Sporeling Coalescence and Intraclonal Variation in Gracilaria Chilensis (Cracilariales, Rhodophyta)'
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/ 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<aluates the hypothesis that spore coales- ation: 1) physiological and developmental differ- cence may cause intraclonal variation. Spore coalescence ences among ramets derived from a single genet, 2) might allow the occurrence of unitary thalli that in fact pathogens differentially affecting some ramets with- correspond to genetically different, coalesced individuals. in a clone, and 3) genetic changes in a given ramet. Plant portions simultaneously derived from these chimeric These factors are recognized as the most important, individuals may exhibit dissimilar growth responses ei'en causing phenotypic variation among ramets derived when incubated under similar abiotic conditions. Testing from the same genet in organisms as diverse as Bryo- ofthe hypothesis included -various approaches. Transmis- zoa, land plants, and seaweeds (Buss 1985, Harper sion electron microscopy observations of early stages of 1985, Watkinson and White 1986, Santelices 1992). .sporeling coalescence indicated that polysporic plantlets A fourth factor, spore coalescence, has been pos- were formed by groups of spores and their derivatives. tulated as exclusive to seaweeds (Santelices et al. Even though adjacent cells in two different groups may 1995) and constitutes the main focus of this study. fuse, these groups maintained an independent capacity to Spores of the orders Ahnfeltiales, Corallinales, grow and form uprights. Laboratory-grown plantlets Gigartinales, and Gracilariales (Rhodophyta) have showed a significant correlation between the initial number the ability to form basal crusts that may grow to- of spores and the total number of erect axes differentiated gether, forming a completely coalesced mass, which from the sporeling. Construction and growth of bicolor subsequently develops into a single plant (for specific individuals indicated the chimeric nature of the coalesced taxa, see Maggs and Cheney 1990). The coalesced individuals. Coalesced, bicolor holdfasts had green and mass exhibits vertical and horizontal alignment of red cells, which subsequently produced green and red up- cells, cuticular continuity, and formation of second- rights, respectively. Individuals fronds were also chimeric, ary pit plugs between cells of confluent individual as indicated by the production of green and red branchlets spores. from single, red uprights. The existence of mixed tissues For Cracilaria, successful spore coalescence has was further substantiated by random amplified polymor- been achieved between spores of similar or different phic DMA analysis. The banding pattern produced by thalli (e.g. G. verrucosa Jones 1956, G. chilen.sis Munoz branchlets of a unisporic thallus was consistently mono- and Santelices 1994), or similar or different karyo- morphic, whereas the patterns produced by the polysporic logical phases, and even between spores of difTerent thallus were polymorphic. Crowth rate.s of polysporic thalU species (G. tikvahiae x G. chilensis Maggs and Cheney had larger data dispersal and variation coefficients than 1990). Therefore, spore coalescence might allow the oligosporic or monosporic thalli. Therefore, all results sup- occurrence of unitary thalli that in fact correspond port the original hypothesis and suggest tbat coalescence to genetically different, coalesced individuals. It might be ecologically more important than prei'iously would be expected that axes of these cbimcric in- thought. dividuals may exhibit intraclonal variation in growth or performance. Key index words: bicolor individuals; Gracilaria chi- Experimental data in the literature partially sup- lensis; intraclonal variation; RAPD; Rhodophyta; spo- port this idea. Cheney (in Maggs and Cheney 1990) reling coalescence obtained bicolored (i.e. red and green) discoid mass- es of cells starting from green (mutant) and red (wild- Experimental field and laboratory studies have type) spores. Subsequently, both cell types produced shown significant intraclonal variation in Cracilaria uprights that displayed a pigmentation identical to chilensis Bird, McLachlan et Oliveira. Plant portions their parental cells, supporting the notion that ge- simultaneously derived from the same clone or suc- netically different fronds could come from a single, cessive generations from the same cut may exhibit coalescing holdfast. However, although red and dissimilar growth responses when incubated under green fronds were produced, individual chimeric similar abiotic conditions (Santelices 1992, Santel- fronds were not observed, suggesting that spore co- ices and Varela 1993). alescence could explain intraclonal variation among primary axes only and not among branchlets of the same axis. ' Received 5 September 1995. Accepted 19 December 1995. ' Author for reprint requests. In this study, we evaluate whether or not spore 313 314 BERNABE SANI ELICES E T AL. 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