Codominant inheritance of polymorphic color variants of Gracilaria domingensis (Gracilariales, Rhodophyta)

Codominant inheritance of polymorphic color variants of Gracilaria domingensis (Gracilariales, Rhodophyta)

Estela M. Plastino, Melina Guimarães, Sergio R. Matioli and Eurico C. Oliveira

Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11461, 05422-970 São Paulo, SP, Brasil. Send correspondence to E.M.P. Fax: [phone]. E-mail: [e-mail]

ABSTRACT

Agar is the most valuable phycocoloid in the world market. Currently, about half of its production is obtained from the red alga Gracilaria (Gracilariales, Rhodophyta). Consequently, this genus has been the subject of many studies worldwide. A common green color variant of G. domingensis (Kützing) Sonder ex Dickie was found in a natural population on the northeastern coast of Brazil. Crosses were performed to determine the mode of color inheritance. The results can be interpreted as the expression of a pair of codominant alleles, where the green and red phenotypes are homozygous, and the heterozygotes present a brownish color. Heterozygous tetrasporophytes, at least until they are 4-5 cm long, exhibit a parental influence on the female gametophyte, since the reddish-brown or greenish-brown color is dependent on the female parent color (red or green). Mixed reproductive phases, as well as specimens with patches of different colors bearing spermatangia or cystocarps, were observed in laboratory cultures. Gametophytes that resulted from in situ germination of meiospores were also observed, and could be easily detected when red or green gametophytes were grown on brown tetrasporophytes.

INTRODUCTION

Agar is the most valuable phycocoloid in the world market. Currently, about half of its production is obtained from members of Gracilariaceae (Gracilariales, Rhodophyta). The most important genus is Gracilaria (Kain, 1995). Consequently, this genus has been the subject of many studies worldwide (e.g. Oliveira and Plastino, 1994).

Recently, we found common green specimens in a natural population of the normally reddish G. domingensis (Kützing) Sonder ex Dickie on the northeastern coast of Brazil. This species produces an unsuitable gel for microbiological purposes, but it is satisfactory in food preparations (Saito, R. and Oliveira, E.C., unpublished results). Besides, the whole alga has a commercial value as food in the Japanese market. Specially for the latter application, these pigment variants are favored, since they can be appealing not only by different colors, but also by different tastes.

Genetic studies of Gracilaria started only in the late 1970s with the isolation and characterization of G. tikvahiae McLachlan mutant strains. These strains arose spontaneously in culture (van der Meer and Bird, 1977; Patwary and van der Meer, 1982; van der Meer et al., 1984) or were induced by chemical mutagenesis (van der Meer, 1977, 1979a).

Color mutants have been useful visual markers in genetic studies. This approach has led to the discovery of inheritance in some G. tikvahiae pigmentation mutants (van der Meer and Bird, 1977; van der Meer, 1978, 1979a,b), and has elucidated some cases of mixed reproductive phases (van der Meer, 1977, 1981, 1986; van der Meer and Todd, 1977; van der Meer et al., 1984). Color mutants have also been useful in studying phycobilisome composition and structure (Kursar et al., 1983a,b).

Like most Rhodophyta species, the life history of Gracilaria can be divided into three phases: two diploid (tetrasporophyte and carposporophyte) and one haploid (gametophyte). Both tetrasporophyte and carposporophyte are free living generations, whereas the carposporophyte is a parasite of the female gametophyte (Figure 1). The gametophytes are dioecious and isomorphic with the tetrasporophytes.

Figure 1
- Life history of Gracilaria spp. (from Oliveira and Plastino, 1994).

Autosomal nuclear transmission in G. tikvahiae mutants was found in the majority of the cases studied (van der Meer and Bird, 1977; van der Meer, 1978, 1979a,b, 1986, 1990; van der Meer et al., 1984), although cytoplasmic transmission was also observed (van der Meer, 1978, 1990). Most of the mutant genes were recessive, although dominant ones have also been found (van der Meer, 1990; Patwary and van der Meer, 1992). Up to now, most genetic studies on Gracilaria, including color mutants, have been carried out on G. tikvahiae, except for some studies on G. foliifera (Forskaal) Børgesen and G. sjoestedtii Kylin (van der Meer and Zhang, 1988; Zhang and van der Meer, 1988).

Here we describe experimental crosses performed to determine the mode of color inheritance in pigment variants of G. domingensis found in natural populations.

MATERIAL AND METHODS

Apical segments of green and red Gracilaria domingensis specimens were collected in Guajirú Beach, Trairí, State of Ceará, Brazil and transported to the laboratory in São Paulo. Voucher specimens have been deposited in the herbarium of the Instituto de Biociências, University of São Paulo (SPF).

Unialgal cultures were established as described by Plastino and Oliveira (1990). Standard culture conditions were 16-8-h light-dark cycle, with alternating 3 h aeration periods, 20 ± 2^oC, 30-35-mmol photons m^-2 s^-1 (Osram 40-W daylight fluorescent lamps), and von Stosch medium (Edwards, 1970) diluted to 50% with sterile seawater (32 ppt salinity).

Cultures were started from carpospores obtained from green cystocarpic thalli. Tips cut from male and female green and red specimens were kept isolated for at least two months, to ensure the absence of fertilized carpogonia, before being utilized in the crosses.

Crosses between male and female plants of the same color (green or red), and between different colors (red male x green female and green male x red female) were performed to determine the color inheritance pattern. During the experimental period, female branches of each color were kept isolated from male branches to check for the existence of hermaphroditic or parthenogenetic plants. For the crosses, male and female branches were incubated together until cystocarps appeared on the female plants. The male branches were then removed, and the female branches were cultured until the carpospores matured and released.

Carpospores from five cystocarps of the same cross type were collected and grown separately, in order to obtain fertile tetrasporophytes, as well as to verify color ratios. One fertile tetrasporophyte originating from each cystocarp was selected, and eight tetraspores from each of these tetrasporophytes were cultured in order to generate fertile gametophytes. The sex ratios were then analyzed.

A chi-square (c²) test with Yates correction (Zar, 1984) was applied to analyze sex and color ratio results in the gametophytic generation.

RESULTS

Color inheritance

All cross combinations were positive. No cystocarps were formed in the absence of males. Carposporophytes resulting from crosses of green or red males with green females produced dark red spore masses inside green pericarps, and released reddish carpospores.

Carpospores developed into small red basal discs, independent of their origin, and soon gave rise to red erect axes. At the age of 57-60 days, all these plants, which were still reddish and about 10 mm long, produced tetrasporangia. Green color differentiation was observed only after tetraspore release, when the tetrasporophytes were longer than 2-3 cm and 153 days old.

Tetrasporophytes, resulting from germination of carpospores produced from crosses between red male and red female gametophytes, remained red. Those which originated from crosses between green gametophytes became green, except for their extreme 4-5-mm tips, which remained reddish. Crosses between green male and red female gametophytes resulted in reddish-brown tetrasporophytes, while crosses between red male and green female gametophytes resulted in greenish-brown tetrasporophytes. However, all the heterozygous tetrasporophytes became brown when they reached a length of 4-5 cm.

Color and sex ratios of plants from different crosses are shown in Table I. Tetraspores resulting from different tetrasporophytes were always reddish and gave rise to red plantlets. This color persisted up to the age of 50-65 days, when it started to change, according to its genetic origin. Tetraspores from red tetrasporophytes produced red gametophytes. Tetraspores from green tetrasporophytes gave rise to green gametophytes, except for their extreme tips which remained reddish. Tetraspores from reddish-brown or greenish-brown tetrasporophytes gave a 1:1 ratio of green to red gametophytes.

Table I
- Summary of the crossing results showing number, sex and color of tetrasporophytic and gametophytic individuals. P > 0.05 for all calculated chi-square values ( c² ) (degrees of freedom =1).

Gametophytes took 77-124 days to become fertile with a sex ratio of 1:1. Color/sex recombinants among heterozygous tetrasporophyte originating gametophytes were observed in a frequency of 50%, showing no evidence of linkage.

Mixed reproductive phases

Mixed reproductive phases were frequently observed in the laboratory. Gametophytes originating from tetraspores germinated in situ were observed, and were more easily detected when red or green gametophytes grew on brown tetrasporophytes. These gametophytes became fertile after 50 days, and developed somewhat more precociously than free-living gametophytes. Spermatangial structures next to cystocarps as well as patches with different colors were also observed. In some thalli, different reproductive structures and colors appeared together.

DISCUSSION

Although Gracilaria spp. mutants have been extensively studied, there have been only a few reports of collection and genetic inheritance studies of spontaneous color mutants (van der Meer, 1990). Pigment differentiation in natural populations of Gracilaria has been reported, but genetic studies have not been performed (e.g. Zhang et al., 1993; Gonzáles et al., 1996).

The present study shows that the green color of some specimens found in a natural population of G. domingensis in northeastern Brazil is stable, and both red and green color phenotypes are codominant. When green and red alleles are expressed together, a different, brown phenotype is produced. Homozygous plants are either green or red. This codominant pattern was reported in some color phenotypes of Gracilaria tikvahiae as incomplete dominance, since the heterozygotes had an intermediate hue between the mutant and wild type (van der Meer and Todd, 1977; van der Meer, 1979b).

The reddish color of all n-or 2n-spore types obtained from red, green or brown forms was also reported in green mutants of G. tikvahiae (van der Meer and Bird, 1977). Color segregation became apparent only in plantlets older than 70 days, when they already had an erect axis. Among tetrasporophytes, color differentiation appeared only after reproductive maturity was attained.

The red color of spores and plantlets and the reddish coloration of all green specimen tips indicate that the green phenotype is expressed only in more mature thalli. It is possible that cell and vacuole size relative to plastid number determine color in tips and young plants.

Heterozygous tetrasporophytes exhibit the influence of the female gametophyte from which they were produced, at least until they are 4-5 cm long, since their reddish-brown or greenish-brown color is influenced by the color of the female parent (red or green). After fertilization in red algae, the reproductive female apparatus incorporates into the 2n phase a relatively large amount of cytoplasmic material, including organelles from the female gametophyte. Besides, as reported to the most of red algae (van der Meer, 1990), spermatial cells contain little but their nuclei, and it is unlikely that male cytoplasmic organelles are introduced into the trichogyne. Both processes can explain the influence of the parent color in the young tetrasporophytes.

A 1:1 sex ratio observed in the gametophytic generation has often been reported for other Gracilaria species in which the life history had been completed in vitro as well as in natural populations (Plastino, 1985). Pinheiro-Joventino and Bezerra (1980), studying the same population of G. domingensis, also found a 1:1 ratio of male to female gametophytes, as did Hay and Norris (1984) for a Panamanian population. However, there are reports of variable sex ratios among some natural populations of other species (Kain and Destombe, 1995).

The occurrence of mixed reproductive phases has been reported for some species of Gracilaria (see Oliveira and Plastino, 1994, for further references). However, when one has color variants, the presence of mixed reproductive phases becomes more conspicuous. Precocious fertile gametophytes when germinated in situ were also observed in other species of Gracilaria, suggesting a possible influence of mature tetrasporophyte on sexual maturation of the gametophytes (Oliveira and Plastino, 1984; Plastino, 1985).

The occurrence of sexually mosaic plants in a dioecious species, like those reported for G. domingensis, could be attributed to a failure of cytokinesis during tetraspore formation, as was demonstrated by van der Meer (1977) using nuclear genetic markers in G. tikvahiae. On the other hand, this phenomenon might even be caused by a mitotic recombination (van der Meer and Todd, 1977).

The stable green color, described here, has been corroborated by successive generations of these algae cultivated in our lab. Descendants of plants used in the crosses have shown the same Mendelian codominant inheritance pattern, even in different light and temperature conditions. This finding, as well as the common occurrence of the green variant in the field, has motivated further studies, such as population genetics, physiology of growth rates, photosynthesis, etc. Some of these subjects, such as color and reproductive phase frequencies in natural populations, have been studied and will be presented elsewhere.

ACKNOWLEDGMENTS

Research supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, No. 91/3590-2; scholarship, No. 90/3431-9). E.C. Oliveira and E.M. Plastino acknowledge scholarships from CNPq. The authors would like to thank Ralph Lewin, Helena Khouri, and N. Rejane W. Lima for reviewing the manuscript. Publication supported by FAPESP.

RESUMO

Dentre os colóides extraídos de algas, o ágar é o mais valioso no mercado mundial, sendo que aproximadamente metade da produção é obtida a partir de algas vermelhas do gênero Gracilaria (Gracilariales, Rhodophyta). Conseqüentemente, o gênero vem sendo alvo de muitos estudos em todo o mundo. Um variante de cor verde de G. domingensis (Kützing) Sonder ex Dickie foi encontrado numa população natural do nordeste da costa do Brasil. Realizaram-se cruzamentos para determinar o modo de herança da cor. Os resultados foram interpretados como a expressão de um par de alelos codominantes, onde os fenótipos de cor verde e vermelha são homozigotos e o heterozigoto apresenta a cor marrom. Tetrasporófitos heterozigotos de até 4-5 cm de comprimento apresentam uma influência do gametófito feminino que lhes deu origem, já que a coloração marrom-avermelhada ou marrom-esverdeada é dependente da cor do gametófito feminino (vermelho ou verde). A ocorrência de diferentes estruturas reprodutoras em um mesmo talo, bem como espécimens com manchas de diferentes cores apresentando cistocarpos ou espermatângios, foi observada em culturas de laboratório. Foi possível constatar a germinação de tetrásporos e conseqüente desenvolvimento de gametófitos in situ, facilmente detectados pela coloração verde ou vermelha sobre tetrasporófitos de coloração marrom.

(Received November 7, 1997)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  07 Nov 1997