/ Phycol. 32, 313-322 (1996)
SPORELING COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA CHILENSIS (CRACILARIALES, RHODOPHYTA)'
Bernabe Santelices,^ Juan A. Correa, Isabel Meneses, Diego Aedo, and Daniel Varela Departamento de Ecologia, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Casilla 114-D. Santiago. Chile
ABSTRACT Three kinds of factors may cause intraclonal vari- This study n
coalescence could be regarded as a factor causing cystocarps. In the latter case, the proportion of spores from each intraclonal variation in Gracilaria chilen.sis. Using cystocarp was approximately the same. Twenty seven cover slips transmission electron microscopy (TEM), we Brst contained unicystocarpic spores, and 18 contained polycystocar- pic spores. All cover slips were incubated in individual glass Petri examined early stages of development to measure dishes (50 x 10 mm), filled with SWM-3 culture medium the extension of the coalescence and fusion pro- (McLachlan 1973), and maintained under standard culture con- cesses. Combining spores from similar and different ditions. Growth was monitored at 3-d intervals during the first 2 cystocarps, we constructed uni- and polycystocarpic weeks and weekly thereafter. To prevent diatom growih, I mL plantlets with initially different numbers of spores of a stock CeOj solution (6 mg L"') was added to each liter of and studied sporeling survival, growth pattern, and SWM-3. Culture medium was changed weekly. external morphology as a function of the initial At the tenth week of growth, cover slips with their thalli were transferred to 250-mL flasks, ea(h containing 200 mL of SWM-3 number of spores. Combining green and red car- culture medium. Gentle air bubbhng was also provided. Other pospores, we obtained bicolor individuals and stud- abiotic conditions were as already described including weekly ied the expression of the chimeric tissues in fully changes of the culture medium. The experiment was started on developed plants. Two plants (one unisporic and the June 1993 and was terminated 30 weeks after, when the first four other polysporic), which survived the experimental experimental individuals became reproductive. period, allowed us to make an exploratory analysis By 7-10 weeks, sporeling masses were large enough to distin- of the genetic variability that could be induced by guish, and the emerging erect shoots could be counted. Elon- spore coalescence. Preliminary DNA comparisons gation rates and number of lateral branches produced by the erect shoots were measured every week between the tenth and were made using the random amplified polymorphic the thirtieth weeks as described by Mufioz and Santelices (1994). DNA (RAPD) technique, which has been used, so Data on growth patterns as functions of the original number far, to fingerprint species and populations of Por- of spores were analyzed using regression and covariance analysis phyra (Dutcher and Kapraun 1994) and Sargassum (Snedecor and Cochrane 1967). (Ho et al. 1995) and individuals ofCelidium (Patwary Assembling and growth of bicolor inidividuals. The experimental etal. 1993, Patwary and van der Meer 1994). Using procedures for this experiment were similar to those already unisporic and polysporic thalli, we then evaluated described. However, we combined carpospores from red, wild- type thallus collected in Niebla with carpospores from a green, whether or not the range of intraclonal variation fertile cystocarpic thallus collected in Mejillones, near Antofa- regarding growth and performance changes as a gasta (23°38'S, 7O''24'VV) in northern Chile. Similar numbers of function of the original number of spores. green and red carpospores were used to construct bicolor indi- viduals. The initial number of spores ranged from 2 to 36, and the total number of assembled plantlets was 25. Plantlets were incubated and handled as those in the preceding experiment. MATERIALS AND METHODS However, because the main interest of producing bicolor plant- All the experiments were performed with thalli collected in lets was to study tissue distribution, quantitative measurements Niebla, in southern Chile (39''51'S, I^'QS'W). Plants were trans- were replaced by recording the development of the surviving ported by air in refrigerated containers to the laboratory in San- plantlets photographically. Observations of these chimeric thalli tiago. Fhey were washed with running tap water and maintained terminated after 75 weeks of growth. for 24 h in filtered seawater under standard conditions of tem- RAPD. Polymerase chain reaction amplification was performed perature (14 ± 2" C), irradiance (20 ± 10 ^tmol photons-m"*' with DNA extracted from branthlets of one unisporic and one s '), and photoperiod (12:12 h LD). Sporulation was induced by polysporic plant. Both types of vegetative thalli were 8 cm long immersing mature cystocacps in microfiltered seauater (0.45 fim). and had been kept growing under standard culture conditions These cystocarps were dehydrated for 24 h at room temperature for 28 weeks prior to the extraction. The unisporic thallus had prior to sporulation. one erect axis supporting numerous lateral branches in a semi- Ultrastructure of germlings originated from coalescing .spores. Car- alternate manner. The polysporic plant came from the coales- pospores shed by the experimental cystocarps were inoculated cence of three spores and had three erect shoots. Shoots a and into holes (1.8 mm diameter, 250-300 ^m deep) drilled in glass c rach had three laterals branchlets, labeled a,, a^, and a, and c,, microscope slides. The number of carpospores in each hole varied Cj, and Cj, respectively. The third axis (shoot b) had four laterals, from 30 to 50, and a total of 30 cover slips was used. Individual named b,, bj. b,, and b^. DNA from these 10 lateral branchlets cover slips were incubated in Petri dishes (50 x 10 mm) contain- as well as from 9 laterals from the unisporic thallus was extracted ing approximately 10 mL of filtered seawater. Growth chambers and analyzed. Before extraction, all branchleis were cleaned un- were set up under the preceding conditions. der the microscope and sonicated to avoid contaminants. Al difFereni times, ranging from 24 h to 30 days after inocu- Total DNA was extracted from fresh tips, each less than 2 mm lation, spores and plantlets at different stages of development long. Individual tips were ground in 200 ;JL of buffer (250 mM were fixed while in the depressions with 1% glutaraldehyde in Tris-HCI, 250 mM NaCI, 25 mM FD'FA, 0.17i SDS) with a silicon 0.45-Mm-filtered seawater for 24 h. Postfixation in We OsO^ te- carbide abrasive (carborundum) in an Eppendorf tube of 1.5 mL. troxide for 3 h was followed by dehydration in an ethanol series. Addition of 400 /.iL of chloroform : isoamyl alcohol (24:1) with Samples were infiltrated and embedded in Spurr"s resin. Semithin occasional vortexing of the mixture for 10 min was followed by sections for light microscopy were stained with 19f Toluidine centrifugation at 13,000 rpm for 15 min in an Fppendorf cen- blue and thin sections for TEM were stained with 4% uranyl trifuge. acetate and lead citrate according to Reynolds (1963). Observa- The upper aqueous phase was then transferred to a new tube, tions were made in a Philips 300 electron microscope operated and an equal volume of lOOS^ ethanol was added to precipitate at 60 kV. the nucleic acids. 'T'he mixture was frozen at —70° C for 30 min, Suni-i'al, growth, and external morphology as a function of the initial and the nucleic acids were pelleted by centrifugation for 15 min number of spores. Carpospores. 1-32, were pipetted into the holes (13,000 rpm). 'Fhe pellet was washed in 7O'-{ ethanoi, dried, and drilled in the glass microscope .slides. Inoculations included car- redissolved in TE buffer (10 mM Tris HCl, pH 8.0. and 1 mM pospores shed by a single cystocarp (generating unicystocarpic EDTA). 'Fhe sample was incubated at 37° C for 20 min and plantlets) or two different (generating polycystocarpic plantlets) centrifuged. Nucleic acids in solution were removed with a mi- COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA 315 cropipette, and further purification was achieved using a Wizard the adjacent groups of cells could occur (Fig. 6). Minipreps DNA purification system (Promega, Madison, Wiscon- However, this was not frequent. In general, where sin). many germlings grew close enough to establish con- Amplifications were performed in 20-(iL solutions containing Taq DNA polymerase buffer (Promega), 100 |tM of each deox- tact, most individuals remained as discrete entities yribonucleoiide triphosphate (Amersham, England), 1,0 nM of (Fig. 7). decamer primers (Operon Technologies, California), 20 ng of The outer cell wall of coalescing individuals ap- template DNA, and 0.5 unit Taq DNA polymerase (Promega). peared as a continuum (Fig. 8), with a finely retic- The amplifications were performed in a Perkin-P^lmer DNA ther- ulate texture that clearly differed from the homo- mal cycler model 480 programmed for 3 initial cycles of 1 min geneous aspect displayed by the immediate cell wall at 95''C, 1 min at 37" C, and 1 min at 72° C, followed by 40 cycles of 30 s at 94" C, 30 s at 40" C, and 1 min at 72" C, with a f^nal (Fig. 9). Secondary pit plugs were established be- run of 5 min at 72° G. Fifteen microlitersof the reaction products tween cells of coalescing germlings at the fusion zone were separated by etectrophoresis through 1.0*^ agarose gels and (Fig. 10). There seems, however, to be no ultrastruc- stained with ethidium bromide. .A.garose gels were photographed tura! differences between these and the primary pit with a 2N Olympus camera with a Wratten gelatin filter 22 (KO- plugs. DAK) over a ultraviolet transilluminator (312 nm) using T-MAX 100 ASA film. Sunnval, growth, and external morphology as a func- tion ofthe initial number of spores. Average carpospore Variability in groirth and pfrjormance. Fhree, 25-week-old plants were used in this experiment. One thallus (plant 2) was unisporic. germination rate was 74.8 ± 9.4%. Germination One (plant 13) developed from the coalescence of three spores, rate of individual spores within a coalescing mass and one (plant 3) developed from 11 coalescent spores. The un- slightly decreased with increasing spore density, but isporic plant had one, the 3-spore plant had two, and the II- the reduction was not statistically significant for ei- spore plant had three erect axes invested with lateral branchlets. ther unicystocarpic and polycystocarpic coalescing Branchlets (9-14) of similar initial weight (3-14 mg) were cut spores. Covariance analysis {F ^ 2.33, df — I, P ^ from each erect axis in each plant and incubated under the stan- dard conditions described earlier. Experimental procedures fol- 0.136) indicated an absence ot significant differ- lowed the protocol described by Santelices and Varela (1993), ences in germination rates between uni- and poly- The branchlets were tagged, and their secondary branchlets were cystocarpic spore assemblages. counted. "The branchlets were photographed under water to avoid Sporeling and sporophyte development followed dehydration and wet-weighed (blotted weight) in a Sartorious the pattern previously described for this species balance. Three ramets from each thallus (= genets) were placed inside each of four replicate l-L beakers filled with 900 mL of (Murioz and Santelices 1994). Uni- and multisporic SWM-3 culture medium. Because four genets (= individuals) were plantlets formed irregular, circular discs that grad- used, each beaker contained a total of 12 ramets, which were ually expanded in diameter and height. Between the kept at the bottom by fastening lo glass slides. The fastening seventh and tenth weeks of development, plantlets order for each ramet was at random, 1 he culture medium was produced erect shoots, the longest of which reached changed every 6 d. Length and blotted weight of each ramet was about 10 mm by the fifteenth week. Four weeks recorded after 30 d. Data dispersal and coefficient of variation later, some of these axes showed differentiation of for each plant and each erect axis within the multi-a.xis thalli were compared. lateral branches, and by the thirtieth week some of the longest erect axes (8-10 cm) became fertile. Survival rates ofthe plantlets during the 30 weeks of growth were essentially similar for uni- and poly- RESULTS cystocarpic plantlets. Three ofthe 27 unicystocarpic Ultrastructure of germlings originated from coalescing and two of the 18 polycystocarpic plantlets died spores. Once released and prior to the first mitosis, within the first 3 weeks. Two more plantlets of each spores were naked (Fig. 1). Immediately after set- type died in the next 2 weeks. The remaining 22 tling, small vesicles containing large concentrations unicystocarpic and 14 polycystocarpic plantlets re- of fibrillar material coalesced into larger ones, lo- mained alive until the end of the experiment. cated at the periphery of the cell (Fig. 2). Subse- The time of appearance of erect axes ranged from quently, the large vesicles located underneath the the seventh to the tenth week of growth. There was plasmalemma began releasing the fibrillar material a tendency for plantlets from a few coalescing spores (Fig. 3), and plantlets with the characteristic double to differentiate uprights earlier in development. cell wall were present at the end ofthe 24-h period However, regression values were not statistically sig- (Fig. 4). nificant for unicystocarpic nor for polycystocarpic Spores that settled close enough to be in contact sporeling masses. to each other during germination showed no evi- The total number of erect axes produced by the dence of plasmalemmal fusion (Fig. 5). On the con- coalescing masses of spores after 19 and 29 weeks trary, each of these spores produced a distinctive of development was a function ofthe original num- cell wall that separated neighbor individuals. The ber of spores (Fig. 11). Unicystocarpic plantlets pro- wall produced by each spore became the outer cell duced more erect shoots than polycystocarpic plan- wall of individual germlings by surrounding the cells tlets. However, although differences were not sta- originated during spore germination (Fig. 5). Dur- tistically significant at the nineteenth week (covari- ing early development, some germlings shared a ance analysis, F = 3.78, df = I, P = 0.06), they common external envelope that reacted strongly with became significant by the end of the experiment Toluidine blue (Figs. 5, 6). In such cases, fusion of (covariance analysis, F = 4.27, df ^ 1, P ^ 0.04). In 316 BERNABE SANTELICES ET AL.
o
FIGS, 1—7. Ultrastructure of germlings originated from coalescing spores. FIG. 1, Naked spore after release. Abundant, small vesicles (arrows) containing fibrillar material appear scattered among sections of the chioroplast. Scale bar = 2 nm. Fic. 2. Settled spore with large vesicles (V) loaded with coarse fibrillar material. Extracellular material (arrows) begins accumulating outside the cell to form the cell wall. Scale bar = 1 *im. FIG. 3. Close-up ofthe cell surface showing the release of coarse material. Scale bar = 0.5 tim. FIG. 4, Double cell wall in an individual spore that has completed settlement. Scale bar = 1 jim. FIG. 5. The first mitotic division yields two cells separated by a low-contrast cell wall. Several of the two-cell groups have coalesced with neighboring groups through a common, external cell wall. Scale bar = 30 nm. FIG. 6. Fused plantlets 24 days after settlement. Both plantlets share a common external cell wall that tends to dissappear at the intercell contact area (arrows). All other cells maintain their individuality. Scale bar = SO ^m. FIG, 7. Coalescent but unfused planilets 24 days after settlement. Scale bar = 50 ixm. COALESCENCE AND INTRACLONAL VARIATION IN GRACJLARIA 317
FIGS. 8-10. Kxiemal cell wall and secondary pit connections between cell groups of difTerent origins. FIG. 8. External cell wall (arrows) common to two-cell groups of different origins (C, and Gj) in 24-d-old plantlets. Scale bar = 3 ^m. FIG. 9, Enlargement showing the immediate cell wall (I) of more homogeneous texture and the external cell wall (E) of fibrillar appearance. Scale bar = 1 Mm, Fic. 10. Fusion zone between two adjacent, 24-d-old plantlets. The dotted lines indicate the limits ofthe coalescing plantlets. The external cell wall does not exist at the fusion zone. Secondary pit connections (circle) may occur between cells of both plantlets. Scale bar = 5
both cases, the relationship between initial number axes of four polysporic thalli, originated from 6, 8, of spores and number of erect axes was logarithmic, 10, and 13 coalescing spores. with a decreasing number of shoots produced by Assembling and growth of bicolor individuals. Ger- plantlets formed by a larger number of spores. mination and growth of bicolor individuals followed The preceding numerical relationship was reflect- the normal developmental patterns although their ed in the external morphology ofthe plants. Thalli mortality rates were much higher than those gen- grown from one or a few spores were elongated erated by coalescence of red spores alone. Only eight plants with a single or few main axes bearing lateral of the 25 initial plantlets (32S?) were alive by tbe branches. Individuals grown from many coalescent time erect shoots began to appear (weeks 8 and 9). spores appeared bushy, with many short, sparsely Of these, only three survived long enough to exhibit branched erect axes arising from a discoid holdfast. initiation and development of lateral branchlets (be- The first reproductive fronds were the longest yond week 15). Twcj of these bleached by week 18, 318 BERNABE SANTELICES ET AL.
20 Nineteenth week 14). The plants, however, persisted as a mixture of red and green axes. Generally, only one type of pig- 18 mentation was observed in each axis, suggesting ab- 16 sence of individual chimeric fronds. However, green and red lateral branchlets differentiating from a sin- 14 gle, all-red axis were detected in the 70-week-old 12 plant (Fig. 15). Green pigmentation persisted as the branchlets enlarged (Fig. 16). 10 Ietraspores produced by the bicolor individual 8 were either red or green. Germination and devel- (D 6 opment of these tetraspores followed the pattern £ already described for the species, generating red or 3 4 green plantlets. 2 ' Unicystocarpic RAPD. A total of 12 different primers were • Polycystocarpic screened to compare banding patterns of one un- 0 isporic (Fig. 17, upper panel) and one polysporic (Fig, 17, lower panel) thalius. All primers were pos- 0 2 4 6 8 10 12 14 16 18 20 itive, each producing 4-10 bands. None of the prim- Number of spores ers revealed polymorphism among the apices from the unisporic thaltus (Table 1), while all but one showed polymorphism among the apices from the polysporic thallus ('Fable 1). 20 Thirtieth week Variability in growih and performance. Plotting weight increase as a function of initial weight in the three 18 experimental plants (Fig. 18) indicated that total to plant variability was greater in individuals formed by a larger number of spores. Furthermore, data dispersal and variation coefficients for an individual o axis within each of the two polysporic plants (plants 10 13 and 3) were heterogeneous. While ramets from some axes exhibited little variation in their re- 8 sponses to culture conditions (e.g. axes 13.2 and 3.2, 6 Fig. 18) others (axes 3.3 and 13.1) bore ramets that E responded very differently to the culture conditions. 3 4 '1 his physiological heterogeneity was highly consis- 2 H A Unicystocarpic tent with the chimeric nature of" the individual axis • Polycystocarpic observed in the bicolor thalli and the genetic poly- 0 morphism among lateral branchlets ofthe same erect 0 2 4 6 8 10 12 14 16 18 20 axis detected by the RAPD technique.
Number of spores DISCUSSION FIG. 11. Total number of uprights at two times of develop- ment as a function of the original number of spores used to Our results support the hypothesis that spore co- construct coalescing plantlets of Gracilaria chilensis. Regression alescence is a factor causing intraclonal variation in equation values are, for the nineteenth week, unicystocarpic plan- species with developmental patterns similar to that tlets (upper line): y = 2.11 + 6.86 log(x), ft* = 0.54, P = 0.0001; of Gracilaria chilensis. Even though adjacent spores polycystocarpic plantlets: y = -1.09 + 8.69 log(x), ii^ = 0.51, P = 0.003. Eor the thirtieth week of growth, the equation for un- or plantlets may c(ialesce during early development, icystocarpic plantlets (upper line) is y = 2.70 + 8.07 log(x), R^ = intergermiing fusions generally involve few cells. 0.53, P = 0.0001: polycysiocarpic plantleis: y = -0.41 + 9.14 Thus, polysporic plantlets are formed by groups of log(x), K* - 0.39. P = 0.017. cell clusters, each with an initial spore and its deriv- atives that, under TEM, appear as discrete units surrounded by a common external cell wall. Cells and the only surviving individual became reproduc- in adjacent clusters may fuse, establishing secondary tive by week 42 and remained alive and healthy for pit plugs, but within each cluster active cell division 75 weeks (Fig. 12). produces numerous derivatives ofthe initial spore. All eight plantlets alive by the time of upright Seemingly, none of the previous studies (Tveter- initiation produced green and red axes from the Gallagher and Mathieson 1980, Maggs and Cheney bicolor holdfast (Fig. 13). With time, tbe surface of 1990) examined early developmental stages of co- the holdfast usually became dominated by either alescing plantlets. Rather, their contribution was to green or red tissue, and the color ofthe new shoots establish the continuity between coalesced, well-de- was that ofthe dominating tissue in the holdfast (Fig. veloped holdfasts. This is also observed in Cracilaria FIGS. 12-16. External morphology and branching details of the bicolor individuals. FIG. 12. Seventy-week-old bicolur individual of Gracilaria cbileiists grown under laboratory conditions. Most uprights are green-pigmented. Arrow indicates red-pigmented uprights. Scale bar = 2 cm. FIG. 13. Twenty-three-week-old bicolor individual oi Gracilaria chilensis growti under laboratory conditions. Uprights with different pigmentation have grown from a bicolorwi holdfast. Scale bar = 2 mm. FIG. 14. The same individual as in Figure 13, 4 weeks older. The .surface ofthe holdfast is dominated by red tissue. New red shoots are being produced. Scale bar = 2 mm. FIG. 15. Green-pigmented and red-pigmented branchlets originating from a red-pigmented upright. The upright was part of the 70-week-old bicolor individual. Scale bar = 2 mm. FIG. 16. The same branchlets in Figure 17, 1 week later. All branchlets are actively growing. Scale bar = 2 mm. 320 BERNABE SANTELICES ET AL.
Ml 2345678 TABLE 1. Presence (+) or absence (—) of genetic pohmorphism in branchlets of unisporic and polysporic thalli with va nous primers. Poh- morphism is here defined as at least one band difference among 10 apices.
23130- Primer Prilysponi Unisporic OPX-01 GTGGGGAGGA OPX-02 TTGCGGGAGC OPX-04 GGGCTACCGA OPX-06 ACGCCAGAGG OPX-07 GAGCGAGGCT OPX-08 CAGGGG IGGA OPX-13 ACGGGAGCAA OPX-18 GACTAGGIGG OPX-20 CCCAGCTAGA OPO-06 CCACGGGAAG OPO-07 CAGGACTGAC OPO-08 CCTCCAGTGT
Tveter and Mathieson 1976). A lower, nonspecific ratio was suggested for unisporic plantlets of these two species. In G. chilensis, we found a logarithmic relationship with proportionately fewer shoots as M12345678 spore number increased. This may indicate that, as the number of spores increases, a proportionately larger number of adjacent spores fuse, not forming derivatives and, eventually, upright shoots. Alter- natively, the logarithmic relationship might be an 23130- artifact in larger plantlets resulting from an increas- ing number of ceil clusters remaining in the slide P''" ^^ wm Wm holes that were prevented from issuing uprights. / / Additional experiments are required to determine whether the numerical relationship is logarithmic, linear, or otherwise. The chimeric nature of the coalesced individuals is evident in the bicolor thalli. Our results are similar to those reported by Maggs and Cheney (1990) in the sense that green and red cells in the coalesced FIG. 17. RAPD patterns in monosporic and polysoric thalli. holdfast gave origin to thalli with pigmentation sim- Upper panel DN.'\ fragment patterns from a mono.sporic plant oi Gracilaria chilensis. Lane numbers indicate different apices of ilar to that of the parental cells. However, in the the same thallus. Lane M corresponds to X DNA digested with case of G. chilensis, even the individual fronds were Hind III. Primer was OPO-06. Lower panel Polymorphic DNA chimeric, as evidenced by the production of green fragment patterns from a polysporic plant of Gracilaria chilensii. and red branchlets produced by a single all-red axis. Lane numbers correspond to different apices of the same thallus. Arrows indicate absence of bands. Lane M corresponds to X DNA Previous studies with bicolor individuals {Maggs and digested with Hind III. Primer was OPO-20. Sample 6 was lost Cheney 1990) did not mention the age or the size during the polymerase chain reaction due to lack of mineral oil. ofthe studied plants: therefore, no comparisons can be made with our experiments. The detection of the chimeric frond in our cultures was done after an chilensis. However, it should be emphasized that this extended cultivation period (70 weeks). Until then, continuity may involve genetically different tissues the group of green cells immersed within the red united into a chimeric holdfast. axis went undetected. The numerical relationship between the initial For species other than Gracilaria, chimeric indi- spore number and the number of erect shoots is viduals have been obtained from the fusion of ge- consistent with the preceding interpretation and netically different protoplasts. Blades consisting of suggests that the capacity to grow as an independent green and red groups of cells developed from the unit and eventually to form uprights is retained by fusion products of green (mutant-type) and red (wild- the spore derivatives in the cluster. The mathemat- type) protoplasts of Porphyra yezoensis Ueda (Fujita ical nature of this relationship may vary from one and Migita 1987, Fujita and Saito 1990). Protoplast species to another. A ratio of five spores to each fusion takes place only during a narrow period of shoot was suggested for large sporeling masses of time, before the potentially merging cells synthesize Gracilaria verrucosa and Chondrus crispus (Jones 1956, their cell walls. Although our TEM observations in- COALESCENCE AND INTRACLONAL VARIATION IN GRACILARIA 321
Plant N" 2. One spore is formed the green tissue is overgrown and hidden by the red tissue. 120-| Variation coefficient of total plant = 34.15 The monomorphic DNA patterns obtained from the unisporic thallus should be considered with cau- 100- tion when compared to the polymorphism recorded for the branchlets from the polysporic thallus. This 80- analysis should be replicated in a number of indi- viduals from both origins (i.e. unisporic and poly- 60- sporic) in order to reach definitive conclusions about the genetic variability of both types of plants. Nev- 40 ertheless, due to the consistency of our results with 20- all the primers, the occurrence of genetically het- erogenous tissues in the branchlets of polysporic I ' I ' 1 ' r ' T thalli is a likely phenomenon. In turn, the presence 2 4 6 8 10 12 14 of genetically heterogeneous tissues can be inter- Initial weight (mg) preted as one of the factors responsible for the high- ly variable growth responses displayed by branchlets Plant N** 13. Three spores ofthe same individual when incubated under similar growth conditions. The large data dispersal and 120n Variation coefficient (v.c.) of total plant=38.81 variation coefficient shown in our experiments by Axis 13.1= • (v.c.=72.53) 100 the polysporic thalH did not reflect high heteroge- Axis 13.2= i(v.c.=28.04) neity in all axes and branchlets. Rather, branchlets 80- of some axes were more homogeneous while others were more heterogeneous, suggesting that genetic 60- heterogeneity does not affect all axes in similar fash- o ion. 40- H B Gracilaria chilensis, as many other species in the 20 genus, is a morphologically variable taxon. Previous studies have attempted to relate morphological vari- ability with environmental factors {for reviews, see I ' I ' I 0 10 12 14 Hoyle 1975 and Bird and McLachlan 1986), with Initial weight (mg) habitat characteristics and mode of reproduction {Meneses 1996) and with genetic polymorphism. Our Plant N" 3. Eleven spores results suggest that the number of coalescing spores producing the thallus may be still another factor. 120 n Variation coefficient (v.c.) of total plant=98.41 Thalli grown from one or a few spores exhibit a Axis3,1=-(v.c, = 1 single or few main axes. I'halli originated from many 100 Axis3.2= -J(v.c.=36.31) spores had a bushy appearance conferred by the Axis 3.3= - (v.c.=110.27) development of many short uprights. It is as yet 80- unknown whether or not this morphological differ- 60- ence persists in the field. If it does, the number of erect axes, alone or combined with the RAPD tech- 40- nique, could help to determine whether field-col- lected individuals are mono-, oligo-, or poiysporic 20- in origin. The frequency of spore coalescence in field pop- r I ulations of species oi Cracilaria and other gregarious 2 4 6 8 10 12 14 Rhodophyta remains to be elucidated. The aggre- Initial weight (mg) gated pattern of propagule release and dispersal, the FIG. 18. Weight increase as a function of initial weight under sticky mucilaginous envelope around the spores, and similar abiotic conditions in ramets of thalli originated from 1, the recruitment on or close to the paternal thaili 3, and 11 coalescing spores. with a resulting aggregated spatial distribution are all features suggesting that coalescence might be more frequent in field populations than previously dicate that spores of G. chilensis are naked when thought. Because several coalescing Rhodophyta are released, spore fusion has not been detected. Thus, among the dominant organisms in intertidal and it seems unlikely that our chimerical thalli resulted shallow subtidal marine habitats {e.g. Chondrus, Gi- from spore fusion. Rather, our TEM observations gartina, Iridaea, Mazzaella, Sarcothalia, Mastocarpus, ofthe early stages of coalescence support the notion Ahnfeltia, Ahnfeltiopsis), coalescence might not only that when the mosaic of wild- and mutant-type cells be frequent but also ecologically important. 322 BERNABE SANTELICES ET AL.
Several adaptive traits have been suggested for Population Biology and Evolution of Clonal Organism.^. Yale Uni- coalescing seaweeds (e.g. Maggs and Cheney 1990); versity Press, New Haven, Connecticut, pp. 1-33. however, relatively few have been quantified {e.g. Ho, C. L., Phang, S-M. Sc Pang, T. 1995. Molecular character- ization o{Sargas-ium polycystum and S. siliquosum (Phaeophyta) Martinez and Santelices 1992, Munoz and Santelices by polymerase chain reaction (PCR) using random amplified 1994). The most important adaptive value of clonal polymorphic DNA (RAPD) primers./ Appl. Phycol. 7:33-41. growth may be based on the possibility that a par- Hoyle, M. 1975. The Literature Pertinent to the Red Algal Genus ticular gene, or gene combination, is repeatedly ex- Cracilaria in Hawaii. Tech. Report 3, Marine Agronomy U.S. Sea Grant Program, University of Hawaii, pp. 1-340. posed to a diversity of selective forces, increasing Jones, W, F. 1956. Effect of spore coalescence on the early de- the possibility of genet survival under at least one velopment of Gran/r!n(7Tc»-f(/ro.!i2 (Hudson) Papeniuss. Nature such environment (Harper 1985). Enhanced phe- (Lond.) 178:426-7. notypic heterogeneity within a genet conferred by Maggs, C. A. &i Cheney, D. P. 1990. Competition studies of spore coalescence and chimerical tissues would re- marine macroalgae in laboratory culture, y. Phycoi 26:18- sult in ramets being different. This would further 24. Martinez, E. & Sanlelices, B. 1992. Size hierarchy and -3/2 increase the possibilities for individual and popula- "Power law" relationship in a coalescent seaweed./. Phycol. tion survival, either by increased variation and re- 28:259-64. sistance to the abiotic environment (Buss 1985), by McLachlan, J. 1973. Growth media-marine. In Stein, J. R, |Ed.j increased competitive capacity (Paine 1990), or by Handbook of Phycological Method.^. Culture Methods and Crowth increased resistance to grazers (Maggs and Cheney Measurement.^. Cambridge University Press, Cambridge, pp. 25-52. 1990). As Buss (1985) and Paine (1990) noticed, Meneses, L 1996, Sources of morphological variation in popu- clonal and chimeric individuals challenge the appli- lations of GrrtnVnJiVi chilemis Qhd, McLachlan Sc Oliveira. flci'. cation of concepts linking Darwinian selection with Chil. Hist. Nat. (in press). population biology, and their study should help to Mufioz, A. A. & Santelices, B. 1994. Quantification of the effects expand traditional ecological theory. of sporeling coalescence on the early development of Grac- ilaria chilensis (Rhoduphyta).y. Phycol. 30:387-92. Paine, B. 1990. Benthic macroalgal competition: complications The authors thank Drs. P. Gacesa and P. Hiiirichsen and the and consequences./. Phycol. 26:12-7. biochemists from Instituto Naciona! de Investigacion Agrope- Patwary, M. U., Mackay, R". M. & van der Meer, J. P. 1993. Revealing genetic markers in Gelidium vagum (Rhodophyta) cuaria (INIA), Chile, for much help with the RAPD technique. through the random amplified polymorphic DNA (RAPD) Our appreciation to Dr. R. We.stermeier and Mr. R. Rojas for technique./ Phyioi 29:216-22. the provision of the red- and green-pigmented fertile materials Patwary, M. U. & van der Meer, J. 1994. Application of RAPD used in this study. Ms. Veronica Floresand Mr. Alejandro Munoz markers in an examination of heterosis in Gelidium vagum helped wiih the ultrastructure studies and the physiological ex- (Rhodophyta)./ Phycoi 30:91-7. periments, respectively. This study was supported by grant FON- Reynolds, F. S. 1963. The use of lead citrate at high pH as a DECYT 1930581 IO B'.S. andJ.C.'grant SAREC-CONICYT 90-7 electron-opaque stain in electron microscopy./ Cell Bioi 17: to B.S.. and a postdoctoral grant EONDECYT 3930014 to LM. 208-12. Santelices, B. 1992. Strain selection of clonal seaweeds. In Round, Bird, C. 8c McLachlan, J. 1986. Gracilaria (Gigartinales, Rho- F. E. &: Chapman, D. J. [Eds.] Progre.'i.^ in Phycological Research, dophyta) and productivity. Aquatic Bot. 26:27-49. Vol. 8. Biopress, Bristol, UK, pp. 85-116. Buss, L. W. 1985. The uniqueness ofthe individual revisited. In Santelices, B., Aedo, D. Sc Varela, D. 1995. Causes and impli- Jackson, J. B. C, Buss, L. W. & Cook, R. C. [¥As.\ Population cations of intra-clonal variation in Gracilaria chilensis. J. .Appl. Biology and Exvlution of Clonal Organisms. Yale University Press, Phycol. 7:283-90. New Haven, Connecticut, pp. 467—505. Santelices, B. & Varela, D. 1993. liitra-clona! variation in the Diitcher, J. A. &: Kapraun, D. F. 1994. Random amplified poly- red seaweed Gracilaria rhilen.sis. Mar. Biol. (Beri) 116:543- morphic DNA (RAl'U) identification oi genetic variation in 52. three species of Porphyra (Bangiales, Rhodophyta}. y. Appl. Snedecor, G. W. & Cochrane, W. C. 1967. Statistical Methods, Phycol. 6:267-73. 6th ed. Iowa State University Press, Ames, 593 pp. Fujita, Y. Sc Migita, S. 1987. Fusion of protoplast from thalli of Tveter, E. &: Mathieson, A. C. 1976. Sporeling coalescence in two different color types in Porphyra yezoensis tJeda and de- Chondrus criipui (Rhodophyceae)./. Phycoi 12:1 10-8. velopment of fusion products.y(i/).y. PhycoL fi'oVuiJ 35:201-8. Tveter-Gallagher, E. & Mathieson, A. C. 1980. An electron mi- Fujita, Y. & Saito, M. 1990. Protoplast isolation and fusion in croscopy study of sporeling coalescence in the red alga Chon- Porphyra (Bangiales, Rhodophyta). Hydrobiologia 204/205; drus crispus. Scanning FAectron Microsc. 1980:571-80. 161-6. Watkinson, H. R. Sc White, J. 1986. Some life-history conse- Harper, J. L. 1985. Modules, branches and the capture of re- quences of modular constructions in plants. Phil. Trans. R. sources, /H Jackson J. B. C, Buss, L. W. & Cook, R. C. [Eds.] Soc. Ser. B 313:31-51.