Biosystematic studies on the genus Isoetes (Isoetaceae) in Japan. II. Meiotic behavior and REPRODUCTIVE MODE OF EACH CYTOTYPE

Meiotic behaviors and reproductive modes of Japanese Isoetes were studied. The hexaploid (2n = 66) and the octaploid (2n = 88) of I. japonica consistently formed 33 and 44 bivalents, respectively, at diakinesis and/or metaphase I in both micro- and megaspore mother cells. The tetraploid (2n = 44) of I. sinensis formed 22 bivalents and its hexaploid made 33 bivalents in both types of spore mother cells. At diakinesis and/or metaphase I of microspore mother cells in I. asiatica with 2n = 22, 11 bivalents were detected. Because behaviors of meiosis in all cytotypes mentioned above were quite regular and plants yielded normal-appearing spores, they should reproduce sexually. Aneuploids of I. japonica with 2n = 87 formed 43 bivalents and one univalent, and I. sinensis with 2n = 65 formed 32 bivalents and one univalent in microspore mother cells. Meiosis of both cytotypes was almost regular and yielded microspores of normal appearance. In the heptaploid (2n = 77) of I. japonica, a configuration of 22 bivalents and 33 univalents was detected in micro- and megaspore mother cells, and various irregularities were observed throughout the meiotic divisions. Therefore, the genomic formula of the heptaploid is symbolized as AABBCDE, and the heptaploid is a sterile F, hybrid between the hexaploid (AABBCC) and the octaploid (AABBDDEE) of I. japonica. Since diploid and even-numbered polyploids regularly formed bivalents and odd-numbered ones displayed irregularities, allopolyploidy should act as a significant speciation mechanism in this genus.     

中国水韭属两个四倍体新种

水韭属(Isoëtes)是起源最为古老的水生维管植物,全属物种均被列为国家一级重点保护植物。通过对全国水韭属植物的调查和研究发现,不同产地的四倍体植株在形态上存在显著差异。基于形态学、孢粉学和细胞学证据,将分布于中国湖南省长沙地区和怀化地区的四倍体居群分别命名为隆平水韭(Isoëtes longpingii)和湘妃水韭(I. xiangfei),并详细描述了其形态特征。隆平水韭形态上与中华水韭(I. sinensis)相似,不同之处在于其大孢子具小的瘤状或冠状纹饰,叶细长而柔弱,长达60 cm; 该种也与六倍体东方水韭(I. orientalis)相似,不同之处在于其染色体44条,大孢子具瘤状或冠状纹饰。湘妃水韭的大孢子纹饰虽与二倍体云贵水韭(I. yunguiensis)相似,但在小孢子纹饰、孢子囊形状和染色体数目方面却不同。隆平水韭仅少数植株生长于湖南省宁乡市一处池塘,完全沉水生长,而湘妃水韭则分布于怀化市通道县和会同县的湿地。由于这两个新种的分布区狭窄,野生居群数量和个体数较少,栖息地环境受到人为干扰,因此根据IUCN红色名录评估标准,将隆平水韭评为极危(CR)等级,湘妃水韭评为易危(VU)等级。所编制的中国已知水韭属物种的分种检索表,为本属物种的鉴定和保护工作提供了重要参考。     

Two new species ofIsoetes (Isoetaceae) from Jeju Island, South Korea

We name and describe two new species,Isoetes jejuensis and I.hallasanensis, from Jeju Island, South Korea. These taxa are compared with related species in East Asia, including I.coreana andI. sinensis. The rugulate megaspore ornamentation ofI. jejuensis differs from those otherIsoetes species. Furthermore,I. hallasanensis varies fromI. asiatica in its microspore ornamentation and chromosome number, although both species have an echinate megaspore ornamentation. These new species are tetraploid (2n = 44), whereasI. coreana is hexaploid (2n = 66).     

Spore morphology of Isoëtes (Isoëtaceae) from China

Micromorphological characters of mega- and microspores of five Isoëtes species from China were observed under scanning electron microscope (SEM). Megaspores of I. hypsophila have a levigate ornamentation, and are 358 µm in diameter (mean, n=30), microspores of this species have a gyrus ornamentation, and are 22 µm in length (mean, n=30); megaspores of I. yunguiensis have a cristate-reticulate ornamentation, with a size of 390 µm in diameter (mean, n=30), while microspores nearly smooth with some fine granulates, and with a size of 22 µm in length (mean, n=30); megaspores of I. taiwanensis are tuberculate and 312 µm in diameter (mean, n=30), echinate microspores are 24 µm in length (mean, n=30); cristate megaspores of I. sinensis are 409 µm in diameter (mean, n=30), and echinate microspores are 28 µm in length (mean, n=30); megaspores of I. orientalis have a cristate-reticulate ornamentation, with diameter of 420 µm (mean, n=30), microspores of this species have an echinate-tuberculate ornamentation with a size of 34 µm in length (mean, n=30). Based on the morphological characters of spores and chromosome numbers among taxa, it was found that mean spore size in each species of Isoëtes generally reflected the ploidy level. I. hypsophila, I. yunguiensis and I. taiwanensis, with smaller mean spore size, are diploidy with 2n=22; I. sinensis (2n=44) and I. orientalis (2n=66), with larger mean spore size, are polyploidy. The spore morphology of Isoëtes still plays an important role in identification and classification of Isoëtes from China.     

Ornamentation ofIsoetes (Isoetaceae, Lycophyta) microspores

More than any other taxonomic character, megaspores have been used in the genusIsoetes (known by the English common name of “quillwort”), despite the fallacy of a single-character taxonomy. Microspores, on the other hand, have been largely neglected in taxonomic schemes. Like megaspores, terms for microspore ornamentation (also known as “sculpturing”) have not been standardized. I examined microspore ornamentation, including both macroornamentation and microornamentation, of 52 taxa from Africa, Asia, Australasia, Europe, North America, and South America with the scanning electron microscope. Macroornamentation is discernible with light microscopy; microornamentation requires scanning electron microscopy. Ornately sculptured spores were much more frequent than were laevigate or psilate patterns: 21 taxa had an echinate pattern; 19 had an aculeate pattern; 6 were cristate; 5 were psilate; and 1 was laevigate. The proximal and distal ridges and surfaces may vary in both the type and density of ornamentation. Distinct macroornamentation patterns characterize certain species groups. Microornamentation types include granulate, bacillate, fimbriate, and filamentose: of the microspores I examined, virtually all were partially granulate; 11 were bacillate; 4 were fimbriate; and 1 was filamentose. Based on this limited sampling, species with a higher ploidy level often have larger microspores, but no clear relationship between microspore ornamentation and ploidy level was established, nor were any geographical or ecological trends clear. Like megaspores, microspore ornamentation is strongly convergent. Although microspores are often attached to megaspores, the role of spore ornamentation in coordinated dispersal remains unclear.     

Biosystematic studies on the genusIsoetes in Japan I. Variations of the somatic chromosome numbers

The somatic chromosome number of three Japanese species ofIsoetes, I. asiatica, I. japonica andI. sinensis, was determined in 199 individuals from 49 populations. The chromosome number ofI. asiatica was 2n=22, confirming previous reports. However,I. japonica andI. sinensis displayed a diversity in chromosome number. Six cytotypes, 2n=66, 67, 77, 87, 88 and 89, were found inI. japonica; 2n=67, 87, 88 and 89 are new counts in the genusIsoetes. The plants with 2n=66 were the most frequent (72% of total individuals examined) and were distributed throughout Honshu and Shikoku. The plants with 2n=88 occurred in western Honshu and a limited region in northeastern Honshu where the plants with 2n=77 were also found. In contrast, four cytotypes, 2n=44, 65, 66 and 68, were found inI. sinensis. The chromosome numbers ofI. sinensis were reported here for the first time. The plants with 2n=44 occurred only in Kyushu, while the plants with 2n=66 were found throughout a large area of western Japan.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Effect of ploidy variation on amino acid composition ofDatura innoxia (Solanaceae)

In haploid, diploid, triploid, tetraploid and hexaploid cytotypes of the scopalamine-yieldingDatura innoxia, the amino acid profiles were studied. The results suggest that the amino acid composition is influenced quantitatively by increasing ploidy levels, but in an irregular way.     

An experimental investigation of life history and plasticity in two cytotypes of Polygonum aviculare L. subsp. aviculare that coexist in an abandoned arable field

Polygonum aviculare subsp. aviculare is an annual selfing weed common in abandoned arable fields where it occurs as a widespread hexaploid cytotype (6x=60) and a rarer tetraploid cytotype (4x=40). The basis of phenological differentiation between the two cytotypes observed in a natural population where they coexist was examined in a greenhouse experiment comprising six soil conditions consisting of factorial combinations of two levels of fertility and three pot sizes. The environmental and genetic component of variation in 11 life history and morphological traits was quantified. Even though all traits except life span were plastic the two cytotypes appear to have evolved contrasting life history strategies and it is inferred that this can account for the temporal niche differentiation observed in the abandoned field during the first year of dereliction. Tetraploids are short-lived plants allocating a high proportion of their biomass to reproduction and completing their life cycle before July when the plant cover is sparse. Hexaploids are larger, later flowering, longer lived, plants with a lower reproductive effort and a higher final seed yield; it is inferred that these traits enable the hexaploids to compete successfully with the dense herbaceous layer of summer annuals that develops in the course of the first year of secondary succession. Differentiation in phenotypic plasticity between the two cytotypes was interpreted as indicative of higher opportunism and lower tolerance of poor soils and restricted rooting space in the hexaploid compared to the tetraploid cytotype.     

Generation of Octaploid Switchgrass by Seedling Treatment with Mitotic Inhibitors

Switchgrass (Panicum virgatum L.) exists as multiple cytotypes with octaploid (8x) and tetraploid (4x) populations occupying distinct, overlapping ranges. These cytotypes tend to show differences in adaptation, yield potential, and other characters, but the specific result of whole-genome duplication is not clear and 8x and 4x switchgrass populations are reproductively isolated with limited genetic exchange. To create new opportunities for population improvement and to study the effects of whole genome duplication on switchgrass, seedling treatment of the tetraploid cultivar Liberty with microtubule inhibitors was used to generate an octaploid population. Resulting octaploids, tetraploids, and cytochimeras were resolved by intercrossing octaploid sectors to produce a population of 19 octaploid families. Fertility of octaploid sectors was significantly reduced relative to tetraploid sectors and caryopsis size significantly increased. Cell size was significantly increased which resulted in quantitative changes to leaf anatomy. During seedling and early vegetative growth stages, no differences in vigor or tillering ability were seen. This technique resulted in efficient genome doubling and was simple to perform. However, aneuploids were also identified with both larger and smaller than expected genome sizes.     

Regeneration of fertile green plants from oat isolated microspore culture

Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set seed after its chromosome complement was doubled with colchicine.     

小花鬼针草的染色体数目及其自然多倍体类型

本文对小花鬼针草的体细胞染色体进行了观察和计数,并对其染色体倍性和有关的细胞学及分类学等问题略作讨论,旨在为药用植物的分类、品种鉴别和育种实践等工作提供必要的细胞学资料。笔者观察到的小花鬼针草的体细胞染色体除了２ｎ＝４８国外已有报道外,其中２ｎ＝２４,３６,７２,８４和９６的二倍体、三倍体、六倍体、七倍体和八倍体等多倍体系列均为首次报道,此外２ｎ＝２２,２６,４２和４７等体细胞染色体的非整倍性变异,迄今也尚未见有报道。     

Development of asparagus microspores in vivo and in vitro is influenced by gametogenic stage and cold treatment

Summary Development of asparagus microspores in cold-treated buds of varying sizes and shed microspores from these buds in in vitro culture were observed cytologically for the G459 genotype. Before cold pretreatment, more than 75% of the microspores in flower buds of the 1.4–1.6, 1.7–1.9, 2.0–2.2, 2.3–2.5, and 2.6–2.8 mm size classes were at the early-, mid-, late-uninucleate, early-, and late-binucleate stages, respectively. After 7 d in cold treatment, percentages of microspores at different stages changed in all flower buds. Most notable was the appearance of binucleate microspores resulting from symmetric rather than asymmetric division. For flower buds of 1.7–1.9, 2.0–2.2, and 2.3–2.5 mm size classes, 4.9%, 27.2%, and 11.4% of the microspores had divided symmetrically, respectively. When microspores from buds of each size category were cultured in androgenesis induction medium, only microspores completing symmetric pollen mitosis I during cold treatment were observed to divide further, and calluses were only obtained from microspores of flower bud size classes where symmetric divisions were observed after several days of cold treatment. Significant correlations existed among microspore callus yield, the percentage of late-uninucleate microspores in vivo before cold treatment, and the frequency of symmetric pollen mitosis I after 7 d of cold treatment. Consequently, asparagus microspore androgenesis may occur through one developmental pathway, where a symmetric first mitotic division is a prerequisite for continued development.     

Meiotic pairing as an indicator of genome composition in polyploid prairie cordgrass (<Emphasis Type="Italic">Spartina pectinata</Emphasis> Link)

The existence of neopolyploidy in prairie cordgrass (Spartina pectinata Link) has been documented. The neohexaploid was discovered coexisting with tetraploids in central Illinois, and has been reported to exhibit competitiveness in the natural environment. It is hypothesized that the natural tetraploid cytotype produced the hexaploid cytotype via production of unreduced gametes. Meiosis I chromosome pairing was observed in tetraploid (2n?=?4x?=?40), hexaploid (2n?=?6x?=?60), and octoploid (2n?=?8x?=?80) accessions and the percentage of meiotic abnormality was determined. Significant differences in meiotic abnormality exist between tetraploid, hexaploid, and octoploid cytotypes. An elevated incidence of abnormal, predominantly trivalent pairing in the neohexaploid suggests that it may possess homologous chromosomes in sets of three, in contrast to the tetraploid and octoploid cytotypes, which likely possess homologous chromosomes in sets of two. Abnormal chromosome pairing in the hexaploid may result in unequal allocation of chromosomes to daughter cells during later stages of meiosis. Chromosome pairing patterns in tetraploid, hexaploid, and octoploid cytotypes indicate genome compositions of AABB, AAABBB, and AABBA′A′B′B′, respectively.     

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

Cytogeography of<Emphasis Type="Italic"> Ixeris nakazonei</Emphasis> (Asteraceae,Lactuceae) in the Ryukyu Archipelago of Japan and Taiwan

The cytogeographical structures of Ixeris nakazonei, a putative hybrid between I. debilis (6x) and I. repens (2x), were investigated in the Ryukyu Archipelago and Taiwan. In the Ryukyus, I. debilis occurs on Miyakojima Island of the southern Ryukyus and northward, while I. repens occurs on all islands except for Iriomotejima and Yonagunijima Islands. I. nakazonei, comprises six polyploid cytotypes, 3x, 4x, 5x, 6x, 7x and 8x, based on x=8. Four cytotypes from 3x to 6x occur in the central Ryukyus, while four cytotypes from 5x to 8x occur in the southern Ryukyus. The higher polyploids of I. nakazonei tend to be distributed in the more southerly area. Tetraploids of I. nakazonei always co-occur with I. debilis and I. repens, supporting the hybrid origin of this cytotype. Considering the chromosome number, octoploids, which predominate in the southern Ryukyus and Taiwan, may have derived directly from hybridization between I. debilis and I. repens. Odd-numbered polyploids of I. nakazonei, 3x, 5x and 7x, are relatively rare. Their chromosome numbers indicate that triploids and heptaploids are hybrids between the tetraploid of I. nakazonei and I. repens, and between the octoploid of I. nakazonei and I. debilis, respectively. Pentaploids of I. nakazonei in the central and southern Ryukyus are, respectively, hybrids between the tetraploid of I. nakazonei and I. debilis and between the octoploid of I. nakazonei and I. repens, indicating that pentaploids of I. nakazonei have at least two independent origins.     

国产毛茛科驴蹄草属两种植物的细胞学研究

为探讨国产毛茛科(Ranunculaceae)驴蹄草属(Caltha L.)植物的细胞学特征,对驴蹄草(C.palustris L.)3个居群和花葶驴蹄草(C.scaposa Hook.f.&Thoms.)5个居群进行了细胞学研究。驴蹄草贵州纳雍居群的染色体数目为2n=32(四倍体),两个云南中甸居群的染色体数目均为2n=64(八倍体)。花葶驴蹄草四川红原、康定、石渠居群的染色体数目均为2n=32(四倍体),该数目为首次报道;西藏林芝和云南德钦居群的染色体数目均为2n=64(八倍体)。驴蹄草的染色体比花葶驴蹄草大。这两种植物的32或64条染色体分别以4条或8条为单位大致能够排列为8组同源染色体,但同一组内的染色体经常具有明显的异形性(heteromorphy),不同居群的核型组成多少具有差异。同时,还分析了驴蹄草和花葶驴蹄草的不同倍性细胞型在我国的地理分布式样。     

Evidence of widespread hybridization among couch grasses (Elymus,Poaceae)

Hybridization, polyploidization, and crop‐to‐wild gene transfer within the agriculturally important tribe Triticeae are well explored experimentally, but the true consequences of both phenomena under natural conditions remain understudied. The present paper reports on an investigation of three species of couch grasses (Elymus hispidus, E. repens, and E. caninus) examining the ploidy levels and absolute genome sizes (1081 plants from 302 natural populations in Central Europe, verified by chromosome counts) and their morphological delimitation. In the present study, the hexaploid level prevailed in E. hispidus and E. repens whereas E. caninus was exclusively tetraploid. Introgressive hybridization between hexaploid species, unidirectionally shifted towards E. hispidus, was indicated by a continual pattern of genome size values. We did not find any evidence for heteroploid hybridization involving tetraploid E. caninus; however, we detected minority cytotypes among both E. caninus plants (hexaploid) and E. repens–E. hispidus hybrids (heptaploid and nonaploid) suggesting the formation of unreduced gametes. Morphometric results (367 plants, redundancy analysis, principal component analysis, and correlation analysis) mirrored the continual homoploid pattern of absolute genome size (including the unidirectional shift), and a significant correlation between absolute genome size and morphology was confirmed. Moreover, morphometric analyses detected additional characteristics for the delimitation of the Elymus taxa under study. Considering the crossability of E. hispidus with Triticum aestivum (bread wheat), the revealed extent of introgressive hybridization has implications for assessing the potential risk of gene flow between crops and troublesome weeds.