Hydrobiologia 398/399: 247–252, 1999. J.M. Kain (Jones), M.T. Brown & M. Lahaye (eds), Sixteenth International Seaweed Symposium, © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Preliminary observations on the development of kelp gametophytes endophytic in red algae David J. Garbary1 , Kwang Young Kim2 , Terrie Klinger3 & David Duggins3 1 Department
of Biology, St. Francis Xavier University, Antigonish, Nova Scotia, B2G 2W5, Canada E-mail:
[email protected] 2 Faculty of Earth Systems and Environmental Science, Institute of Marine Sciences, Chonnam National University, Kwangju 500-757, Korea 3 University of Washington, Friday Harbor Laboratories, Friday Harbor, WA 98250, U.S.A. Key words: Agarum, endophytism, gametophytes, Laminariales, Phaeophyceae, Pleonosporium, reproduction, symbiosis
Abstract The development of kelp gametophytes is described from field collections from the San Juan Islands, Washington from November, 1997 to March 1998. All gametophytes were endophytic in the cell walls of red algae, especially species with filamentous or polysiphonous construction. Gametophyte density ranged from a few to many hundreds of distinct individuals per host plant. Gametophytes formed extensive vegetative growths of irregularly branching filaments, mostly parallel to the host surface, consisting of up to 50 or more cells. Antheridia were formed at/or just above the surface of the host thallus. The stalked egg apparatus was perpendicular to the host surface. Following presumed fertilization, the zygotes developed with typical kelp embryology to form small epiphytic blades. The specific identity of the gametophytes is unknown, although the host plants were collected from three sites where the dominant kelp species were: a) Agarum fimbriatum, b) Nereocystis luetkeana and c) Alaria marginata, Costaria costata and Laminaria groenlandica.
Introduction The life history pattern of Laminariales with its large, diploid sporophytes and microscopic, haploid gametophytes was established early in this century by Sauvageau (1915, 1918). Since then, numerous culture studies on most genera of Laminariales have been carried out. The basic pattern of the life history is thus well established (e.g. Hollenberg, 1939; Kain, 1964; Cole, 1968; Lüning & Neushul, 1978; Lüning, 1980). There is an apparent paradox in that although there are many billions of spores produced per year (Chapman, 1984), the gametophytic stages have only sporadically been described from nature. Examples of kelp gametophytes described from nature include Parke (1932), Sakai & Funano (1964), Funano (1969) and Kaneko (1973) (review by Kain, 1979). All of these reports are based on observations from beds of Laminaria, except for Parke’s observations, which were made
on gametophytes scraped from a floating buoy and described as Laminaria and Saccorhiza. These gametophytes were typically free-living, although Funano (1969) described the substratum for the gametophytes in his study as being coralline red algae. These observations were presumed to be sporadic because of the microscopic size of the gametophytes. In addition, the presumed rock substratum would hamper direct observation of the gametophytes. We initially observed kelp gametophytes in situ endophytic in the cell walls of Pleonosporium vancouverianum (J. G. Agardh) J. G. Agardh and Callithamnion acutum Kylin collected in November 1997 from a bed of Agarum fimbriatum Harvey at 5–10 m depth off San Juan Island. This observation was extended to other hosts and sites through March 1998. Here we describe the vegetative and reproductive development of the kelp gametophytes based on the development of the gametophytes in situ.
248 Materials and methods General collections of red algae were made between November, 1997 and March, 1998, at: 1. Cantilever Point (5–10 m depth), 2. Reed Rock (at 5–10 m depth), and 3. attached to the floating dock at Friday Harbor Laboratories, all in the San Juan Islands of Washington. Red algae were epilithic, epiphytic, epizoic (on shells) or epicanthic (i.e. on car tires) (attached to dock). The primary kelp species at these sites were, respectively: 1. Agarum fimbriatum, 2. Nereocystis luetkeana (Mertens) Postels et Ruprecht, and 3. Alaria marginata Postels et Ruprecht, Laminaria groenlandica Rosenvinge and Costaria costata (C. A. Agardh) Saunders. Potential host plants were returned to the laboratory and maintained in a running seawater table prior to observation. Whole thalli or portions of thalli were examined using light microscopy at 100× or 400× magnification. Kelp gametophytes were identified based on the occurrence of diagnostic oogonia and antheridia, and the presence of juvenile kelp sporophytes (e.g. Bold & Wynne 1985) attached to endophytic filaments. Numbers of individual kelp gametophytes in a host plant were determined by counting only discrete filaments as individuals. The terms oogonium and egg are retained for female gametangial structures in Laminariales even though flagella have been reported in eggs from Laminaria (Motomura & Sakai, 1988).
Results We initially observed endophytic thalli in plants of Pleonosporium vancouverianum that were growing as epiphytes on the holdfasts of Agarum fimbriatum. Subsequent inspection of other red algal species at this and other sites showed that numerous, diverse taxa served as hosts for endosymbiotic gametophytes. During a five month period, over 15 host species were observed including mostly filamentous [e.g. Pleonosporium vancouverianum, Callithamnion acutum, Scagelia pylaisaei (Montagne) Wynne], polysiphonous [e.g. Herposiphonia plumula (J. G. Agardh) Hollenberg, Pterosiphonia spp.] and membranous species [e.g. Polyneura latissima (Harvey) Kylin].
These red algal hosts supported epiphytic juvenile sporophytes (Figure 1); further inspection showed that these sporophytes were associated with microscopic filamentous gametophytes embedded in the host cell wall (Figures 2–7). The endophytic filaments were common in the thicker cell walls near the base of the plant (10–25 µm thick), and absent or poorly developed in younger parts of thalli with thin cell walls (2-4 µm thick). Inspection of numerous individual thalli of P. vancouverianum and C. acutum showed that endosymbiotic gametophytes occurred in most host plants examined from our sampled habitats (over 85% of ca. 50 plants), and in all life history phases of the host (male and female gametophytes, and tetrasporangial and polysporangial thalli). Even plants less than 1 cm tall often had kelp endosymbionts. Each host thallus had tens to more than 200 individual endophytic kelp gametophytes. These varied from separate unbranched filaments comprising one or only a few cells, to much larger, irregularly branching filaments (Figures 2, 4, 6). Filaments did not grow through the wall into the host cell cytoplasm. In none of the hundreds of host plants observed was there apparent cell death or signs of pathology of the host in response to the presence of the endophytes. Even in host filaments with a high density of endophytic cells (e.g. Figure 4), neither the cell walls nor the protoplast of the host became modified (based on bright field microscopy). Endophytic cells were 8–28 µm long and 4–10 µm wide. They were typically longer than wide and cylindrical to barrel-shaped or somewhat irregular. Cells associated with oogonial development were more quadrate than vegetative cells. Wider cells were conspicuously flattened in the plane of the cell wall surface. Cells of male plants were typically narrower than those in female plants (4–8 versus 6–10 µm). All cells except antheridia contained four to many discoid, parietal chloroplasts without pyrenoids. Hairs were absent. Oogonial development was associated with the larger, more quadrate cells. These cells were densely pigmented and contained numerous discoid chloroplasts; they developed darkly pigmented swellings above the host surface (Figure 7). Oogonia became elongate and were oriented roughly perpendicular to the host surface (Figures 7, 8). Prior to oogonial division (and presumed fertilization) the egg moved up in the stalk leaving an empty space at the base (Figure 9). The zygote divided transversely several times to form a distinctive filament before initiating par-
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Figures 1–6. Figure 1. Juvenile sporophytes developing from endophytic gametophytes in cell walls of Pleonosporium vancouverianum. Note transverse divisions in early stage sporophyte on left. Figure 2. Portion of kelp gametophyte in P. vancouverianum showing cells with multiple discoid chloroplasts without pyrenoids. Figures 3–4. Optical sections showing (Figure 3) cell wall surface with epiphytic cyanobacteria (arrow) and debris and (Figure 4) gametophytic filaments within the host cell wall. Arrows and arrow heads show equivalent positions in the two focal planes. Figure 5. Optical section of host cell wall with gametophytic filament clearly under the surface (arrow heads) of cell wall. Asterisk indicates corner of host cell protoplast. Figure 6. Group of gametophyte filaments in portion of host cell. Note; dark mass in background is partially plasmolysed protoplast of host cell. Scale bars in µm.
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Figures 7–10. Figure 7. Early stage of oogonial development showing cell densely packed with chloroplasts and bulging above surface of cell wall (arrow heads). Figure 8. Later stage of oogonial development with dome shaped apex well above surface (arrow heads indicate upper limit of cell wall on oogonium). Figure 9. Cleaved zygote (arrow heads) developed from endophytic filament with distict oogonial stalk and clear space at base (asterisk). Arrow indicates apex of stalk. Figure 10. Optical section of host with antheridia of kelp gametophyte at host wall surface with two clusters of antheridia (arrows) and emergent antheridium (arrow head) developing from endophytic gametophyte. Clear band in upper portion of figure is transverse septum of host Griffithsia pacifica Abbott. Note scattered debris on wall surface.
enchymatous growth to form a flattened blade and differentiating a stipe (Figure 1). Juvenile sporophytes were commonly observed on the surface of the host (Figure 1), and where direct observation was possible, these sporophytes were always associated with endophytic filaments in the host. Antheridia occurred singly or in clusters (Figure 10), but were often difficult to resolve because of their small size and the presence of other organisms and debris. Biflagellate motile cells 2–6 µm long were observed in the slide preparations, and some of these may have been spermatozoids. Fertilization was not observed.
Discussion The endophytic habit is widespread in Phaeophyceae. In Ectocarpales and Chordariales, many species and even genera are endophytic [e.g. Streblonema (Abbott & Hollenberg, 1976), Laminariocolax (Burkhardt & Peters, 1998)]. Henry (1987) described Verosphacela (Sphacelariales) as an endophyte in Spatoglossum. In the Desmarestiales, an order closely related to Laminariales (Tan & Druehl, 1996), Desmarestia antarctica has endophytic gametophytes in the thalli of the red alga Curdiea racovitzae De Wildeman (Moe & Silva, 1989). In addition, Dube & Ball (1971) reported gametophytic filaments of Desmarestia sp. endozoic in a sea pen (Ptilosarcus). The prior observations of
251 laminarialean gametophytes in no way indicates the occurrence of the endophytic habit. However, given the taxonomic distribution of endophytic species or life history stages in brown algae, it is not surprising, in retrospect, that endophytism should also occur in Laminariales. Our observations on the vegetative and reproductive morphology of the brown algal endophytes in red algae are definitive in demonstrating that these filamentous thalli are members of the Laminariales. We know of no other group of brown algae that has the following group of features: (a) filamentous gametophytes with multiple discoid chloroplasts devoid of pyrenoids; (b) distinct unicellular antheridia either single and scattered or clustered in groups; (c) an oogonium that differentiates from an intercalary (or terminal) cell in a filament to produce a stalked egg apparatus in which the egg protoplast migrates to the top of the stalk; (d) unisexual gametophytes; and (e) zygote embryology in which there is an initial filament that later forms a parenchymatous thallus (without the formation of hairs), that differentiates into a stipe and blade (Bold & Wynne, 1985). The identification of our filamentous thalli as members of the Laminariales is certain; however, there is no taxonomy of laminarialean gametophytes that would allow identification at specific, generic or family level. Some species of Laminariales produce bisexual gametophytes (e.g. Chordaceae, Phyllariaceae, Pseudochordaceae: Maier, 1984; Henry, 1987; Kawai & Kurogi, 1985, respectively), however, all gametophytes that we observed were unisexual as is typical of most Laminariales (Bold & Wynne, 1985; Kawai & Kurogi, 1985). Thus it remains to be demonstrated how many species of Laminariales are involved with the endosymbiosis reported here. The occurrence of the kelp symbiosis at multiple sites in the San Juan archipelago where different kelp species were dominant (e.g. members of Laminariaceae, Alariaceae and Lessoniaceae), allows us to infer that more than one species of Laminariales is capable of forming such a symbiosis. Using the microphotometric technique of Graham & Mitchell (1999), Graham (personal communication) has suggested to us that the variation in our samples is also consistent with our contention that several species can occur as endosymbionts. Preliminary observations on the zoospores of Nereocystis luetkeana and Lam-
inaria groenlandica show that zoospores can attach to red algal hosts and germinate, and that some of these become endophytic in the walls of two red algal species [Pleonosporium vancouverianum and Antithamnionella pacifica (Harvey) Wollaston] (Garbary, unpublished). The complete development of these gametophytes was not followed. Laboratory culture studies of all members of the Laminariales studied to date (e.g. Lüning & Neushul, 1978; Henry & Cole, 1982), as well as the use of spore settlement plates in nature (Reed et al., 1988) or the outplanting of laboratory settled zoospores (Hsiao & Druehl, 1973), have demonstrated that the symbiosis is not obligatory. The relative importance of the freeliving versus endophytic gametophytes to successful re-establishment of the sporophytic phase, remains to be demonstrated. On the one hand, it is possible that the endophytic habit is an accident of spore settlement, and that it is of little or no adaptive significance. On the other hand, endophytism may represent a facultative strategy of primary importance to gametophyte growth and reproduction, at least in some species or in some environments. The irregular branching pattern and absence of heterotrichous development in the kelp gametophytes is analogous to that found in many endophytic red algae (e.g. in Acrochaetiales, Garbary et al., 1982) or endophytic green algae [e.g. Acrochaete, Endophyton, Phaeophila (Nielsen & McLachlan, 1986)]. These common features suggest a morphological convergence in red, brown and green algae based on the common adaptation for endophytism.
Acknowledgments We thank the Director and Staff of Friday Harbor Laboratories for providing the research environment in which this work was performed. Dr Dianna Padilla provided encouragement, helpful discussion and access to the ‘free car’. Bruno Pernet, Brian Allen & Sarah Carter kindly provided diving assistance. This work was supported by research grants from the Natural Sciences and Engineering Research Council of Canada to DJG and the Korean Science and Engineering Foundation to KYK.
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