Comparative development of gametophytes ofOsmunda lancea andO. japonica (osmundaceae): Adaptation of rheophilous fern gametophyte

Development of the gametophytes of a rehophytic fernOsmunda lancea and a related dryland speciesO. japonica was examined, and adaptation ofO. lancea gametophytes to rheophytic habitat was discussed. The spores ofO. lancea were larger in size and contained larger oil droplets than those ofO. japonica. The germination rate was similar. In agar culture,O. lancea gametophytes grew more rapidly and reached reprudctive maturity earlier thanO. japonica, although there was no prominent gross-morphological difference in developing and mature gametophytes. Sporophyte formation ofO. lancea took place at roughly two times higher rate than that ofO. japonica. TheO. lancea gametophytes might be adapted to the periodically flooded habitat by shortening its life, which is a different strategy from that of the sporophytic generation.     

Comparative leaf development ofOsmunda lancea andO. japonica (osmundaceae): Heterochronic origin of rheophytic stenophylly

Comparative development of the narrow pinnules of rheophyticOsmunda lancea and of the broad pinnules of a related dryland species,O. japonica, was examined and the origin of rheophytic stenophylly was discussed. The mature leaves and their various parts ofO. lancea are smaller and narrower than those ofO. japonica. The young pinnules ofO. lancea at the initiation of cell expansion are smaller than those ofO. japonica. The growth pattern of the pinnules is fundamentally the same in the two species, but pinnule growth period is shorter inO. lancea than inO. japonica. While the largest growth rate in pinnule length is quite similar, inO. lancea the pinnules are less elongated and much less broadened during ontogeny. Cell expansion in the mesophyll and epidermis proceeds acropetally and toward the margin along the axes of costules and veins. Although the numbers of mesophyll and epidermal cells between two adjacent veinlets are almost the same inO. lancea andO. japonica, during the subsequent growth period inO. lancea, the cells expand to a smaller extent and the veinlets become more narrowly oblique to the costule. This oblique distortion of laminar segments framed by veins causes stenophylly, an allometric modification. The stenophylly ofO. lancea is believed to have arisen by heterochronic evolution, in particular, progenesis.     

Genetic population structure of <Emphasis Type="Italic">Osmunda japonica</Emphasis>, rheophilous <Emphasis Type="Italic">Osmunda lancea</Emphasis> and their hybrids

Rheophilous Osmunda lancea often hybridizes with a dryland ally, Osmunda japonica, to produce O. × intermedia, forming zonation in riverbanks and the adjacent dryland along flooding frequency clines. This study examined the genetic structure of populations consisting of O. × intermedia and the two parental species by analyzing ten nuclear DNA markers [six cleaved amplified polymorphic sequence (CAPS) markers and three simple sequence repeat (SSR) markers developed from an expressed sequence tag (EST) library, and the sequence of the glyceraldehyde-3-phosphate dehydrogenase gene GapCp] and chloroplast DNA sequences. The results suggest that the nuclear genes of O. japonica and O. lancea are genetically differentiated despite shared polymorphism in their chloroplast DNA sequences. This discrepancy may be attributable to natural selection and recent introgression, although it is not evident if introgression occurs between O. japonica and O. lancea in the examined populations. Our findings of putative F2 hybrids in O. × intermedia support its partial reproducibility, and also suggest that formation of later-generation hybrids generates morphological variation in O. × intermedia. O. lancea plants collected from geographically distant localities were genetically very similar, and it is suggested that O. lancea originated monotopically.     

Ploidy chimeras induced in haploid sporophytes of <Emphasis Type="Italic">Osmunda claytoniana</Emphasis> and <Emphasis Type="Italic">Osmunda japonica</Emphasis>

Haploid sporophytes of Osmunda claytoniana (2n = x = 22) were apogamously produced from calli when cultivated on a hormone-free medium. Flow cytometric analysis showed that ploidy chimeras were spontaneously produced in a haploid sporophyte of O. claytoniana and those of O. japonica that were obtained in the previous study. In the haploid sporophyte of O. claytoniana, a diploid pinnule and a partially diploid terminal segment were produced in a haploid pinna. In O. japonica, a haploid sporophyte yielded a diploid pinna in a haploid frond, and another haploid sporophyte yielded a diploid pinnule in a haploid pinna. Diploid chimeras were large in size and could be readily distinguished from other haploid parts of the fronds. It is likely that the chimeras were produced clonally from a single diploid cell that established chromosome doubling.     

Transmission ratio distortion of molecular markers in a doubled haploid population originated from a natural hybrid between Osmunda japonica and O. lancea

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.     

Co-occurrence of the cirolanid isopodEurydice nipponica Bruce & Jones and the ghost shrimpCallianasa japonica Ortmann on an intertidal sand flat

On a moderately protected intertidal sand flat in west Kyushu, Japan, most of the population of the cirolanid isopodEurydice nipponica Bruce & Jones (89%–100%) was found in the zones occupied by the thalassinidean ghost shrimpCallianassa japonica Ortmann in July, August and December 1980.C. japonica later extensively expanded its habitat, and in July 1984, when almost the whole sand flat had been densely populated byC. japonica, the range of the distribution ofE. nipponica was the same as that ofC. japonica with a density about 10 times greater than in July 1980. The occurrence ofE. nipponica has previously been recorded from several exposed sandy beaches as well as their adjacent subtidal areas of well oxygenated sands along the coast of Kyushu, but not on more protected shores like the present sand flat. It is suggested thatC. japonica, through its bioturbating activities, produces sediment characteristics approximating those of the exposed sandy beaches which are the preferred habitat ofE. nipponica. It is supposed thatE. nipponica is a facultative commensal ofC. japonica. In a field experiment to excludeC. japonica and to detect its positive influence onE. nipponica, the densities ofE. nipponica were found to be lower in the experimental plots than in the intact plots. Statistically, however, the difference was only weakly significant, and the possible reasons for this are discussed.     

Reticulate evolution of the hybrid produced artificially by crosses between Osmunda banksiifolia and Osmunda lancea

Although ferns have been developed by hybridization and chromosome doubling, no natural polyploidy has yet been recorded in Osmundaceae. So, we produced hybrids artificially by crosses between Osmunda banksiifolia (2n = 2x = 44) and Osmunda lancea (2n = 2x = 44), and investigated their sporogenesis. From the O. banksiifolia × O. lancea hybrid with 44 univalent chromosomes, allotetraploids with 44 bivalent chromosomes were produced by chromosome doubling, and allotriploids with 22 univalent chromosomes and 22 bivalent chromosomes were then produced by back crosses. The results show when and how chromosome doubling occurs in hybrids. The success of artificial hybridization between O. banksiifolia and O. lancea, did not, however, reflect any product of natural hybridization between the two species.     

Expression of the<Emphasis Type="Italic"> lacZ</Emphasis> reporter gene in sporophytes of the seaweed<Emphasis Type="Italic"> Laminaria japonica</Emphasis> (Phaeophyceae) by gametophyte-targeted transformation

The seaweed Laminaria japonica (Phaeophyceae) has a two-generation life cycle consisting of haploid gametophytes and diploid sporophytes. Female and/or male gametophytes were transformed using particle bombardment and the histological LacZ assay was performed on sporophytes generated by either parthenogenesis or inbreeding. Female gametophyte-targeted transformation resulted in similar lower efficiencies in both parthenogenetic and zygotic sporophytes, and only a chimeric expression pattern was observed. Male gametophyte-targeted transformation led to a higher efficiency, with 3.5% of the zygotic sporophytes stained completely blue (all-blue), implying the integration of lacZ at the one-cell stage. Polymerase chain reaction analysis using primers specific for a lacZ-vector juncture fragment and subsequent blotting indicated the presence of the introduced gene in the sporophytes. The method reported here has a potential for seaweed transformation using spore-based bombardment followed by the developmental process.Abbreviations DPR Detected positive rate - ER Expression rateCommunicated by F. Sato     

OSMUNDA WEHRII,A NEW SPECIES BASED ON PETRIFIED RHIZOMES FROM THE MIOCENE OF WASHINGTON

Silicified rhizomes from Miocene strata near Yakima, Washington represent a new species of Osmunda. The stems are 8–13 mm in diameter and are surrounded by a thick sheath of adherent leaf bases, each of which shows stipular expansions typical of the Osmundaceae. The new species has an ectophloic siphonostele in which the xylem cylinder is dissected by leaf gaps with 12–14 strands being visible in a given stem cross section. Such sections also show 12–16 leaf traces in the cortex. The xylem of each leaf trace diverges from the xylem cylinder of the stem as an adaxially concave strand with its protoxylem organized into a single medial adaxial cluster. Initial bifurcation of the leaf-trace protexylem occurs as the leaf trace passes through the outer cortex of the stem. In the basal part of the stipular region of the petiole base, thick-walled fibers form an arch on the abaxial side of the sclerenchyma ring around the petiolar bundle. This arch persists throughout most of the length of the stipular region, with the thick-walled fibers becoming reorganized into two lateral masses in the distal part of the stipular region. Similar thick-walled fibers form an elongate strip of tissue in each wing of the stipule along with several small clusters scattered near the sclerenchyma ring. The new species belongs to the subgenus Osmunda and shows that during the Neogene, the latter existed as a group of closely related species much as it does today. Furthermore, Osmunda wehrii combines features of the modern O. regalis, O. japonica, and O. lancea with those of O. claytoniana and thus supports the inclusion of the latter species in the subgenus Osmunda.     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Induced apogamous sporophytes inPteris dispar andP. semipinnata,and the meiotic behavior in their sporocytes

Spores ofPteris dispar andP. semipinnata were aseptically cultured in flasks for apogamous sporophyte induction. Calli or cell colonies similar to calli were induced in cultures supplemented with hormones. Sporophytic leaves subsequently developed from them in hormone-free medium and the young sporophytes were raised into plants with sporangia. Since the wild-type plants having 116 chromosomes are tetraploid, the sporophytic plants originating from spores would appear to be diploid (dihaploid). In induced sporophytes ofP. semipinnata, non-homologous chromosomes (58 univalents) were found during the meiotic process in sporocytes. InP. dispar, however, the meiotic cells showed many bivalent chromosomes (maximum 29ll). These results suggest thatP. semipinnata is allotetraploid, whereasP. dispar is autotetraploid.     

Anatomical study on the shoot apex ofOsmunda japonica thunb

The ontogenetic and seasonal variations in the organization of the shoot apical meristem ofOsmunda japonica Thunb. were investigated. The meristem is composed of an apical segmentation zone (SZ), a mother cell zone of the stele (MS) and a periphereal zone (PZ). A single apical cell is mostly discernible in all sesons throughout the whole process of ontogeny observed in the present study. The paical cell is usually four-sided, nearly triangular, with a regular segmentation pattern in transverse view. However, it is sometimes acccurately three-sided with a highly regular segmentation pattern in the active season, while it is often four-sided, nearly trapezoid or five-or six-sided, with a less regular segmentation pattern in the inactive season. The size of the apical cell represented by its free surface are increases with the increase in size of the plant body in the young plants. However, in the adult plants, the size of the apical cell is smaller in the active season and larger in the dormant season. The organization pattern of the shoot apical meristen ofO. japonica does not show an intermediate type between the eusporangiate and the leptosporangiate ferns, but the leptosporangiate fern type.     

Adaptation of stomatal response ofCamellia rusticana to a heavy snowfall environment: Winter drought and net photosynthesis

The adaptation ofCamellia rusticana, an evergreen broad-leaved shrub found in areas of heavy snowfall in Japan, to heavy snowfall environments, and the mechanisms by which it is damaged in winter above the snow, were investigated. The stomatal response and photosynthetic characteristics ofC. rusticana were compared to those ofCamellia japonica found in areas of light snowfall. In field conditions, the mean net photosynthesis ofC. rusticana at photon flux density (PFD) over 200 μmol m⁻²s⁻¹ (Pn(_>200). was 50% larger than that ofC. japonica, but in both light saturated and CO₂ saturated conditions, the O₂ evolution rate (Pc) ofC. rusticana was not different from that ofC. japonica. Mean leaf conductance at PFD over 200 μmol m⁻²s⁻¹ (gl(_>200)) was about 100% larger than that ofC. japonica in the field. The Pn(_>200)) was 50% ratio ofC. rusticana was 37% higher than that ofC. japonica which suggests thatC. rusticana's larger Pn(_>200) can be explained by its larger gl(_>200). WhenC. rusticana trees wintering underneath the snow were projected above it, the leaves of these plants showed serious drought within five days in non-freezing conditions. Their Pc and the maximum stomatal conductance decreased by half and did not recover. The leaves ofC. rusticana showed larger gl(_>200) and a less sensitive stomatal response to the decrease of leaf water potential than that ofC. japonica. The stomata characteristics ofC. rusticana caused larger net photosynthesis than that ofC. japonica during the no snow period, and caused the need for snow cover in winter as protector from winter drought.     

Host habitat finding and host selection of theDrosophila parasitoidLeptopilina australis (Hymenoptera,Eucoilidae), with a comparison of the niches of EuropeanLeptopilina species

Summary Decaying petioles of giant hogweed,Heracleum mantegazzianum Sommier & Levier, are used as a breeding site by six species ofDrosophila and the drosophilidScaptomyza pallida. The most numerous parasitoid species associated with this community isLeptopilina australis. BecauseL. australis was previously unknown in western Europe, we present the characters to distinguish it form its close relativeL. clavipes. Experiments on host species selection and survival ofL. australis showed that this parasitoid mainly usesD. limbata as host. Olfactometer experiments showed thatL. australis is attracted by the odour of decaying hogweed stalks, especially when these contain larvae ofD. limbata. L. australis is also strongly attracted by the odour of stinkhorns, a habitat in which it has never been found in nature.D. phalerata is the dominant fly species in stinkhorns, and is not a host ofL. australis. We offer a possible functional explanation for this unexpected habitat choice, by showing thatD. transversa andD. kuntzei, both species found to breed in fungi, are also suitable hosts forL. australis. We also discuss habitat choice with regard to a proposed phylogeny of theLeptopilina species in temperate Europe. Finally, we discuss niche overlap ofL. australis with the otherLeptopilina species.     

Growth and survival rates of large-type sporophytes of Ecklonia cava transplanted to a growth environment with small-type sporophytes

Stipe lengths of sporophytes of Ecklonia cava Kjellman have been reported to be longer along the southeast than southwest coast of the Izu Peninsula, central Japan. Two bays in this region that have natural populations of E. cava, but with different stipe lengths, were chosen for transplant experiments to examine if stipe length was an environmentally controlled trait. Transplant experiments were carried out in order to determine whether large-type sporophytes of E. cava with long stipes growing in Nabeta Bay (southeast Izu Peninsula, Japan) would turn into small-type sporophytes with short stipes when transplanted to Nakagi Bay (southwest Izu Peninsula). Ten juvenile sporophytes of E. cava (stipe length < 5 cm) were collected from Nabeta Bay (large-type habitat) and transplanted to Nakagi Bay (short-type habitat) in December 1995. As a transplant control, ten juvenile sporophytes of E. cava growing in Nakagi Bay were also transplanted to the same artificial reefs. Growth and survival rates of the sporophytes were monitored monthly for 3 y until December 1998. The transplanted sporophytes showed an increase in their stipe length and diameter from winter to spring, whereas almost no increase was observed from summer to autumn. However, the elongation was greater in Nabeta sporophytes than in Nakagi sporophytes. The primary blade length increased mainly from winter to early spring and decreased largely in autumn. Average primary blade lengths were similar in both Nabeta and Nakagi sporophytes from the end of the first year of transplanting. Although ca. 70% of both Nabeta and Nakagi sporophytes survived during the first 2 y after transplantation, no Nakagi sporophytes and only two Nabeta sporophytes survived to the end of the 3 y study period. Despite transplantation to Nakagi Bay, where short sitpes are naturally present, the sporophytes from Nabeta Bay persisted in having longer stipes, which suggests that stipe length is genetically, rather than environmentally, controlled.     

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of <Emphasis Type="Italic">Laminaria japonica</Emphasis>

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.     

内生真菌与苍术粉对连作花生根际微生物区系和微量元素的影响

通过盆栽试验,研究了内生真菌拟茎点霉B3(Phomopsis liquidambari)及苍术(Atractylodes lancea)粉联合施用对连作花生根际土壤微生物区系、酶活性及有效态微量元素(Mo、B、DTPA-Fe、Zn、Cu、Mn)含量的影响。结果表明:内生真菌B3和苍术粉复合处理比内生真菌B3处理的荚果和秸秆产量分别增加10.28%和14.11%,内生真菌B3处理与正常施肥相比显著提高了根瘤数量、荚果和秸秆产重,各处理组与正常施肥对照相比分枝数和根长无显著差异。B3处理与对照相比显著提高了种子期、结荚期和成熟期根际土壤可培养细菌和放线菌数量,B3和苍术粉复合处理与对照相比显著提高种子期、花期和成熟期可培养真菌和放线菌数量;细菌DGGE指纹图谱聚类分析表明,B3和苍术粉复合处理相对于正常施肥处理,显著改变种子期、苗期、花期和成熟期花生根际土壤细菌群落结构,同时苗期、花期和结荚期的细菌条带数和香农指数也有所提高,真菌DGGE指纹图谱聚类分析表明,B3和苍术粉复合处理对真菌群落影响较大,除种子期以外的生育期真菌条带数和香农指数都有明显提高,花期真菌群落结构变化最大,相似度仅为49.6%。花生关键生育期(花期和结荚期)根际土壤脲酶和蔗糖酶活性B3处理和复合处理都显著高于正常施肥对照,促进了连作花生生态系统的物质循环和能量流动。B3和苍术粉复合处理促进了花生生长发育必需微量元素Mo、B、Fe、Zn、Mn的活化,花生叶片和籽粒中微量元素Mo、B、Fe的积累显著增加。研究结果表明,内生真菌和苍术粉联合施用能有效改善连作花生根际微生物区系,提高土壤酶活性,促进微量元素的活化和吸收,对缓解花生连作障碍具有重要意义。     

Habitat preferences of four chironomid species associated with aquatic macrophytes in an irrigation reservoir

Habitat preferences of four chironomid species associated with aquatic macrophytes were studied during the vegetation season from April to October at an irrigation reservoir located in Nagoya, Japan.The two smaller sized species,Corynoneura cuspis Tokunaga andPentapedilum tigrinum Hashimoto, were found to be abundant in two floating-leaved plants,Nymphoides indica O. Kuntze andTrapa japonica Flerov. Phytophages,C. cuspis larvae were observed freely moving on the surfaces of leaves and stems within both of these floating-leaved plant communities. Although found within both plant communities,P. tigrinum larvae, as facultative leaf miners, were more concentrated on the surfaces and within the leaves ofN. indica, which have a thin epidermis and thick mesophyll layer.The two larger sized species,Glyptotendipes viridis (Macquart) andPentapedilum sordens (van der Wulp), were found to be living principally within the stems ofN. indica and the petioles ofT. japonica. These parts of the plants, with thin epidermis and many lacunae in the parenchyma, are favorable for miners.     

Amylostereum laevigatum associated with the Japanese horntail,Urocerus japonicus

The fungus associated with the Japanese horntail,Urocerus japonicus, in Kochi, Kagawa and Ehime Prefectures was studied. Cultures isolated from the mycangia of 113 adult females of the horntail showed the same cultural characteristics. Four of basidiocarps found on felled logs ofCryptomeria japonica were identifieds asAmylostereum laevigatum based on morphological characteristics. This was the first record ofA. laevigatum from Japan. The cultures isolated from the basidiocarps had the same cultural characteristics as those from the mycangia ofU. japonicus. One mycangial isolate produced basidiocarps on artificially inoculated stem segments ofCr. japonica after a 6-mo incubation and was identified asA. laevigatum. One isolate from the basidiocarps ofA. laevigatum and one from the mycangium ofU. japonicus were artificially inoculated into five trees each ofChamaecyparis obtusa andCr. japonica. The wood of all inoculated trees showed discoloration, with no difference in shape and pattern of discoloration between the two isolates. The inoculated fungi were reisolated from the areas of discoloration in the inoculated trees.     

A taxonomic study of KoreanSmilacina

Several taxonomic problems of KoreanSmilacina were examined by statistical methods, based on morphological data. Consequently,S. davurica described in Korean flora differs greatly from originalS. davurica, and closely related toS. bicolor morphologically. Therefore, the present authors treated this new taxon as a variety ofS. bicolor, S. bicolor var.flavovirens N. S. Lee et J. Y. Kim var. nov (“Yondusomdae” in Korean). The distribution in South Korea ofS. bicolor var.bicolor is known as only Mt. Chiri, but this species is collected in Mt. Dukyou, also. The bounds of morphological variations ofS. japonica var.japonica, the short plant size and no hairs on the stem, were revealed. The morphological characteristics ofS. japonica var.japonica andS. japonica var.mandshurica is obscure to distinguish. According to the present study,S. japonica var.mandshurica differs fromS. japonica var.japonica in plant size, size of adaxial leaf epidermal cell, ratio of style/ovary length and shape of stigma. The unrecorded taxon in Korean flora,S. robusta, was given a new Korean name as “Kunsomdae.” AlthoughS. japonica var.mandshurica is the variety ofS. japonica var.japonica, it is more closely related toS. robusta than toS. japonica var.japonica.