Satoko Iida

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES, RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.

Molecular Adaptation of rbcL in the Heterophyllous Aquatic Plant Potamogeton

Background Heterophyllous aquatic plants show marked phenotypic plasticity. They adapt to environmental changes by producing different leaf types: submerged, floating and terrestrial leaves. By contrast, homophyllous plants produce only submerged leaves and grow entirely underwater. Heterophylly and submerged homophylly evolved under selective pressure modifying the species-specific optima for photosynthesis, but little is known about the evolutionary outcome of habit. Recent evolutionary analyses suggested that rbcL, a chloroplast gene that encodes a catalytic subunit of RuBisCO, evolves under positive selection in most land plant lineages. To examine the adaptive evolutionary process linked to heterophylly or homophylly, we analyzed positive selection in the rbcL sequences of ecologically diverse aquatic plants, Japanese Potamogeton. Principal Findings Phylogenetic and maximum likelihood analyses of codon substitution models indicated that Potamogeton rbcL has evolved under positive Darwinian selection. The positive selection has operated specifically in heterophyllous lineages but not in homophyllous ones in the branch-site models. This suggests that the selective pressure on this chloroplast gene was higher for heterophyllous lineages than for homophyllous lineages. The replacement of 12 amino acids occurred at structurally important sites in the quaternary structure of RbcL, two of which (residue 225 and 281) were identified as potentially under positive selection. Conclusions/Significance Our analysis did not show an exact relationship between the amino acid replacements and heterophylly or homophylly but revealed that lineage-specific positive selection acted on the Potamogeton rbcL. The contrasting ecological conditions between heterophyllous and homophyllous plants have imposed different selective pressures on the photosynthetic system. The increased amino acid replacement in RbcL may reflect the continuous fine-tuning of RuBisCO under varying ecological conditions.

Genetic diversity and origin of Potamogeton anguillanus (Potamogetonaceae) in Lake Biwa, Japan

Abstract We analyzed the genetic variation in Potamogeton anguillanus Koidz. and its putative parents, P. malaianus Miq. and P. perfoliatus L., at five allozyme loci of four enzymes to test the hypothesis of a hybrid origin for P. anguillanus, collected in Lake Biwa, Japan. Alleles diagnostic for either P. malaianus or P. perfoliatus were present at four loci. Of 13 single locus phenotypes (SLPs) of P. anguillanus, eight were phenotypes that were expected in F1 hybrids between P. malaianus and P. perfoliatus. Two SLPs were different from those expected in F1 hybrids but could have resulted from segregation of parental alleles in later generation hybrids. Each of the remaining three SLPs possessed one allele unique to P. anguillanus. Allozyme analyses thus supported the view that P. anguillanus was derived from hybridization between P. malaianus and P. perfoliatus. It seems likely that the genetic diversity of P. anguillanus found previously originated through multiple hybridizations and sexual processes in P. anguillanus. Other processes such as intragenic recombination, mutation, or hybridization with another lineage are also discussed with reference to the origin of unique alleles.

Inherited maternal effects on the drought tolerance of a natural hybrid aquatic plant, Potamogeton anguillanus

We tested whether maternal effects have led to the adaptive divergence of strains of the natural hybrid Potamogeton anguillanus, whose putative parents show contrastingly divergent ecologies. To examine the correlation between phenotypic characters and maternal types, we conducted drought experiments and DNA typing using nuclear and chloroplast genes. In the field, we investigated the distribution of the maternal type along the depth and the inshore-offshore gradient. Hybrids of P. malaianus mothers (M-hybrids) and those of P. perfoliatus mothers (P-hybrids) could not be distinguished morphologically under submerged conditions, but differed in drought tolerance. M-hybrids and P. malaianus formed more terrestrial shoots and exhibited higher survival than P-hybrids and P. perfoliatus in drought experiments. The distribution survey clarified that M-hybrids were dominant in shallow and inshore areas, whereas they were almost absent in deeper and offshore areas. These results indicate that the natural hybrid P. anguillanus differs in adaptive values depending on the maternal type. Bidirectional hybridization and heritable maternal effects may have played important roles in its phenotypic adaptation to local environmental conditions.

Comparative studies of thermotolerance: different modes of heat acclimation between tolerant and intolerant aquatic plants of the genus Potamogeton

BACKGROUND AND AIMS Molecular-based studies of thermotolerance have rarely been performed on wild plants, although this trait is critical for summer survival. Here, we focused on thermotolerance and expression of heat shock transcription factor A2 (HSFA2) and its putative target gene (chloroplast-localized small heat shock protein, CP-sHSP) in two allied aquatic species of the genus Potamogeton (pondweeds) that differ in survival on land. METHODS The degree of thermotolerance was examined using a chlorophyll bioassay to assess heat injury in plants cultivated under non- and heat-acclimation conditions. Potamogeton HSFA2 and CP-sHSP genes were identified and their heat-induction was quantified by real-time PCR. KEY RESULTS The inhibition of chlorophyll accumulation after heat stress showed that Potamogeton malaianus had a higher basal thermotolerance and developed acquired thermotolerance, whereas Potamogeton perfoliatus was heat sensitive and unable to acquire thermotolerance. We found two duplicated HSFA2 and CP-sHSP genes in each species. These genes were induced by heat shock in P. malaianus, while one HSFA2a gene was not induced in P. perfoliatus. In non-heat-acclimated plants, transcript levels of HSFA2 and CP-sHSP were transiently elevated after heat shock. In heat-acclimated plants, transcripts were continuously induced during sublethal heat shock in P. malaianus, but not in P. perfoliatus. Instead, the minimum threshold temperature for heat induction of the CP-sHSP genes was elevated in P. perfoliatus. CONCLUSIONS Our comparative study of thermotolerance showed that heat acclimation leads to species-specific changes in heat response. The development of acquired thermotolerance is beneficial for survival at extreme temperatures. However, the loss of acquired thermotolerance and plasticity in the minimum threshold temperature of heat response may be favourable for plants growing in moderate habitats with limited daily and seasonal temperature fluctuations.

The D1 and D2 proteins of dinoflagellates: unusually accumulated mutations which influence on PSII photoreaction

Plastid encoded genes of the dinoflagellates are rapidly evolving and most divergent. The importance of unusually accumulated mutations on structure of PSII core protein and photosynthetic function was examined in the dinoflagellates, Symbiodinium sp. and Alexandrium tamarense. Full-length cDNA sequences of psbA (D1 protein) and psbD (D2 protein) were obtained and compared with the other oxygen-evolving photoautotrophs. Twenty-three amino acid positions (7%) for the D1 protein and 34 positions (10%) for the D2 were mutated in the dinoflagellates, although amino acid residues at these positions were conserved in cyanobacteria, the other algae, and plant. Many mutations were likely to distribute in the N-terminus and the D–E interhelical loop of the D1 protein and helix B of D2 protein, while the remaining regions were well conserved. The different structural properties in these mutated regions were supported by hydropathy profiles. The chlorophyll fluorescence kinetics of the dinoflagellates was compared with Synechocystis sp. PCC6803 in relation to the altered protein structure.

Circumnutation on the water surface: female flowers of Vallisneria

Circumnutation, the helical movement of growing organ tips, is ubiquitous in land plants. The mechanisms underlying circumnutation have been debated since Darwins time. Experiments in space and mutant analyses have revealed that internal oscillatory (tropism-independent) movement and gravitropic response are involved in circumnutation. Female flower buds of tape grass (Vallisneria asiatica var. biwaensis) circumnutate on the water surface. Our observations and experiments with an artificial model indicated that gravitropism is barely involved in circumnutation. Instead, we show that helical intercalary growth at the base of peduncle plays the primary role in all movements in Vallisneria. This growth pattern produces torsional bud rotation, and gravity and buoyancy forces have a physical effect on the direction of peduncle elongation, resulting in bud circumnutation on the water surface. In contrast to other water-pollinated hydrophilous plants, circumnutation in Vallisneria enables female flowers to actively collect male flowers from a larger surface area of water.

Genetic diversity of Potamogeton anguillanus in Lake Biwa, Japan

Abstract The genetic structure of Potamogeton anguillanus in Lake Biwa, Japan was surveyed by enzyme electrophoresis. Polymorphic banding patterns were observed in four of eight enzymes investigated. Based on the combination of the single enzyme phenotypes, 27 multi-enzyme phenotypes (MEPs) were determined in the whole lake based on 258 shoot samples collected from 14 local populations. Most of the local populations were polymorphic and involved one to eight unique MEPs. A mapping survey of MEPs in selected quadrats showed establishment of ramets with different genotypes and the clonal spread of the species. The origin of genetic diversity in the species may partly be due to recurrent hybridization. Sexual reproduction may also be involved.

Population genetic structure of Potamogeton anguillanus in Lake Shinji, Japan

Abstract Enzyme electrophoresis was performed to investigate the genetic variation of Potamogeton anguillanus Koidz. in Lake Shinji, Shimane Prefecture, Japan. The collection was made in six local populations along the coast of the lake, and a total of 78 shoot samples were obtained. No variation among samples occurred in the six enzymes analyzed, and the whole population in Lake Shinji was proved to consist of only a single multienzyme phenotype. Clonal growth without sexual reproduction may have resulted in the genetic uniformity. The implication of the lack of genetic variation is briefly discussed from the viewpoint of conservation of the species in Lake Shinji.

Maternal effects and ecological divergence in aquatic plants: a case study in natural reciprocal hybrids between Potamogeton perfoliatus and P. wrightii

When parental taxa are adapted to different habitats, hybrid genotypes are often highly heterogeneous, such that habitat or ecological factors influence hybrid fate and ecological performance. Trait expression in hybrids is not always intermediate between the parents, but may instead be either parental-like or extreme (transgressive) depending on genetic control of the phenotypes. Maternal effects arising from interspecific interaction between cytoplasmic and nuclear genomes are widely recognized as playing a role in character expression of natural hybrids. Such interaction often leads to hybrid sterility or inviability. When hybrids are viable, however, cytonuclear interaction may contribute to hybrid persistence through its influence on trait expression. To date, maternal influence on hybrid performance has been examined primarily in experimentally produced hybrids, or in natural hybrids without identification of the cross direction owing to difficulty in obtaining species-specific molecular markers. In aquatic plants, many hybrids persist by extensive clonal growth and are important components of aquatic communities. Many such hybrids are known in Potamogeton (pondweeds), the largest aquatic genus. Because Potamogeton species are ecologically highly diverse and maternal lineages are readily distinguished using molecular markers, natural hybrids of Potamogeton are well-suited for studies of maternal effects, especially those affecting vegetative performance. As a case study, we have focused on maternal effects on drought tolerance and depth distribution in the natural hybrid P. ¥ anguillanus derived from the closely related species P. perfoliatus and P. wrightii.