Siro Kurita

Development of primer sets for PCR amplification of the PgiC gene in ferns

Abstract Polymerase chain reaction (PCR)-based nuclear DNA markers were developed for fern species. We first determined the partial nucleotide sequence of cDNA of the pgiC gene encoding cytosolic phosphoglucose isomerase from Dryopteris caudipinna, and then PCR primers for exon-primed, intron-crossing (EPIC) amplifications were designed. The EPIC primers are universally applicable to the most derived indusiate fern families such as Dryopteridaceae, Thelypteridaceae, and Woodsiaceae. The PCR products of primers 14F/16R containing two introns are moderate in size (534 bp–ca.1000 bp) and are possibly of value in phylogenetic reconstruction at specific and generic levels. Codominant nuclear DNA markers applicable to the estimation of mating systems and other population genetic studies were also developed by a combination of single-strand conformation polymorphism (SSCP) and EPIC amplification using primers 14F/15R and 15F/16R. In order to provide a case study using these markers, allelic variation of PCR products using 15F/16R was examined in populations of Arachniodes standishii (Dryopteridaceae).

Phylogenetic Relationships of Amaryllidaceae Based on matK Sequence Data

matK gene, which is located in the chloroplast genome and evolves more quickly than the rbcL gene. A total of 31 species representing 31 of the 59 genera in the family were examined in this study. We also used 21 species from another ten families of Asparagales, four species from three families of Liliales and Acorus as outgroups. We obtained partial sequences of matK with lengths of 1,109–1,148 bp, corresponding to positions 230 to 1,343 of the Oryza sativa matK gene. The pairwise percentage sequence divergence ranged from 0 to 19.1% for all the species examined except Acorus, and 0 to 4.6% within Amaryllidaceae. Two methods of phylogenetic analysis, the Maximum Parsimony and Neighbor-Joining methods, were used. The trees obtained from these two analyses were fundamentally consistent. In both trees, the Amaryllidaceae sensu Dahlgren et al. formed a well-supported monophyletic clade with 100% bootstrap support. Amaryllidaceae were included in the Asparagales; however, its phylogenetic position within the Asparagales was not clearly resolved. Judging from the NJ tree, Agapanthus might be a sister group of the Amaryllidaceae, although bootstrap support for this was low. Character-state mapping was used to infer a center of origin and the biogeographic history of Amaryllidaceae. The result supports the hypothesis that the family evolved in Africa and subsequently spread to other continents, further suggesting that South America is the center of secondary diversification.

Chloroplast DNA Phylogeny of Asian Bamboos (Bambusoideae, Poaceae) and Its Systematic Implication

The bamboo is usually classified as a subfamily Bambusoideae of Poaceae, and includes approximately 20 genera and 300 species. To estimate phylogenetic relationships among these genera, we examined restriction site mutations of cpDNA for 16 Asian genera.In the cladogram obtained, the Bambosoideae was divided into two major lineages, one includingPleioblastus, Pseudosasa, Semiarundinaria, Shibataea, Phyllostachys, Sasa, Sinobambusa, Chimonobambusa, Arthrostylidium, andYushania, and the other consisting ofBambusa, Gigantochloa, Dendrocalamus, Thyrostachys, Melocanna, andSchizostachyum. Monophylly of each clade was supported by 83% and 98% bootstrap probability, respectively. The present result supports monophylly of Arundinarieae of Potztals (1964) classical system, but does not support his treatment to recognize Dendrocalameae.

Diplazium subsinuatum and Di. tomitaroanum should be Moved to Deparia According to Molecular, Morphological, and Cytological Characters

rbcL sequence data revealed that the putative intergeneric hybrid, Diplazium tomitaroanum Masam. belongs in Deparia, as also does Diplazium subsinuatum (Wall, ex Hook, et Grev.) Tagawa, one of the putative parents. An examination of rachis, scale and spore morphology, and chromosome data provide support for this placement. We propose a new taxonomic treatment of the two Diplazium species as Deparia.

Ultrastructural study of spore wall morphogenesis inOphioglossum thermale kom. var.nipponicum (Miyabe et Kudo) nishida

Spore wall morphogenesis ofOphioglossum thermale var.nipponicum was examined by transmission electron microscopy. The spore wall of this species consists of three layers: endospore, exospore, and perispore. The spore wall development begins at the tetrad stage. At first, the outer undulating lamellar layer of the exospore (Lo) is formed on the spore plasma membrane in advance of the inner accumulating lamellar layer (Li) of the exospore. Next, the homogeneous layer of the exospore (H) is deposited on the outer lamellar layer. Both lamellar layers may be derived from spore cytoplasm; and the homogeneous layer, from the tapetum. Then the endospore (EN) is formed. It may be derived from spore cytoplasm. The membranous perispore (PE), derived from the tapetum, covers the exospore surface as the final layer. Though the ornamentation of this species differs distinctly from that ofO. vulgatum, the results mentioned above are fundamentally in accordance with the data obtained fromO. vulgatum (Lugardon, 1971). Therefore, the pattern of spore wall morphogenesis appears to be very stable in the genusOphioglossum.

Species relationships of Lycoris endemic to Korea evaluated by RAPD and SNPs of nrDNA-ITS regions

This study was performed to investigate the species relationships and variation of Lycoris Herb. (Amaryllidaceae) species using random amplification of polymorphic DNA (RAPD) markers. Also, single nucleotide polymorphisms (SNPs) of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 regions in Lycoris sanguinea var. koreana were analyzed. All accessions formed 6 major clusters; cluster A with all L. sanguinea and L. chejuensis; cluster B with 3 accessions of L. flavescens; cluster C with 8 accessions of F. flavescens var. flavescens; cluster D with 10 accessions of L. uydoensis; cluster E with L. chinensis var. sinuolata and 4 accessions of L. uydoensis; and cluster F with all L. radiata. Five haplotypes were observed; L. sanguinea and L. chejuensis having the haplotype 1 with bases of CTTATATATAT; L. chinensis var. sinuolata and all L. flavescens. Lycoris incarnata and L. aurea, non-endemic to Korea had haplotype 2 and 5, respectively. Genetic variations in L. flavescens, L. chinensis var. sinuolata, and L. uydoensis are revealed based on the analysis of molecular variances (AMOVA) and haploid types analyzed by sequence analysis. It is suggested that L. chejuensis may result from hybridization involving L. sanguinea var. koreana due to a close affinity between L. sanguinea complex and L. chejuensis. Nomenclature for L. chejuensis and L. flavescens whether they should be described as a hybrid origin should be discussed in the future.

Intraspecific polymorphism of pollen sculpture inParis tetraphylla A. Gray (Liliaceae)

Paris tetraphylla has a greater diversity of pollen exine sculpture than was previously thought. On the basis of 357 plants from 52 localities of Japan, we distinguished seven sculpture types that are tentatively referred to here as G1, G2, G3, R1, R2, R3 and S. G1, G2 and G3 have gemmate sculpture in which the gemmae are characteristically large, medium and small in size, respectively; likewise, R1, R2 and R3, have coarsely to finely ruglate sculpture; and S, scrobiculate sculpture. In general, any one of the seven types always or predominantly occurs in each plant, thus allowing us to make comparisons among plants and further among local populations. We thus found that G2 and G3, respectively, are the most and the second most widespread and occur in plants of 41 and 22 respectively, of the localities examined, although their frequencies differ with locality. Generally R3 occurs in plants distributed at higher latitudes, while R2 is rather widespread and common to plants growing on high mountains. G1 and R1 are restricted to plants occurring south of Ibaraki Pref. and north of Gunma Pref., respectively. Such localized distributional patterns of some types may reflect the reproductive nature and the history of geographical isolation inParis tetraphylla over the Japanese Islands.