it strongly suggests that sexual reproduction occurs. Observations of actual fusions should be made, zygotes should be isolated and cultured, and ploidy level of swarmers and apparent zygotes determined. Acknowledgements If sexual reproduction does occur in Petalonia fascia, its fully expressed life history is probably very similar to that of P. zosterifolia or Scylosiphon lomentaria. The life histories of these species include an upright frond stage with plurilocular reproductive organs, the swarmers from which, at least in some geographies and at certain times of the year, can function as gametes (Clayton 1981 a, b). They also include a crustose or cushion-like stage which can produce unilocular sporangia, give rise directly to the upright stage, or both (Edelstein et al. 1970, Roeleveld et al. 1974, The authors would like to express their appreciation to Or C. A. Penniman for constructive comments on the manuscript, and to Dr T. D. Lee for advice during the study and constructive comments on an earlier draft of the manuscript. This research was from a thesis submitted by the senior author to the Graduate School at the University of New Hampshire in partial fulfillment of the requirements for the M.S. degree, and was supported by grant number 2-11073 from the Central University Research Fund (CURF) of the University of New Hampshire. Kristiansen and Pedersen 1979). Apparently there are both haploid and diploid crustose stages in the Scytosiphonaceae (Nakamura and Tatewaki 1975). Haploid crusts are the products of parthenogenetically developing swarmers from fronds or of swarmers from other crusts; no meiosis occurs in the unilocular sporangia of the haploid crusts (Nakamura and Tatewaki 1975). Diploid crusts are the products of zygotes, with meiosis occuring in their unilocular sporangia; such crusts represent the sporophyte generation (Nakamura and Tatewaki 1975). References Bold, H. C. and M. J. Wynne. 1985. Introduction to the Algae: Lee, R. E. 1980. Phycology. Cambridge University Press, CamStructure and Reproduction. Second ed. Prentice Hall, Inc., bridge. 478 pp. Engelwood Cliffs, NJ. 720 pp. Müller, D. G. 1975. Experimental evidence against sexual fusions of spores from unilocular sporangia of Ectocarpus Clayton, M. N. 1980. Sexual reproduction — a rare occurrence siliculosus (Phaeophyta). Br. Phycol. J. 10: 315-321. in the life history of the complanate form of Scytosiphon (Scytosiphonaceae, Phaeophyta) from southern Australia. Nakamura, Y. and M. Tatewaki. 1975. The life history of some Br. Phycol. J. 15: 105-118. species of the Scytosiphonales. Sei. Pap. Insi. Algol. Res. Clayton, M. N. 1981 a. Correlated studies on seasonal changes Fac. Sei. 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Life history and systematic studies of some and seasonal variation of Scytosiphon lomentaria (FucophyPacific North American Phaeophyceae (brown algae). Univ. ceae, Scytosiphonales) in Denmark. Bot. Tidsskr. 74: Calif. Publ. Bot. 50: 1-88. 31-56. Kunieda, H. and S. Arasaki. 1947. On the life history of flea Wynne, M. J. and S. Loiseaux. 1976. Recent advances in life history studies of the Phaeophyta. Phycologia 15: 435—452. fascia and Punctaria sp. Seibutsu 2: 185 — 188. Brought to you by | University of Queensland - UQ Library Authenticated Download Date | 7/14/15 1:40 PM Botanica Marina / Vol. 31 / 1988 / Fasc. 6 Brought to you by | University of Queensland - UQ Library Authenticated Download Date | 7/14/15 1:40 PM