Microsporidian xenomas in fish seen in wider perspective

The history of understanding xenoparasitic complexes or xenomas provoked in the host cell by various protists and especially by microsporidia is outlined. Microsporidia have been known to produce xenomas in oligochaetes (e.g., genera Bacillidium, Burkea, Hrabyeia, Jirovecia, species of the collective group Microsporidium), crustaceans (e.g., Abelspora, Mrazekia), insects (e.g., Polydispyrenia, Thelohania) and poikilothermic vertebrates, mostly fish (Alloglugea, Amazonspora, Glugea, Ichthyosporidium, Loma, Microfilum, Microgemma, Neonosemoides, Pseudoloma, Spraguea, Tetramicra). An overview of characters of xenomas caused by species of these genera is presented. The study of microsporidia causing xenomas in fish offers an insight into cell pathology and is of interest since many of these species are important agents of diseases in commercial fish. Xenomas produced from a few types of target cell display a complete change of organisation of the host cell and differ, according to the agent, in their structure. Recent data show that proliferation of the parasite may have already started in the cells transporting the parasites to the final site of xenoma formation. However, these are preliminary revelations and most of the facets of the life cycle are still to be clarified. Curiously, xenoma-forming microsporidia do not seem to be strictly host specific. The salient features of fish microsporidian xenomas are discussed, such as role of the xenoma, whether its features are host- or microsporidium-dependent, development and demise of the xenoma in the course of time, and host reaction phenomena. The need of further research is emphasised.     

Review of the sequential development of Loma salmonae (Microsporidia) based on experimental infections of rainbow trout (Oncorhynchus mykiss) and Chinook salmon (O. tshawytscha)

Loma salmonae is a common gill parasite of salmonids, and essentially all species in the genus Oncorhynchus are susceptible. Infections occur in both fresh and salt water. Loma salmonae is directly transmissible by ingestion of spores or infected tissue. The parasite infects the wall of blood vessels of various organs, but the gill is the primary site of infection. Initial infection occurs in the intestine, and xenomas are easily detected in the gills by standard histology at 4-6 wk post-exposure. A few presporogonic stages of the parasite are found in the heart endothelium prior to xenoma formation in the gills. Ultrastructure studies of early infections demonstrated that wandering blood cells transport the meronts to the gills, and that merogony occurs in pillar cells and other cells underlying the gill endothelium. Xenomas develop in these cells, resulting in hypertrophied host cells filled with spores. Xenomas ultimately rupture, and are associated with severe inflammation in which free spores are found in macrophages. The parasites are most pathogenic during this phase of the infection, resulting in severe vasculitis and clinical disease. Both rainbow trout (Oncorhynchus mykiss) and Chinook salmon (Oncorhynchus ishawytscha) recover from infections, but free spores persist in kidney and spleen phagocytes for many months after xenomas are absent in Chinook salmon. Fish that have recovered from the infection show strong immunity against the parasite, lasting up to 1 year. Fish are susceptible to infection by other routes of exposure by spores; co-habitation, anal gavage, and intramuscular, intraperitoneal and intravascular injection. Autoinfection probably occurs following release of spores in blood vessels after xenomas rupture. The optimal temperature for L. salmonae infections is 15-17 degrees C, with a permissive range of 11-20 degrees C.     

Multinucleate host cells induced by Vittaforma corneae (Microsporidia)

The microsporidium Vittaforma corneae develops within the target cell cytoplasm. In the present study, green monkey kidney (E6) cells infected at 30 degrees C, 35 degrees C or 37 degrees C with V. corneae developed enlarged multinucleate structures of up to 200 microm in any horizontal dimension made up either of a single cell or of multiple fused cells. A number of epithelial cell types (SW-480, HT-29, Caco-2 and HCT-8) were infected with V. corneae but did not induce the same highly organized structures, suggesting that for the structure to develop, the host cell must be capable of continued mitosis, and not be differentiated or be detaching from the surface matrix. Live cell imaging of infected E6 cells revealed large, multinucleate infected cells characterized by a central focus from which radiated parasite stages and host cell mitochondria. Immunocytochemistry identifying gamma and alpha tubulin suggested that a single centrally-located microtubule organizing centre governed the distribution of parasite stages and host cell organelles, with mitochondria and parasites being eventually transported towards the periphery of the structure. Whole cell patch clamp analysis of infected cells indicated an average five-fold increase in total membrane capacitance, consistent with an enlarged single cell. Scanning electron microscopy revealed cell-like protrusions around the periphery of the structure with the intervening space being made up of parasites and cell debris. Clearly in the case of V. corneae-infected E6 cells the parasite-host cell relationship involves subverting the host cell cytoskeleton and cell volume control, providing the parasite with the same protected niche as does a xenoma.     

Evidence for Antibiosis and Induced Host Defense Reactions in the Interaction Between Verticillium lecanii and Penicillium digitatum, the Causal Agent of Green Mold

ABSTRACT Chronological events of the intercellular interaction between Verticillium lecanii and the postharvest pathogen Penicillium digitatum were investigated by transmission electron microscopy and gold cytochemistry. Growth inhibition of P. oligandrum as a response to V. lecanii attack correlated with striking host changes including retraction of the plasma membrane and cytoplasm disorganization. Such changes were associated with the deposition on the inner host cell surface of a chitin- and cellulose-enriched material which appeared to be laid down as a structural defense reaction. The accumulation of chitin in the newly formed material correlated with a decrease in the amount of wallbound chitin. However, the deposition of cellulose appeared to correspond to a de novo synthesis, as evidenced by the occurrence of cellulose-containing vesicles which released their content in the space between the invaginated plasma membrane and the host cell wall. Results of the present study provide the first ultrastructural and cytochemical evidence that antagonism, triggered by V. lecanii, is a multifaceted process in which antibiosis, with alteration of the host hyphae prior to contact with the antagonist, appears to be the key process in the antagonism against P. digitatum.     

Structural and functional changes in mouse fibroblast cells treated with 2,3-dichloro-1,4-naphthoquinone (dichlone)

When strain L mouse fibroblasts were treated with the substituted quinone pesticide dichlone (2,3-dichloro-1,4-naphthoquinone), a rapid loss of intracellular potassium, ATP, and protein with a concomitant increase in intracellular water and volume occurred. Electron microscopic examination showed a nonreversible swelling of the cells associated with the formation of large protruding blebs. The findings indicate that dichlone can alter the permeability of the cell membrane which may be one of the sites of action of the pesticide in the fibroblast cells. Menadione (2-methyl-1,4-naphthoquinone), a synthetic vitamin K, also a substituted quinone, was found to be less effective in short-term incubation at corresponding concentrations, althought it did produce loss of K⁺ ions and protein from the cell.The action of dichlone was compared with that of model compounds which are known to act on plasma membranes (p-chloromercuribenzene sulfonate—PCMBS) and those which interfere with intracellular energy metabolism (combination of antimycin A plus iodoacetate). From the results reported in this paper it appears that dichlone acts in most respects, like PCMBS, by a direct interaction with membrane components, but unlike PCMBS, dichlone enters the cell rapidly and stimulates oxygen uptake probably by constituting a bypass of electron transfer.     

Host-parasite relationships of Zootoca vivipara (Sauria: Lacertidae) in the Pyrenees (North Spain)

The helminths infesting the common lizard, Zootoca vivipara (Jacquin, 1787), were studied with special attention to the relations between the number of nematodes, Oswaldocruzia filiformis (Goeze, 1782), and the size, sex and age class of the host. The possible seasonality of the parasite intensity and the relationship with the feeding habits of the host were also tested. Helminth infracommunities of Z. vivipara were depauperate with lizards harbouring only two species, the trematode Plagiorchis molini (Lent et Freitas, 1940) and the nematode O. filiformis. A positive correlation between host size and the number of O. filiformis was found for female Z. vivipara. However, no correlation was detected between intensity and sex or age class. The feeding habits of Z. vivipara, the isolation of the population studied and the low level of interaction with other reptilian or amphibian species are suggested as the causes of the depauperate helminth infracommunities found in this lacertid lizard.     

Toxic effects of novel organophosphorus insecticide (RPR-V) on certain biochemical parameters of euryhaline fish, Oreochromis mossambicus

The euryhaline fish, Oreochromis mossambicus was exposed to sub-lethal concentration (0.017 mg L⁻¹) of a novel phosphorothionate, 2-butenoic acid-3-(diethoxy phosphinothionyl) ethyl ester (RPR-V) for 30 days and allowed to recover for 7 days. Important biomarker enzymes were assayed in plasma, brain, gill, liver, kidney, and muscle during exposure tenures of day-3, -7, -15, -30, and also at 7 days (withdrawal) after stopping treatment. Acetylcholinesterase (AChE) activities of brain, gill, and muscle were strongly inhibited by 67, 75, and 66%, respectively, on day-30. Exposure (time) dependent increases in alanine aminotransferase (ALAT), and aspartate aminotransferase (ASAT), acid phosphatase (AcP), and alkaline phosphatase (AkP), activities in plasma and kidney; AcP and AkP activities in gill were noticed. However, significant decrease in ALAT, ASAT, AcP, and AkP activities in liver was observed. The depletion of glycogen was observed in liver, brain, and gill tissues, an indication of typical stress related response of the fish with pesticide. A significant increase in lactate dehydrogenase (LDH) activity in gill and brain was observed and decreased in liver and muscle, indicating tissue damage and muscular harm. Depletion of glutathione (GSH) was observed in the above tissues, there by enhancing the lipid peroxidation resulting in cell damage. The induction in hepatic glutathione-S-transferase (GST) levels indicates the protection against the toxicity of xenobiotic-induced lipid peroxidation. There was a significant recovery in all the above biochemical parameters, in all the tissues of fish after a recovery period of 7 days. These results revealed that RPR-V affects the intermediary metabolism of O. mossambicus and the increase of biomarker enzymes in plasma, might be due to the necrosis of liver.     

Monoclonal antibodies to specific components of the Dutch elm disease pathogen Ophiostoma ulmi

The potential of polyclonal antisera and monoclonal antibodies to differentiate the EAN and NAN aggressive subgroups of Ophiostoma ulmi was explored. Polyclonal antisera, when tested by ELISA, cross-reacted widely with unrelated species and failed to distinguish between the two aggressive subgroups but small quantitative differences were found, particularly between antigens secreted overnight, by EAN and NAN germlings. Monoclonal antibodies were raised in mice against mycelial homogenates. From two fusions, 33 cell lines were raised that secreted antibodies positive for O. ulmi. Approximately one third were non-specific; 11 were specific either to species or subspecies. Two cell lines differentiated mycelial antigens of the aggressive isolates of O. ulmi from those of the non-aggressive subgroup, but not antigens from surface washings. Only quantitative differences were detected between the EAN and NAN aggressive subgroups. Almost all the monoclonal antibodies and antiserum recognized antigens present in surface washings of cultures on solid medium, in cell-free extracts of mycelial homogenates, in cell-free culture fluids, and in substances secreted overnight by germinating spores. Specific detection of such molecules promises to provide a highly sensitive mechanism for studying early pathogen/host plant interactions. Most of the monoclonal antibodies appeared to have potential diagnostic value; they gave readings twofold to tenfold higher with extracts from diseased than from healthy tissue. However, one cell line that secreted antibodies specific to O. ulmi cross-reacted strongly with extracts of healthy tissue.     

柿树炭疽菌侵染柿树叶柄的超微结构观察

 利用透射电镜技术研究了柿树炭疽菌侵染柿树叶柄的超微结构。结果表明:病原菌侵入寄主细胞后,产生细胞内的初生菌丝,其表面沉积凹凸不平的电子不透明物质。一层界面基质(interfacial matrix)把表初生菌丝细胞壁和凹陷的寄主原生质膜分开。随着初生菌丝定殖下一个细胞,原先细胞中的细胞膜消失,形成许多泡囊,随后叶绿体消失,内质网和高尔基体也逐渐降解,最后细胞内物质全部被降解成电子不透明的颗粒,降解的物质沿着初生菌丝和细胞壁表面沉积。初生菌丝穿透细胞壁的过程中,菌丝顶端接触细胞壁后膨大,并在中部产生一个隔膜,然后顶端细胞产生一个较细的穿透菌丝,穿透寄主细胞壁。穿透菌丝在寄主细胞壁中的狭窄处产生一个隔膜,一旦穿透寄主细胞壁后,迅速膨大。次生菌丝在细胞间和细胞内扩展,通过菌丝体对细胞壁施加的机械压力引起寄主细胞壁破裂,或同初生菌丝一起使细胞壁解体。侵染90 h后,形成垫形分生孢子盘。在分生孢子盘周围的表皮细胞中,次生菌丝不断形成子座组织,使原来的子座扩大,子座不断分化形成产梗细胞,产梗细胞产生分生孢子梗,分生孢子梗生长和发育对角质层和表皮细胞壁组织折叠处施加机械压力,使角质层和表皮细胞壁组织进一步折叠,分生孢子盘也相应扩大。     

Permeability of the haustorium-host interface in powdery mildews

The extent to which uranyl ions and peroxidase permeated haustorial complexes isolated from Pisum sativum infected with Erysiphe pisi was determined by transmission electron microscopy (TEM).Freshly isolated complexes were treated with uranyl acetate, either directly, or after treatment with cell wall degrading enzymes, or with Triton X-100 and the uranyl ion, precipitated with phosphate. The complexes were then fixed with glutaraldehyde and examined by TEM to determine the distribution of uranyl phosphate crystals.Two independent experiments, each involving the examination of 80 complexes, gave similar results. After direct uranyl treatment, crystals were excluded from 38% of the complexes, were present in the extrahaustorial matrix only in 19% and occurred throughout in 43%. Breaks were observed in the extrahaustorial membrane of the last two categories. Uranyl crystals bound to the outer (host cytoplasmic) surface of the extrahaustorial membrane and also to the matrical surface, where this was accessible. In addition, the few necrotic haustorial complexes observed were completely permeated.Following pre-treatments with pectinase and cellulase, or Triton X-100, uranyl crystals were present throughout the complexes in all or 96%, of the complexes respectively.In similar experiments involving treatment with horseradish peroxidase (instead of uranyl acetate) followed by the addition of hydrogen peroxide and 3,3-diaminobenzidene, electron opaque deposit occurred around the complexes but never internally in any part of them. Even where breaks in the extrahaustorial membrane were visible the extrahaustorial matrix was not permeated.It is concluded that the extrahaustorial membrane and haustorial plasma membrane are semipermeable in many of the isolated haustorial complexes but that some are damaged during isolation and purification procedures; that a barrier to diffusion exists where the extrahaustorial membrane is joined to the wall of the haustorial neck and in other parts of the neck wall so that the whole boundary of the extrahaustorial matrix restricts diffusion of solutes; and that the extrahaustorial matrix is a gel which precludes the passage of large molecules through it. The physiological implications of the results are discussed.     

Cytological Investigation of Resistance to Leptosphaeria maculans Conferred to Brassica napus by Introgressions Originating from B. juncea or B. nigra B Genome

ABSTRACT Introgressions into Brassica napus from the B genome, either the B. nigra chromosome B4 or the B. juncea fragment carrying the Jlm1 gene, have given rise to the B. napus-B. nigra addition line (LA4+) and the B. napus-B. juncea recombinant line (MXS), respectively. The resistance of these two lines to Leptosphaeria maculans is characterized by a hypersensitive reaction (HR) on both the cotyledons and leaves, while the collar displays a high degree of resistance. Responses induced in cotyledons of the two lines by L. maculans inoculation were investigated with emphasis on cytological events underlying the HR and on host defense reactions. Features of host cell changes including condensation and lobing of nuclei, fragmentation of chromatin, disruption of the nuclear membranes, and plasma membrane withdrawal were reminiscent of HR cell death in MXS and LA4+ plants. Restriction of pathogen growth to the infection areas in LA4+ was correlated to reinforcement of cell wall barriers, including wall apposition, papillae, and vessel plugging. In MXS, the lower expression of resistance was associated with a delay in plant responses. These results indicate that mechanisms underlying the HR in the B. napus recombinant and addition lines are differently controlled according to the introgressed genes.     

An ultrastructural study of the effect of metalaxyl on Phytophthora capsici infected stems of Capsicum annuum

Stems of pepper seedlings were inoculated with zoospores of Phytophthora capsici 18 h after a soil-drench with metalaxyl solution (5 μg ml^?1) or water. Infected stem tissues were examined by electron microscopy 24 h after inoculation. Compared with untreated controls, in which the fungal cells were generally normal in shape and ultrastructure, the most conspicuous effects of metalaxyl treatment on the fungal ultrastructure in the stem tissue were an abnormal shrinking of the fungal cell, a separation of the plasma membrane from the hyphal wall, a peculiar invagination and breakdown of the plasma membrane, the presence of vesicles in the invaginated spaces and within the damaged fungal cells, and an indistinct structure of cell organelles. In metalaxyl-treated stems, an electron-dense material was apposed in those sites of the host cell wall in most intimate contact with the fungal cell wall, indicating that the deposition of these substances from the host cell walls may function as a plant defence reaction to the fungus, whereas in untreated controls, the dark-stained host cytoplasm did not aggregate around the sites of fungal contact. The haustorium was encased by the extra-haustorial matrix in treated stems. These results suggest that metalaxyl treatment not only changes the fine structure of P. capsici, but may also induce the plant defence reaction.     

Ralstonia (Pseudomonas) solanacearum Race 3 (Biovar 2) in Surface Water and Natural Weed Hosts: First Report on Stinging Nettle (Urtica dioica)

The population dynamics of the brown rot bacterium Ralstonia (Pseudomonas) solanacearum in surface water of two selected water-areas were monitored over a two-year period. In some cases during summer, high bacterial numbers (up to 10⁶ cfu l^–1) were observed. In a host plant survey a few plants of stinging nettle (Urtica dioica) were found to be a natural host of the bacterium when plants were growing with their roots in contaminated water. The significance of U. dioica in the epidemiology of the brown rot bacterium is not yet known and subject to further investigation. Pathogenicity of R. solanacearum to stinging nettle (U. dioica) and bittersweet (Solanum dulcamara) was demonstrated in a greenhouse experiment.     

The role of primary germ tubes (PGT) in the life cycle of Blumeria graminis: The stopping of PGT elongation is necessary for the triggering of appressorial germ tube (AGT) emergence

The conidia of Blumeria graminis f. sp. hordei (Bgh), following contact with a host surface, first form a short germ tube, called the primary germ tube (PGT), and then an elongating germ tube emerges. It differentiates into an appressorial germ tube (AGT), and then the AGT elongates and swells. It forms a hooked, appressorial lobe that penetrates the epidermal cell wall of the host. In a series of infections, the positive role of PGT in the morphogenesis of the fungus is unclear except for the possibility reported by Carver and Ingerson that the growth of a long germ tube, with the potential to differentiate into an appressorium, seems to be dependent on the perception of a suitable host surface through contact with the PGT. Therefore, the aim of the present study is to further clarify the role of PGT in the morphogenesis of the fungus. When the conidia of Bgh were inoculated onto the coleoptile surface whose cuticle was removed with cellulose acetate, the emergence of the AGT was delayed. This delay was related to the length of the PGT. That is, on the cuticleless coleoptile surface the PGT tended to continue elongating without stopping. If there were gaps on the coleoptile surface such as a cell border on the more hydrophilic substratum like cuticleless coleoptile surface, the PGT stopped elongating there and after that the AGT emerged. Moreover, the length of PGT in the beginning of AGT emergence was same as that of the PGT after appressorium formation. This means that PGT elongation had stopped when AGT began to emerge. Therefore, it is necessary to stop the PGT elongation for the triggering of AGT emergence.     

The haustorial host-parasite interface in rust-infected bean leaves after high-pressure freezing

The haustorial fine structure of the bean rust fungus, Uromyces appendiculatus var. appendiculatus, was studied within the cells of its host, Phaseolus vulgaris. Results were obtained after high-pressure freezing and subsequent freeze-substitution or freeze-fracturing. Good preservation of leaf tissue after freeze-substitution needed cryoprotection with 8% methanol. For freeze-fracturing, no chemical treatment was applied. In addition to the organelles which are generally found in fungi after cryo-fixation, tubular-vesicular complexes were found in the cytoplasm. Both techniques revealed an extrahaustorial matrix of even width, surrounding the haustorial body. The extrahaustorial membrane was not undulated, and the side facing the plant cytoplasm was lined with a delicate fringe of well-stained material. The extrahaustorial membrane was nearly devoid of intramembrane particles. The host plasma membrane in infected tissue, especially the protoplasmic face, had fewer intramembrane particles than those in uninfected tissue. The haustorial plasma membrane contained many intramembrane particles.     

Intoxication effects of lindane (γ-BHC) on the carbohydrate metabolism in the climbing perch, Anabas testudineus (Bloch)

The effects of an acute lethal (0.59 mg/liter) and a sublethal (0.075 mg/liter) concentration of lindane on the levels of glycogen (liver and muscle), glucose (blood), and lactic acid (liver, muscle, and blood) of a freshwater fish, Anabas testudineus (Bloch) were determined. A marked decrease in the liver glycogen and significant increase in blood glucose and muscle glycogen at 1 hr of exposure to 0.075 mg/liter lindane was noted. These results indicate extensive utilization of energy stores in the liver and simultaneous buildup of fuel reserves in the muscle. After 3 hr of exposure, glycogenesis possibly becomes operative in liver through the Cori cycle. The activity of the cycle was observed in the fish in all the subsequent periods of exposure studied. At 120 hr, the decreases observed in muscle and liver glycogen indicate near exhaustion of glycogen stores. Though changes in glycogen content of both muscle and liver were similar in 0.59 mg/liter lindane, they have less adaptive value since mortality of fish was significantly high at all three peroids studied.     

Public health importance of Brachiola algerae (Microsporidia)--an emerging pathogen of humans

Brachiola algerae, a parasite of Anopheles mosquitoes, has also been isolated from a human cornea, a cutaneous nodule and deep muscle tissue. All three human isolates of B. algerae are morphologically, serologically, and genetically similar to the mosquito-derived isolates including the original isolate of Vavra and Undeen. All of these isolates grew well in mammalian cell cultures at 37 degrees C and produced spores. Transmission electron microscopy revealed that all developmental stages including meronts, sporoblasts and spores were diplokaryotic and developed in direct contact with the host cell cytoplasm, a feature characteristic of the genus Brachiola. Spores of all isolates reacted well, in the immunofluorescence assay, with the rabbit anti-B. algerae serum. In the immunoblot assay, although the overall banding patterns of the human and mosquito isolates were similar, minor differences could be discerned. Sequencing of the PCR products of the amplified SSU rRNA gene revealed the existence of two distinct genotypes; the original mosquito (Undeen) isolate belonged to genotype 1 and the isolate from cornea and that from the deep muscle biopsy to genotype 2, whereas the isolates from a mosquito and one of the other two human isolates (one from skin abscess) had both genotypes, 1 and 2. It is known that spores of mosquito-derived B. algerae can not only proliferate in mammalian cell cultures at 37 degrees C but also can infect mice when injected into footpads or deposited on the corneal surface. These observations indicate that the spores have potential to be a risk factor for humans, especially those with immunodeficiency.     

Xenoma-like formations induced by Soricimyxum fegati (Myxosporea) in three species of shrews (Soricomorpha: Soricidae), including records of new hosts

In South Bohemia, Czech Republic, 178 shrews, including 98 common shrews, Sorex araneus L., 70 pygmy shrews, Sorex minutus L., and 10 lesser white-toothed shrews, Crocidura suaveolens (Pallas), were examined for Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007 infections, using squash preparations of unfixed tissues, histological sections and molecular methods. The infection was found in 51 (52%) S. araneus, 14 (20%) S. minutus and 1 (10%) C. suaveolens. The records of the latter two species extend host range of S. fegati. Lesions associated with S. fegati infections in the liver, the organ of specific localisation of the parasite, were found to be induced by proliferative stages migrating toward lumina of bile ducts. In other organs of these three host species, xenoma-like formations (XLFs) were found that severely injured blood vessels. XLFs contained presporogonic stages of S. fegati, whose species identity was evidenced using molecular methods.     

Phylogeography based on the nuclear ribosomal DNA internal transcribed spacer region of native Miscanthus sinensis (Poaceae) populations in Japan

Nucleotide sequence variations were investigated with respect to the geographical distribution patterns of Miscanthus sinensis populations that were sampled from 26 Japanese national parks and three populations of the Ryukyu Islands. Twelve homozygous sequences in the nuclear ribosomal DNA internal transcribed spacer region were detected. The populations of M. sinensis in mainland Japan mainly were composed of a monophyletic group with a symapomorphic character, whereas those in the Ryukyu Islands included a polyphyletic group. Only an internal transcribed spacer haplotype with a plesiomorphic character was found in both mainland Japan and the Ryukyu Islands. Thus, no clear geographical isolation was observed in this species. These facts might be caused by the ability of M. sinensis as a pioneer plant to have a high migration potential and high gene flow by outcrossing. On the basis of the results of this study (nuclear ribosomal DNA) and the previous study (chloroplast DNA), a phylogeographical history of M. sinensis was presumed where the ancestral and derived lineages were distributed in the southern and northern regions of Japan, respectively, without strict geographical isolation.