Life history , morphological variability and growth rates of the life phases of Gracilaria tenuistipitata ( Rhodophyta : Gracilariales ) in vitro

Gracilaria tenuistipitata, a species of commercial interest, is becoming a model organism for studies on red algal physiology and molecular biology as it can be grown easily in vitro under a broad range of conditions. Most of the experiments carried out around the world have been based on a tetrasporophytic clone isolated in our laboratory from a specimen collected in China. Here we describe the life history of this species, give anatomic details of the reproductive structures, illustrate the morphological variability of tetraspore progeny and compare the growth rate of gametophytic and sporophytic thalli. Tetrasporophytic branches showed higher growth rates than gametophytic branches.


INTRODUCTION
The genus Gracilaria (Rhodophyta: Gracilariales) has been intensively investigated due to its economic importance, high diversity and taxonomic problems (e.g.Bellorin et al., 2002).Among the many species recognised in the genus, Gracilaria tenuistipitata C.F. Chang et B.M. Xia, a species originally described from China, has been a favourite alga for different types of investigations because it is easily kept in vitro under a broad range of growing conditions (e.g.Macchiavello et al., 1998;Carnicas et al., 1999;Hagopian et al., 2004).In addition to the academic interest, G. tenuistipitata is cultivated in China as a source of agar and as fodder in abalone (Haliotis sp.) farms (Chiang, 1981;Lin and Liao, 1999).
Most studies on G. tenuistipitata have used a clone of a tetrasporophytic lineage isolated in our laboratory.Nevertheless, there is very little information on the life history of this species, or informa-tion on its morphology.Moreover, the growth rate and the performance of the gametophytic generation have never been mentioned in the literature.
Here we describe the life history of G. tenuistipitata, completed in vitro, show the morphological variability of specimens derived from tetraspore progeny and compare the growth rates of the sporophytic and gametophytic phases.

Algal material
The material used in our experiments is derived from a tetrasporophytic branch of a specimen collected in Haikou, Hainan Island, China, by E.C. Oliveira in 1990.This material was isolated by J. Macchiavello and has been kept in unialgal culture (photoperiod of 14 h, 25ºC and 30±10 µmol photons m -2 s -1 ) in the Gracilaria germoplasm bank at the "Edison J. de Paula" laboratory, University of São Paulo.

Life history
Tips of a tetrasporophytic specimen from our germoplasm bank were incubated in the standard culture conditions in Erlenmeyer vials with 500 ml of medium.Tetrasporangia were noticed after five weeks.Ten branches bearing tetrasporangia were transferred to Petri dishes with sterile seawater and observed under a stereomicroscope for the presence of released tetraspores.Tetraspores were transferred with a Pasteur pipette to four Petri dishes with 50 ml of enriched seawater until plantlets of 2-3 mm were produced.Thirty five plantlets were detached from the bottom of the plates and cultivated in Erlenmeyer flasks with 200 ml of the growing medium until cystocarps became visible to the naked eye.Branches with cystocarps were isolated and kept in the grow-ing medium until carpospores were released.Carpospores were isolated and grown in the same conditions described for tetraspores.Fertile material was hand-sectioned and stained with 1% aqueous aniline blue to study the reproductive structures.Voucher specimens were deposited in the herbarium of the Biosciences Institute, University of São Paulo (SPF) numbered as SPF 56193 (fertile tetrasporophytes), SPF 56194 (female gametophytes with cystocarps) and SPF 56195 (fertile male gametophytes).

Growth rates
Apical branches (length 1 cm, total fresh weight ca. 1 mg) of female gametophytes and of unfertile tetrasporophytes (n=4) were cultivated for 6 weeks in 400 ml of enriched seawater in the standard conditions described above.The biomass was recorded weekly.Branches were gently blotted dry and weighted weekly on an analytical balance Mettler -Toledo AE 200S.Growth rates (GR) were assessed weekly according to Lignell and Pedersén (1989).A two-way analysis of variance was performed using the software STATISTICA 7.0 (StatSoft, Inc.), considering the time and life-history stages as independent variables and the growth rates as dependent variables.This analysis was followed by a Newman-Keuls a posteriori test.

RESULTS
The life history of Gracilaria tenuistipitata is of the Polysiphonia-type and was completed in 135 days.Tetrasporophytic branches cultivated in standard culture conditions produced tetrasporangia in ca.20 days (Fig. 1A).Tetrasporangia are scattered on the cortex in small numbers (Fig. 1B), and are produced from the most external subcortical cells.They are ovoid, measure 24-33 x 45-48 µm and are divided according to a decussate pattern (Fig. 1C).Released tetraspores sunk, attached to the bottom of the vial, produced a cell wall and started the germination process.Around 15 days later, an erect cylindrical axis developed from a basal disk.Erect axes were initially unbranched, but later became sparsely branched.Secondary erect axes were formed from the disc-like base.At the age of 45 days, cystocarps and spermatangial conceptacles could be observed.
The morphology of gametophytes at the age of 53 days is shown in Figure 2. The differences in size and branching of the tetraspore progeny, kept under the same conditions, are obvious, and there is large morphological variability resulting from the segregation process in the tetrasporangia.
Female gametophyte thalli kept together with male ones developed cystocarps, which are domeshaped, with a light constriction at the base, and measure ca.300 µm in diameter near the base when mature (Fig. 3A-B).Carpospores are released through a conspicuous ostiole at the tip of the cystocarp (Fig. 3C).The gonimoblasts are organized in a single gonimolobe; the pericarp has 7-10 layers of anticlinal rows (Figs.3C, 3E); connecting filaments between the gonimoblasts and the pericarp are present at the cystocarpic base (Fig. 3D-E); carposporangia are produced in short series at the tips of the gonimoblast rows (Fig. 3C-E); a star shaped fusion cell can be seen at the base of the carposporophyte (Fig. 3F).
Released carpospores germinated in the same pattern as tetraspores (Dumontia-type) (Chemin, 1937).The resulting tetrasporophytes took around 20 days to become fertile and showed similar morphology to the stock plants, without much variation like that observed for gametophytes.
Male gametophytes could be easily detected under a stereomicroscope due to the presence of lighter spots under light transmitted from below (Fig. 4A).Spermatangial conceptacles (37 to 46 µm) are densely distributed all over the male specimens (Fig. 4B).In cross sections they appear as small depressions   in the cortical region and are of the "textorii-type".The conceptacles are bordered by elongated flanking cells (Fig. 4C-D).Thallus anatomy shows a macrocystiated arrangement of cells (Fig. 4D).The growth rates were significantly influenced by time and life history phases (F=13.154;p<0.01 and F=76.021; p<0.01, respectively).Tetrasporophytic branches showed higher growth rates than gametophytic ones (24.01±0.68%FW.day - for tetrasporophytes and 21.1±0.89for gametophytes).Absolute values of growth rates ranged from 6.36 to 27.7% FW.day -1 (gametophytes) and from 5.88 to 35.5% FW.day -1 (tetrasporophytes).Growth rates decreased over time, regardless of the life-history phase.After three weeks, the growth rates started to decrease, and reached the lowest values after six weeks (Fig. 5).

DISCUSSION
Since the isolation of G. tenuistipitata in our laboratory this species has been taken to other laboratories in the world and is becoming a "model organism" for different kinds of investigations (e.g.Israel et al., 1999;Mercado et al., 2001).Nevertheless, we have not found reports concerning its in vitro life history or studies on the performance of the gametophytic phase.This is the first record of male gametophytes of G. tenuistipitata obtained in vitro.The only description of male structures for G. tenuistipitata is the one given by Chang and Xia (1976) based on specimens from natural populations.Bellorin (2002) interpreted the structures described by these authors as being of the textorii-type, which agrees with our observations.
The first life history of a Gracilaria sp. was completed by Ogata et al. (1972) with material from Japan identified as G. verrucosa (Hudson) Papenfuss, a name that was later abandoned.Since then the life histories of a few other species have been completed in vitro (McLachlan and Edelstein, 1977;Bird et al., 1977;Guimarães et al., 1999;Oliveira and Plastino, 1984;Plastino and Oliveira, 1988;Yamamoto, 1991;Costa and Plastino, 2001).This study confirms the hypothesis that G. tenuistipitata also has a "Polysiphonia-type" life history as described for other species, although deviations have been reported in some species (Plastino and Oliveira, 1984;Kain and Destombe, 1995).
It is well known that species of red algae of commercial interest which are propagated vegetatively are mostly infertile and seem to lose the capacity to reproduce sexually (Bird et al., 1986).Therefore, it is worth noting that this G. tenuistipitata has kept its potential to form reproductive structures intact and completed its life history after being replicated vegetatively in our lab for more than 15 years.The failure of Oliveira and Plastino (1984) to complete the life history of some Gracilaria spp.may be due to the lack of optimal conditions for triggering the formation of reproductive cells rather than the loss of reproduction potential.
The life history of G. tenuistipitata was completed in about 4.5 month, which is a short period when compared to G. foliifera (Forsskål) Børgesen (around 8 months, McLachlan and Edelstein, 1977), G. debilis (Forsskål) Børgesen (5 months), Gracilaria sp.(9 months) (Oliveira and Plastino, 1984) and G. domingensis (Kützing) Sonder ex Dickie (6 months) (Guimarães et al., 1999).The time necessary to complete the life history of G. tenuistipitata was similar to wild strains of G. birdiae Plastino and Oliveira, which completed the in vitro life history in 4 months (Costa and Plastino, 2001).However, as mentioned by other authors, the time needed to complete a life history depends on different factors, such as temperature, nutrients and the amount of photosynthetic active radiation, and so should not be taken exclusively as a specific attribute.
Tetraspore progeny yielded a range of morphologically different individuals that had not yet been seen for a Gracilaria species, although they had been registered for other red algae, such as Kappaphycus alvarezii (Doty) Doty ex P.C. Silva (Paula et al., 1999).Morphological differences between gametophytic and tetrasporophytic generations were observed in some Gracilaria spp., and the gametophytic phase was usually smaller than the sporophytic phase (Oliveira and Plastino, 1984;Kain and Destombe, 1995).This large variability restricts the possibility of describing taxonomical varieties based on gross morphology, as is the case of G. tenuistipitata var.liui Zhang et Xia.However, this again shows that it is a very effective method for producing cultivars for commercial farming, as has been demonstrated for other seaweeds, including for instance, G. tikvahiae polyploids and mutants (Patwary and van der Meer, 1983), G. chilensis C.J. Bird, McLachlan and E.C. Oliveira intra-clonal strains (Santelices and Varela, 1993) and K. alvarezii tetraspore progeny (Paula et al., 1999).
The growth rates of tetrasporophytes were higher than those of female gametophytes but mostly in the first week.Considering the following weeks, growth rates were similar comparing both phases.The relative performance of the two phases varies with the species.Santelices and Varela (1995) observed higher growth rates in fertile female gametophytes in comparison to fertile tetrasporic plants of G. chilensis.Ursi and Plastino (2001) observed higher growth rates of female gametophytes of G. birdiae compared with male gametophytes and tetrasporophytes, whereas Barufi (2004) obtained the opposite results for the same species.This indicates that the performance of gametophytes is a matter of genetic diversity.Furthermore, the differences with respect to growth rates can be related to the different nutritional needs of the reproductive stages, which are advantageous for the survival of the species in heterogeneous environments (Ursi and Plastino, 2001).
The results presented in this work on the life history and on the variability of tetraspore progeny give further evidence that G. tenuistipitata is a model organism among the florideophyte red algae and are also relevant for strain selection for commercial cultivation of this species.

Fig. 2 .
Fig. 2. -Gracilaria tenuistipitata.Gametophytes at the age of 53 days, originated from tetraspores and cultivated in the same conditions, showing a large variability in thalli morphology.Scale bar = 1 cm.

Fig. 5 .
Fig. 5. -Growth rate (GR) of tetrasporophytes and female gametophytes of Gracilaria tenuistipitata over six weeks of cultivation.Bars signify standard deviations.N=4.The different letters over bars mean statistical differences observed after the Newman-Keuls a posteriori test.