Genetic diversity and differentiation in six species of the genus Rhinanthus (Orobanchaceae)

The intricate genus Rhinanthus L. contains many taxonomically unresolved taxa. The assessment of genetic diversity may clarify the differentiation and taxonomic relationships between species and help to determine their mating system, which is important for understanding the future prospects of populations and species. Isozymes were used to assess the mating system, genetic diversity, and differentiation in R. rumelicus, R. osiliensis, R. wagneri, R. alectorolophus, and R. angustifolius from sect. Cleistolemus and R. minor from sect. Rhinanthus. Genetic diversity at species level was found to be higher in widespread R. angustifolius and R. rumelicus compared with insular endemic R. osiliensis. Positive inbreeding coefficient revealed dominant inbreeding in endemic R. osiliensis and R. wagneri. We also detected a significant departure from Hardy–Weinberg equilibrium in some populations of R. rumelicus and R. angustifolius. The common R. alectorolophus and R. minor and several populations of R. rumelicus and R. angustifolius displayed a mixed mating mode. Six studied species divided into three distinct clusters. Rhinanthus alectorolophus was the most differentiated taxon among the investigated species. The species of sect. Cleistolemus formed two distinct groups. The first cluster consisted only of R. alectorolophus while another comprised R. rumelicus, R. osiliensis, R. wagneri, and R. angustifolius, indicating their close affinity. Thus, our isozyme results call into question the monophyly of section Cleistolemus and the taxonomic position of R. alectorolophus.     

Analysis of Genetic Variation in Rare Endemic Species Oxytropis chankaensis Jurtz. (Fabaceae) Using RAPD Markers

The method of polymerase chain reaction with random primers (RAPD) was used to assess genetic variation and population differentiation in the rare endemic plant Oxytropis chankaensis Jurtz. (Fabaceae). DNA samples from plants of two isolated populations were compared at 133 loci detected by use of ten primers. Both populations examined were characterized by high polymorphism levels (P ₉₅ = 72.9%; A = 1.92 and P ₉₅ = 74.4%; A = 1.88, respectively). They were also statistically significantly different in the frequencies of most of the amplicons. For each of the plants, unique multilocus RAPD phenotype was established using 17 to 20 RAPD markers. Diagnostic markers were not revealed. The populations were poorly differentiated. On average, the between-population component accounted for about 8% of the variation, while 92% of the variation was detected within populations. High variation along with the low degree of differentiation characteristic of two most geographically remote populations of O. chankaensis can have several explanations, among which a polyploid origin of the species seems to be most important.     

Ecotypes and genetic structure of Rhinanthus alectorolophus (Orobanchaceae) in southwestern Germany

European annual species of the genus Rhinanthus often exhibit seasonal ecotypic variation, a phenomenon also known from related genera of hemiparasitic Orobanchaceae. Populations with different flowering times exist, correlated with differences in a number of morphological characters. The present study evaluates the correlation of morphological characters and genetic differentiation of populations of Rhinanthus alectorolophus. Thirty-nine populations of three different subspecies from southwestern Germany were sampled. A total of 798 individuals were used for morphological analyses and 187 of these for AFLP analyses. Principal component analysis showed that morphological variation is mostly continuous. In a discriminant analysis based on morphological characters, only 89.7 % of all individuals were correctly assigned to their previously determined subspecies, indicating that subspecies identification is ambiguous for some populations. Using AFLP data and Bayesian assignment analysis, the sampled individuals could be grouped in three genetic clusters which do not correspond to the three subspecies. Instead, the clustering shows a clear geographic pattern and a Mantel test likewise revealed a significant correlation between genetic and geographic distances. Correlations of genetic distances with differences in morphological characters were weak and mostly insignificant. The results indicate that the subspecies of R. alectorolophus do not form discrete entities and that the character combinations distinguishing them are homoplastic.     

Seed Germination Biology of the Narrowly Endemic Species Lesquerella stonensis (Brassicaceae)

Abstract Lesquerella stonensis (Brassicaceae) is an obligate winter annual endemic to a small portion of Rutherford County in the Central Basin of Tennessee, where it grows in disturbed habitats. This species forms a persistent seed bank, and seeds remain viable in the soil for at least 6 years. Seeds are dormant at maturity in May and are dispersed as soon as they ripen. Some of the seeds produced in the current year, as well as some of those in the persistent seed bank, afterripen during late spring and summer; others do not afterripen and thus remain dormant. Seeds require actual or simulated spring/summer temperatures to come out of dormancy. Germination occurs in September and October. Fully afterripened seeds germinate over a wide range of thermoperiods (15/6–35/20°C) and to a much higher percentage in light (14 h photoperiod) than in darkness. The optimum daily thermoperiod for germination was 30/15°C. Nondormant seeds that do not germinate in autumn are induced back into dormancy (secondary dormancy) by low temperatures (e.g., 5°C) during winter, and those that are dormant do not afterripen; thus seeds cannot germinate in spring. These seed dormancy/ germination characteristics of L. stonensis do not differ from those reported for some geographically widespread, weedy species of winter annuals and thus do not help account for the narrow endemism of this species.     

濒危物种裂叶沙参及其近缘广布种泡沙参的遗传多样性研究

根据对１２个形态性状的统计分析和１０个基因位点的等位酶检测,探讨了濒危植物裂叶沙参及其近缘广布种泡沙参的遗传多样性水平,对３个裂叶沙参和６个泡沙参天然群体的遗传分析表明,两种沙参属植物均具有很高的遗传变异水平,这种变异性既体现在形态学水平上,也体现在酶位点水平上,在２个茎叶形状以及１０个花果和种子形状上,濒危种裂叶沙参的变异性均与广布种相当,同样,根据７个酶系统１０个等位酶位点的度量,裂叶沙参群体     

Heterotrophic carbon gain by the root hemiparasites, Rhinanthus minor and Euphrasia rostkoviana (Orobanchaceae)

Hemiparasitic plants gain virtually all mineral nutrients and water from their host plant whilst organic carbon is provided, at least in part, by their own photosynthetic activity, although their rates of assimilation are substantially lower than that found in non-parasitic plants. Hence, hemiparasites must gain at least some of their organic carbon heterotrophically from the host plant. Despite this, heterotrophic carbon gain by root hemiparasites has been investigated only for a few genera. We investigated heterotrophic carbon gain by two root hemiparasites, Rhinanthus minor L. and Euphrasia rostkoviana Hayne (Orobanchaceae), using natural abundance stable isotope (δ¹³C) profiles of both parasites attached to C₃ (wheat) and C₄ (maize) hosts coupled to a linear two-source isotope-mixing model to estimate the percentage of carbon in the parasite that was derived from the host. Both R. minor and E. rostkoviana attached to maize hosts were significantly more enriched in ¹³C than those attached to wheat hosts with R. minor becoming more enriched in ¹³C than E. rostkoviana. The natural abundance ¹³C profiles of both parasites were not significantly different from their wheat hosts, but were less enriched in ¹³C than maize hosts. Using a linear two-source isotope-mixing model, we estimated that R. minor and E. rostkoviana adult plants derive c. 50 and 25% of their carbon from their hosts, respectively. In light of these results, we hypothesise that repeatedly observed negative effect of competition for light on hemiparasites acts predominantly in early ontogenetic stages when parasites grow unattached or the abstraction of host nutrients is less effective.     

Low Genetic Diversity in the Rare Madeiran Endemic Armeria maderensis (Plumbaginaceae)

The genetic diversity and possible geographic structure of the Madeiran endemic Armeria maderensis have been assessed with AFLP. Its scarce distribution (less than 3 km between the two most distant localities) and restricted habitat (vertical pastures on the highest elevations of Madeira), at least in part due to grazing by goats, suggest an assessment of its conservation status. Diversity estimates obtained for A. maderensis were evaluated through comparison with reference values of AFLP diversity for outcrossing plants and, in order to correct for phylogenetic constraints, with a widespread congener analyzed with the same AFLP markers. Our results reveal that low levels of genetic diversity and a weak intraspecific genetic structure underlie the restricted distribution of A. maderensis.     

珍稀濒危植物距瓣尾囊草种子散布及萌发特性

采用野外调查和室内实验相结合的方法,对距瓣尾囊草(Urophysa rockii)种子散布途径、散布方式和种子萌发特性进行初步研究.结果表明:距瓣尾囊草种子千粒重为0.6684±0.0038 g,以果实和种子为扩散单元进行扩散,具有自体扩散和水媒扩散两种方式.温度对距瓣尾囊草种子萌发有显著影响,20℃为最适萌发温度,发芽率达67.78％.低温(4℃)层积可有效提高种子发芽率和发芽势,随层积天数增加,发芽率和发芽势增加,但层积30 d和40 d发芽率和发芽势无显著性差异.光照和黑暗对种子萌发的影响差异不显著,光照不是其种子萌发的必需条件.实验证明,与用湿砂做发芽基质相比,用滤纸做发芽基质,其发芽率和发芽势均比前者高,因此滤纸是距瓣尾囊草种子萌发实验中更为适宜的发芽基质.     

Seed morphological diversity of Pedicularis (Orobanchaceae) and its taxonomic significance

Mature seeds of 109 species of Pedicularis representing 13 groups according to Tsoong’s infra-generic classification were investigated using light microscopy (LM) and scanning electron microscopy (SEM). The largest seeds occurred in P. superba (4.82 ± 0.32 mm × 2.18 ± 0.18 mm) and the smallest in P. crenata (0.95 ± 0.16 mm × 0.72 ± 0.08 mm). The individual seed weight ranged from 0.20 to 9.52 mg. There were two types of primary ornamentation, i.e. reticulate and undulate. Reticulate seeds were found in all examined species except for P. pantlingii and P. confluens. The reticulate seeds were divided into three subtypes, i.e. cristate-reticulate, regular-reticulate and membranous-reticulate. Among them, the cristate-reticulate and the undulate primary ornamentation are firstly reported. The inner tangential wall ornamentation was categorized into granulate, ruminate or striate. Seed characters seem to support the transfer of Pedicularis to Orobanchaceae from traditional Scrophulariaceae. Seed length, individual seed weight, primary ornamentation and inner tangential wall ornamentation were considered as potentially useful characters for infra-generic classification. Non-metric multidimensional scaling (NMDS) analyses indicated a clustering of some species in group 1 (Sceptrum) and all species in group 6 (Cyathophora), respectively. However, seed data were not highly congruent with Tsoong’s classification for delimitation of existing groups. Some characters, such as seed shape, primary ornamentation, inner tangential wall ornamentation and epidermal cell shape could be applied to identification of some species in this genus.     

Germination and development of the soma in Boschniakia hookeri (Orobanchaceae)

Germination of the holoparasitic angiosperm Boschniakia hookeri differs from the usual pattern among the Orobanchaceae. In most members of the family, the germinating seed develops a radix which establishes contact with the host root; in contrast, in B. hookeri the host root makes contact with the parasite seed, penetrates its testa, and association between host root and parasite embryo tissues is then established inside the testa.
The germination and development of the perennial vegetative body, the soma, can be divided into ten stages, with characteristic changes from stage to stage. The first four stages range from the pre–germination through the germination proper, including physical contact between parasite and host root; formation of the soma, through development of the first stem bud, occurs in stages five to ten. The processes for the first six stages occur completely within the testa.
The mature soma apparently produces flowers when it reaches a size around 15 mm in diameter and continues to grow. Maximum seems to be around 50–60 mm, where dense clusters of 10–12 inflorescences can occur.     

Genetic Structure and Conservation Considerations of Rare Endemic Abeliophyllum distichum Nakai (Oleaceae) in Korea

Abeliophyllum distichum , was assessed using starch gel electrophoresis. Although A. distichum maintained relatively high genetic diversity probably due to floral heteromorphism and preferred outcrossing, their genetic variation, where small effective size of population and genetic drift are of utmost importance, was lower than the other outbreeding plants. The calculated G_sT indicated a high level of genetic differentiation among populations, suggesting that gene flow was limited. The dendrogram based on Nei's genetic distance showed that the Pu-an and the Koe-san populations fell into two distinct groups, which were consistent with geographic distance and distribution. It was probable that in the populations of A. distichum sexually reproduced individuals rarely become established within the populations. From observations, Lassioglossum bee was found to be the most effective pollinator of A. distichum. Artificial crossing experiment indicated that pollinators contributed considerably to natural population and that pollinator limitation could potentially reduce fitness of A. distichum in wild populations. The management of rare plant species should include improvement of genetic exchanges among populations by artificial gene flow. From each of the four subpopulations, fresh twigs from 20 trees should be collected and propagated as the cutting for ex situ conservation. Received 30 April 1999/ Accepted in revised form 23 January 2000     

Comparative Phylogeography of the Atlantic Forest Endemic Sloth (Bradypus torquatus) and the Widespread Three-toed Sloth (Bradypus variegatus) (Bradypodidae, Xenarthra)

The comparative phylogeographic study of the maned sloth (Bradypus torquatus) and the three-toed sloth (Bradypus variegatus) was performed using a segment of mitochondrial DNA (mtDNA) control region. We examined 19 B. torquatus from two regions and 47 B. variegatus from three distant regions of Atlantic forest. This first characterization of molecular diversity indicates a great diversity (B. torquatus: h = 0.901 ± 0.039 and π = 0.012 ± 0.007; B. variegatus: h = 0.699 ± 0.039 and π = 0.010 ± 0.006) and very divergent mitochondrial lineages within each sloth species. The different sampled regions carry distinct and non-overlapping sets of mtDNA haplotypes and are genetically divergent. This phylogeographic pattern may be characteristic of sloth species. In addition, we infer that two main phylogeographic groups exist in the Atlantic forest representing a north and south distinct divergence.     

一种简易的研究寄主中国柽柳刺激管花肉苁蓉种子萌发的技术

以组织培养方式建立了寄主中国柽柳的离体快速繁殖体系.用溶有200 mg·L-1亚甲基蓝指示剂的MS培养基研究指示剂与寄主试管苗根分泌物反应的显示,亚甲基蓝在有寄主根生成的培养基中褪色,而在未生根的培养基中仍为蓝色,说明管花肉苁蓉的种子萌发刺激物质为还原性的氢醌类物质.     

RAPD Profiling in Detecting Genetic Variation in Endemic Coelonema (Brassicaceae) of Qinghai-Tibet Plateau of China

Random amplified polymorphic DNA (RAPD) markers were used to measure genetic diversity of Coelonema draboides (Brassicaceae), a genus endemic to the Qilian Mountains of the Qinghai-Tibet Plateau. We sampled 90 individuals in 30 populations of Coelonema draboides from Datong and Huzhu counties of Qinghai Province in P.R. China. A total of 186 amplified bands were scored from the 14 RAPD primers, with a mean of 13.3 amplified bands per primer, and 87% (161 bands) polymorphic bands (PPB) was found. Analysis of molecular variance (AMOVA) shows that a large proportion of genetic variation (84.2%) resides among individuals within populations, while only 15.8% resides among populations. The species shows higher genetic diversity between individuals than other endemic and endangered plants. The RAPDs provide a useful tool for assessing genetic diversity of rare, endemic species and for resolving relationships among populations. The results show that the genetic diversity of this species is high, possibly allowing it to adapt more easily to environmental variations. The main factor responsible for the high level of differentiation within populations and the low level of diversity among populations is probably the outcrossing and long-lived nature of this species. Some long-distance dispersal, even among far separated populations, is also a crucial determinant for the pattern of genetic variation in the species. This distributive pattern of genetic variation of C. draboides populations provides important baseline data for conservation and collection strategies for the species. It is suggested that only populations in different habitats should be studied and protected, not all populations, so as to retain as much genetic diversity as possible.     

青阳参种子的萌发

青阳参（Cynanchum otophyllum）种子在11月成熟时有休眠习性。收获后将其种子种植在自然温室内,到第二年的春天种子才会萌发,且大多数种子在3月28至4月4日间萌发,这期间的日平均最高和最低温度分别为19．0℃和9．9℃。层积能有效地打破青阳参种子的休眠,休眠种子通过大约1周的层积便能萌发。种子在有光的条件下层积1周后转移到25／15℃的黑暗条件下萌发率可达到75．4％。青阳参种子不论在有光的条件下还是在黑暗环境中层积2～3周后转入30／20和25／15℃进行变温处理,其萌发率最低能达到66．4％,而转入20／10℃变温处理其萌发率最多只能达到20．1％,但若层积6周,即便在20／10℃变温处理的情况下其萌发率也可以达到65．3％以上。     

Genetic Variation in the Endangered Endemic Species Cycas fairylakea (Cycadaceae) in China and Implications for Conservation

Cycas fairylakea is an endangered endemic species in China. Genetic diversity within and among four natural populations of this species in China was investigated using amplified fragments length polymorphism (AFLP). A moderate to low level of intraspecific genetic diversity was detected in this species (at population level: P = 39.57 %, H₀ = 0.244; at species level: P = 60.22%, H₀ = 0.356). The among-population component accounted for, respectively, 25.7 and 31.5% of the genetic variation, according to AMOVA and Shannon’s index, indicating most of the genetic variation was found between individuals within populations. All four populations have opposite pyramid age structure, and few coning individuals, which is still decreasing. Possibly because of habitat degradation and environmental pollution, plant diseases and insect pests in the populations were extremely serious, suggesting that the main factors threatening the survival of C. fairylakea populations were not genetic variation, but human activities and the breeding system of this species.     

Seed ultrastructure and water absorption pathway of the root-parasitic plant Phelipanche aegyptiaca (Orobanchaceae)

Background and Aims

Obligate root parasitic plants of the Orobanchaceae do not germinate unless they chemically detect a host plant nearby. Members of this family, like Orobanche, Phelipanche and Striga, are noxious weeds that cause heavy damage to agriculture. In spite of their economic impact, only a few light microscopical studies of their minute seeds have been published, and there is no knowledge of their ultrastructure and of the role each tissue plays during the steps preceding germination. This paper describes the ultrastructure of Phelipanche seeds and contributes to our understanding of seed tissue function.

Methods

Seeds of P. aegyptiaca were examined under light, scanning electron, transmission electron and fluorescence microscopy following various fixations and staining protocols. The results were interpreted with physiological data regarding mode of water absorption and germination stimulation.

Key Results and Conclusions

The endothelium, which is the inner layer of the testa, rapidly absorbs water. Its interconnected cells are filled with mucilage and contain labyrinthine walls, facilitating water accumulation for germination that starts after receiving germination stimuli. Swelling of the endothelium leads to opening of the micropyle. The perisperm cells underneath this opening mediate between the rhizosphere and the embryo and are likely to be the location for the receptors of germination stimuli. The other perisperm cells are loaded with lipids and protein bodies, as are the endosperm and parts of the embryo. In the endosperm, the oil bodies fuse with each other while they are intact in the embryo and perisperm. Plasmodesmata connect the perisperm cells to each other, and the cells near the micropyle tightly surround the emerging seedling. These perisperm cells, and also the proximal embryo cells, have dense cytoplasmic contents, and they seem to represent the two seed components that are actively involved in transfer of reserve nutrients to the developing seedling during germination.     

Allozyme Variation and the Genetic Structure of Populations of the Rare Sedge Carex misera (Cyperaceae)

Abstract Carex misera is a rare sedge, endemic to rocky outcrops and mountain summits within the southern Appalachian Mountains from northern Georgia to northern North Carolina. We assessed allozyme diversity for 406 individuals from nine populations over most of the geographic range. Twenty-seven putative loci were assayed and eight (30%) were found to be polymorphic. Nei's gene diversity statistics ( H _T =0.043, H _S =0.019, G _ST =0.551) indicated low levels of variation but relatively highly differentiated populations, suggesting little gene flow. Significant deviations from genotypic expectations under Hardy-Weinberg equilibrium, high positive fixation indices, and the existence of small genetic neighborhoods within populations suggest that at least some inbreeding occurs. Cluster analysis of Nei's genetic identity statistics and principal component analysis of allele frequency data showed high similarity among the six southern populations with the two northern populations more differentiated from them and from each other. These results suggest that preservation of the northern populations is necessary to conserve the already low levels of genetic diversity within the species.     

寄生药用植物管花肉苁蓉种子的离体萌发与吸器形成的形态学研究

研究了管花肉苁蓉(Cistanche tubulosa(Schenk)R.Wight)种子脱离寄主条件下的离体萌发和吸器形成的形态学,为其高产栽培提供参考.将经过层积处理的种子浸泡于赤霉素(1000 mg·L-1)36小时,然后培养于1/2 MS(加500mg·L-1酵母提取物、250 mg·L-1氯化钠和100mg·L-1甘露醇,pH5.83)培养基上.培养两周后种子开始萌动,首先胚根从软化的内种皮的种孔端伸出,接着形成芽管状物,之后芽管状物逐渐伸长;17天后初生吸器开始发育,厚的带状表皮分化出若干吸根毛;在初生吸器顶端带状表皮围成窝状,表皮上的褶皱和窝内分布有乳头状突起.芽管状物延伸至4mm就停止发育.吸根毛和乳突是黏着寄主根、刺穿寄主皮层发育为内生吸器最终建立寄生关系的基础.