John P. van der Meer

MEIOSIS, BLADE DEVELOPMENT, AND SEX DETERMINATION IN PORPHYRA PURPUREA (RHODOPHYTA)1

The discovery in the early 1980s that meiosis occurs during germination of conchospores of Porphyra yezoensis Ueda suggested that the sexually divided fronds of Porphyra purpurea (Roth) C. Agardh might similarly originate from meiotic segregation of a pair of sex‐determining alleles during early sporeling development. After establishing conditions suitable for propagating P. purpurea in culture, observations on developing sporelings demonstrated that meiosis takes place during the first two divisions of the germinating conchospores. In the first division, the spore is split into an upper and lower cell. In the second, an anticlinal division in the upper cell yields two daughter cells situated one beside the other, and a periclinal division in the bottom cell gives two cells arranged one above the other. Thus, during normal development, the first four cells of the sporeling constitute a meiotic tetrad whose cells are arranged in a characteristic fashion. Stable color mutants of P. purpurea were isolated, genetically characterized, and used as genetic markers to follow the fate of individual cells of the tetrad during subsequent frond development. Nearly the entire blade of the mature thallus is derived from the two upper cells of the tetrad, with the two lower cells mostly giving rise to the rhizoidal holdfast region. Cell lineage boundaries laid down by the segregation of color alleles at meiosis corresponded perfectly with those later defined by sexual differentiation on the same fronds, strongly supporting the hypothesis that sex determination in P. purpurea is controlled by alleles at a segregating chromosomal locus.

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)‐ and (G + C)‐rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)‐rich primers was positive (i.e. band‐producing). Of the (G + C)‐rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair‐group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out‐group for the UPGMA analysis, showed a high degree of dissimilarity.

A PHYSIOLOGICAL TEST OF THE THEORY OF COMPLEMENTARY CHROMATIC ADAPTATION. I. COLOR MUTANTS OF A RED SEAWEED1

The physiological behavior of phycoerythrin‐deficient mutants of the red seaweed Gracilaria tikvahiae (Mc‐Lachlan 1979) is compared to that of their wild types. The mutants are phenotypically green while the wild types are red. Cloned scions were grown factorially at irradiances saturating and limiting to growth, and spectral distributions which were broadband (white) and narrowband (green). The green light field complements the absorptance spectrum of phycoerythrin. Experiments were performed in an outdoor continuous flow system. Physiological measurements included light‐harvesting pigment composition, instantaneous photosynthesis‐light relationships and growth. In all cases, the mutants performed as their wild type progenitors. Further, physiological responses occurring in no less than 8 days were dependent solely on irradiance (“intensity”), and were independent of spectral distribution (“color”). The data do not conform with the predictions of the theory of complementary chromatic adaptation for seaweeds.

APPLICATION OF RAPD MARKERS IN AN EXAMINATION OF HETEROSIS IN GELIDIUM VAGUM (RHODOPHYTA)1

Four morphologically indistinguishable isolates of monoecious Gelidium vagum Okamura were crossed reciprocally to obtain hybrids for a study on heterosis in this alga. Approximately 50% outcrossing was achieved by adding a small fragment of the designated female parent to a much larger quantity of designated male thallus in the crossing dish. Hybrids in the mixed population of isomorphic hybrid and inbred sporelings were identified by the presence of male‐specific random amplified polymorphic DNA markers. Growth performance of hybrid tetrasporophytes was compared to that of their gametophytic parents and with inbred tetrasporophytes at near‐optimum and sub‐optimum temperature and density. In general, the hybrids showed growth superiority over inbred lines, particularly under sub‐optimum conditions. In these experiments, the hybrid plants from cross 129 × 130 exhibited a 9.5–130% higher growth rate as compared to the mid‐value of the related inbred tetrasporophytes, strongly suggesting the presence of heterosis.

GENETICS OF GRACILARIA TIKVAHIAE (RHODOPHYCEAE). X. STUDIES ON A BISEXUAL CLONE12

A male done of the red alga Gracilaria tikvahiae McLachlan spontaneously produced a bisexual frond which remained bisexual in subsequent subcultures. Both male and female components of bisexual fronds were functional; however, some unusual results were obtained in crosses. When bisexual fronds were crossed with a normal haploid male, the resulting carpospores all developed into diploid male gametophytes. When bisexual plants were self fertilized, all the carpospores yielded diploid bisexual gametophytes. Only when bisexual plants were crossed to normal haploid females, did carpospores develop into diploid tetrasporophytes as they normally do. The F1 gametophyte generation obtained from these tetrasporophytes, however, included not only females and males but also bisexual plants, in a 2:1:1 ratio. These results are consistent with the interpretation that bisexual plants have a recessive mutation of a gene other than the primary sex determining locus, and that this mutation is expressed only in male plants. It is suggested that the altered gene may ordinarily have a regulatory function in the maintenance of the dioecious condition.

ISOLATION AND CHARACTERIZATION OF PHASE-SPECIFIC COMPLEMENTARY DNAs FROM SPOROPHYTES AND GAMETOPHYTES OF PORPHYRA PURPUREA (RHODOPHYTA) USING SUBTRACTED COMPLEMENTARY DNA LIBRARIES1

The red alga Porphyra purpurea (Roth) C. Agardh has a life cycle that alternates between shell‐boring, filamentous sporophytes and free‐living, foliose gametophytes. The significant morphological differences between these two phases suggest that many genes should be developmentally regulated and expressed in a phase‐specific manner. In this study, we prepared and screened subtracted complementary DNA (cDNA) libraries specific for the sporophyte and gametophyte of P. purpurea. This involved the construction of cDNA libraries from each phase, followed by the removal of common clones through subtractive hybridization. Sampling of the subtracted libraries indicated that 8–10% of the recombinant colonies in each library were specific for the appropriate phase. Of 20 putative phase‐specific cDNAs selected from each subtracted library, eight unique clones were obtained for the sporophyte and seven for the gametophyte. After confirming their phase‐specificities by hybridization to gametophyte and sporophyte messenger RNA, these 15 phase‐specific cDNAs were sequenced, and the deduced amino acid sequences were used to search protein databanks. Two proteins encoded by the sporophyte‐specific cDNAs and two by the gametophyte‐specific cDNAs were identified by their similarity to databank entries.

Similar unstable mutations in three species of Gracilaria (Rhodophyta)

Unstable mutants with similar variegated pigmentation were genetically characterized in the red algae. Gracilaria tikvahiae (McLachlan), G. foliifera (Forsk.) Børg. and. G. sjoestedtii (Kylin). All three mutants were green plants with flecks of red tissue where cells had reverted to wild type. The mutant green phenotypes were all recessive, and their genetic behavior in crosses indicated that each was the result of a single, unstable, nuclear gene. Wild‐type revertant tissue was stable one it arose. Revertant plants obtained from spores and revertant fronds taken from variegated plants could not be distinguished from the normal wild type, either phenotypically or genetically. Reversion to wild type occurred during all phases of the life cycle. In crosses between the mutants and wild type, most of the F1 tetrasporophytes were heterozygous wild‐type plants, an observation consistent with the recessive nature of the mutations; however, a low frequency of homozygous unstable‐green F1 tetrasporophytes was also obttained from these crosses. The molecular basis of neither the mutant instability, i.e. the reversion to wild type, nor of the process producing the unstable green F1 tetrasporophytes can yet be deduced, but the phenotype of the plants and genetic results suggest the involvement of transposable genetic elements.

Genetic modification of Gracilaria tikvahiae (Rhodophyceae). The production and evaluation of polyploids

Abstract The agar-producing red alga Gracilaria tikvahiae is being examined to determine its potential for mariculture. We are studying the effects of various genetic modifications on the performance of this alga, and present here an overview of our work on polyploids, which are readily constructed in crosses utilizing diploid or triploid gametes. In general, 3 n plants appear to be the most robust but the response to polyploidy is not uniform in all genetic backgrounds. The most heterozygous polyploids appeared to be the most vigorous both at the 3 n and 4 n level. It appears that the response to polyploidy is complex and more work is necessary before a general understanding of their usefulness emerges.

GENETICS OF GRACILARIA TIKVAHIAE (RHODOPHYCEAE). XI. FURTHER CHARACTERIZATION OF A BISEXUAL MUTANT

A spontaneous bisexual mutant of Gracilaria tikvahiae McLachlan has been further characterized. Female plants that are carriers for the mutation, but do not themselves express bisexuality, have been identified among progeny derived from the original bisexual male plant. In crosses to normal males these carrier females yielded normal tetrasporophytes which in the subsequent gametophyte generation produced a 2 female: 1 male: 1 bisexual segregation. In crosses to bisexual males the carrier females produced unusual tetrasporophytes that formed cystocarps in addition to tetraspores. The gametophyte generation obtained from the tetraspores of these tetrasporophytes included only females and bisexuals, these being present in a 1:1 ratio. Other crosses, using bisexual male progeny, indicated that these have the same characteristics as the original bisexual mutant. All of the results are consistent with the genetic interpretation made previously that bisexuality results from a single recessive mutation, designated bi, in a gene distinct from the mt locus controlling male vs. female differentiation. From the phenotypes that have been observed it appears that the mutation does not cause bisexuality per se but rather results in unregulated female expression in males and tetrasporophytes where female‐specific genes are normally repressed. It is suggested that the normal bi+ allele plays an important role in that repression process.

Improvement of Gracilaria tikvahiae (Rhodophyceae) by genetic modification of thallus morphology

Abstract In this paper we compare the growth of two promising, mutant clones MP-40 and MP-44 of Gracilaria tikvahiae with a related wild type, and with the best wild-type clone in our collection. In both comparisons the mutants proved superior. Neither mutation adversely affects agar content as both mutants had slightly more agar than wild type. Agar content of MP-40 exhibits the same sensitivity to nutritional status as wild plants. Although these studies indicate that some mutant lines have excellent potential, their performance on a larger scale must still be evaluated.