Dimorphism and Monomorphism in the Plumbaginaceae: III. Correlation of Geographical Distribution Patterns with Dimorphism and Monomorphism in Limonium

In the normally self-incompatible Limomum, as in the relatedgenus Armeria, there appears to be an important positive correlationbetween establishment after long-distance dispersal and self-compatibility.Thus a seed-reproducing colony may be produced in a self-compatiblespecies even though only a single disseminule overcomes thehazards of long-distance dispersal. With this in mind, the distributionand possible migratory routes of various sections of the genusare traced from the Old World. Particular attention is paidto Atlantic and amphi-Atlantic distributions (in relation toboth ‘Continental Drift’ and ‘Permanent Continent’theories of recent geological history) and also to the specialproblems of Limonium in Australia.     

The Limonium binervosum aggregate (Plumbaginaceae) in the British Isles

A taxometric analysis of 153 colonies of the agamospermous Limonium binervosum aggregate in the British Isles has revealed an ordered hierarchy of variation. A taxonomic treatment of the group describes five new species, redefines four others and describes other infraspecific variants as subspecies or varieties. Each species is morphologically distinct and has a defined geographic range. There are four widespread and morphologically variable species and five geographically restricted species (four monotypic). Limonium recurvum , previously described as a microspecies endemic to a small area of the Dorset Coast, has been widened to include all triploid (2 n = 27) L. binervosum agg. variants found also in W and N Ireland, SW Scotland and Cumbria. All other taxa are tetraploid, usually with 2 n = 35.     

Pattern of variation of agamospermous Limonium (Plumbaginaceae) in the British Isles

An investigation of cytological, palynological, and taxometric variation in Limonium binervosum (G. E. Sm.) C. E. Salmon has revealed an extensive pattern of hierarchical relationships within this agamospermous group in the British Isles. The group is either triploid (2n = 27) or aneuploid tetraploid (usually 2n = 35) with a highly jumbled karyotype. Pollen is either not produced or of very low stainability (in the range 0–38%). Well stained pollen grains are usually abnormally large resulting from a failure of a meiotic division. These facts as well as the presence of only a single self-incompatible polledstigma morph in the majority of the group suggest that it is obligately agamospermic. The very strong correlation of taxometric relationship and geographical distance between variants suggests that evolution in the group has occurred by the gradual accumulation of mutations giving rise to morphologically and geographically related seed clones. The hierarchical pattern of variation as revealed by cluster analysis has been used as the basis of a hierarchical classification of the group.     

Molecular phylogenetics of Limonium and related genera (Plumbaginaceae): biogeographical and systematic implications

Phylogenetic relationships within Limonium (Plumbaginaceae) are evaluated using sequence data from three plastid regions (rbcL, the trnL intron, and the trnL-trnF intergenic spacer). Sixty-six species representing the major genera of Staticoideae, including representatives of all sections and genera formerly included in Limonium, have been analyzed using four species of Plumbaginoideae as an outgroup. Analyses of each separate and combined data set yield similar results. Afrolimon is embedded in Limonium and related to L. vulgare, the type of Limonium. Limonium is split into two major clades corresponding to subgenera, but otherwise the current infrageneric classification proved to be artificial. Some groups restricted to particular areas can be recognized, and their synapomorphies are discussed. The presence of an isolated taxon in the Canary Islands is used as a calibration point for age estimates of the major events in the genus, including migrations to the Southern Hemisphere, the Canary Islands, and Asia. The rapid radiation of Limonium in the Mediterranean basin appears to coincide with the desiccation of the Mediterranean Sea in the Messinian (late Miocene).     

Biosystematic study of the genus Limonium (Plumbaginaceae) in the Aegean area, Greece. EO. Limonium on the islands Kithira and Antikithira and the surrounding islets

The genus Limonium is represented on Kithira, Antikithira and the surrounding islets by the following nine species: L. aphroditae and L. cythereum , recently described from Kithira; L. runemarkii and L. ocymifolium , endemic to Greece; L. graecum and L. sieberi , distributed in E Mediterranean; L. virgatum, L. sinuatum and L. echioides , having a wider Mediterranean distribution. For L. runemarkii , considered so far as endemic to SE Ewia, a new extended distribution range is given from Ewia to NW Kriti, through Peloponnisos and Kithira, showing the phytogeographic relationship of these areas. Moreover, the treatment of L. pigadiense as synonym of L. ocymifolium is proposed. Key and diagnostic characters for the species studied are given, while their affinities to related Aegean and Mediterranean Limonium taxa are discussed. Data on the cytology and breeding system are given. The following chromosome numbers were found: 2n=3x=27 for L. aphroditae and L. virgatum , 2n=6x=52 for L. cythereum , 2n=6x=51 for L. sieberi (firstly reported), 2n=5x=43 for L. runemarkii (firstly reported), L. ocymifolium and L. graecum , 2n=2x=16 for L. sinuatum and 2n=2x=18 for L. echoides. Concerning pollen-stigma combination, L. aphroditae, L. ocymifolium and L. virgatum are monomorphic with the self-incompatible combination B, while L. cythereum and L. runemarkii are also monomorphic with the self-incompatible combination A. According to these data the above species can be considered as apomictic. The facultative apomictic L. graecum was found monomorphic with the combination A, while L. sieberi , has the combination A or B within different populations. The autogamous species L. echioides has the self-compatible combination C, while the allogamous L. sinuatum is dimorphic having both combinations A and B in each population.     

Pollen Dimorphism and Androgenesis of Paeonia in Vivo

It was observed that anthers of Paeonia lactiflora cultivar “Da-Fu-Gui” produced dimorphic pollen grains, i.e. the normal pollen garins and anomalous pollen grains, frequencies of which were 75.8% and 24.2% respectively. Some of the anomalous pollen grains carried out extra divisions (in vivo) and so the multinueleat or multicellular pollen grains were formed. It seem to indicate that there are several different pathways including asymmetric and symmetric divisions and fusion of the nuclei in the androgenesis.     

Molecular and morphological differentiation in Limonium dufourii (Plumbaginaceae), an endangered Mediterranean plant

We have analyzed the morphological andmolecular variation in individuals from aLimonium dufourii population in which we hadpreviously described the presence of twomarkedly different molecular haplotypes bymeans of RAPDs and AFLPs. Ten differentmorphological variables were scored in each of72 individuals and their molecular haplotypegroup was established by RAPD analysis. Thevariation observed in the 10 morphometricvariables was explained by four dimensions in aprincipal components analysis, and a plot ofeach individual in the plane defined by the twofirst dimensions did not show any significantgrouping until the molecular haplotype wasincorporated into the plot. A discriminantanalysis performed using the molecularhaplotype as the grouping variable resulted in88.9% of correctly classified cases, thusreflecting a high correlation betweenmorphometric and molecular variation in theseindividuals. We discuss the relevance of thiscorrelation for the conservation strategypreviously proposed for this species.     

beta-Alanine betaine synthesis in the Plumbaginaceae. Purification and characterization of a trifunctional, S-adenosyl-L-methionine-dependent N-methyltransferase from Limonium latifolium leaves.

beta-Alanine (beta-Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, beta-Ala betaine is synthesized by S-adenosyl-L-methionine-dependent N-methylation of beta-Ala via N-methyl beta-Ala and N,N-dimethyl beta-Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of beta-Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent K(m) of 45 microM S-adenosyl-l-methionine and substrate inhibition was observed above 200 microM. The apparent K(m) values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mM for beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-hydroxymercuribenzoic acid.     

Lack of genetic variability in the rare and endangered Limonium cavanillesii (Plumbaginaceae) using RAPD markers

Limonium cavanillesii is an extremely endangered plant species endemic to the east Mediterranean region of Spain. Regarded as extinct for several years, the recent discovery of a small population (only 29 individuals) has prompted the adoption of measures for its conservation by official agencies. As part of this effort, we have analysed genetic variation in this population by means of random amplified polymorphic DNA (RAPDs). The analysis of 29 individuals with 11 different primers produced 131 monomorphic bands. To our knowledge, this is the lowest level of genetic variation detected in plants using RAPD markers. This result could be explained both by the apomictic reproductive system of this species and by the passage through a severe bottleneck in recent times, after which there has been no chance for mutation to restore detectable genetic variation.     

Karyology of Limonium (Plumbaginaceae) species from the Balearic Islands and the western Iberian Peninsula

Somatic chromosome numbers, conventional karyotype features and idiograms are reported for 27 Limonium species inhabiting the Western Mediterranean basin (Iberian Peninsula and the Balearic Islands). The chromosome numbers of Limonium barceloi (2 n  = 36), L. ejulabilis (2 n  = 24), L. inexpectans (2 n  = 26), L. interjectum (2 n  = 24), and L. scopulorum (2 n  = 25) were determined for the first time. In addition, new aneuploid and/or polyploid cytotypes are reported in L. alcudianum (2 n  = 26), L. bonafei (2 n  = 26), L. camposanum (2 n  = 26) , L. companyonis (2 n  = 26), L. dufourii (2 n  = 26), L. geronense (2 n  = 36), L. marisolii (2n = 54), L. migjornense (2 n  = 50), and L. pseudodictyocladon (2 n  = 16). A group of polyploid species showed karyotypes comprising homologous chromosomes in groups of three ( L. antonii-llorensii, L. ejulabilis, L. interjectum, L. virgatum, and L. wiedmanii ) , four ( L. geronense ), or six ( L. marisolii ), which suggests an autopolyploid origin. Other polyploid species were characterized by the presence of two different chromosome sets ( x  = 8 and x  = 9) in the genome. The species L. alcudianum , L . bonafei , L. camposanum , L. companyonis , L. dufourii , L. gibertii , L. girardianum , L. inexpectans , L. leonardi-llorensii , L. magallufianum , L. migjornense , L. minoricense , and L. scopulorum showed various combinations of paired and unpaired x  = 8 and x  = 9 chromosome sets, suggesting that they are allopolyploids. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 257–272.     

异型花柱植物喀什补血草的传粉者功能群与花粉转移效率

异型花柱是受遗传控制的花柱多态现象, 被达尔文认为是植物通过在传粉者体表不同部位滞落花粉以促进型间花粉准确传递的一种适应。该现象虽已受到广泛关注, 但在一些花型变异较大且不稳定的传粉系统中, 不同传粉者对各花型繁殖所产生的影响仍知之甚少。该研究以分布于新疆天山南坡的一个有同长花柱共存的异型花柱植物喀什补血草(Limonium kaschgaricum)种群为研究对象, 对其花型构成及频率、传粉者及花粉转移效率等进行了调查分析。结果表明: 1)种群中除了存在雌/雄蕊长度交互对应的长(L)/短(S)花柱型花外, 还有雌/雄蕊同长的花(H型), 且各花型花的花冠口直径、花冠筒长及花粉量等参数间无差异, 但花粉纹饰和柱头乳突细胞形态具二型性。其中, H型花的花粉和柱头形态与L型花(或S型花)的一致。2)花型内和自花授粉均不亲和; 型间授粉时, 花粉和柱头形态不同的花型间亲和, 反之不亲和。3)种群内存在长/短吻两类传粉昆虫。在以短吻传粉者为主的盛花初、中期, L和H型花柱头上的异型花粉数均显著高于S型花的, 且L和S型花高位性器官间的异型花粉传递效率高于低位性器官间的; 而在以长吻传粉者为主的盛花后期, L和S型花的柱头间异型花粉数无显著差异, 且高/低位性器官间具有相同的异型花粉转移效率; 与传粉者出现时期相对应的、在花期不同阶段开放花的结实率也明显不同。4)长/短吻昆虫具明显不同的传粉功能, 短吻昆虫只能对L和H型花进行有效传粉, 且访花频率和型间花粉转移效率较低, 为低效传粉者; 而长吻昆虫对各花型均能有效传粉, 具高的访花频率和型间花粉转移效率, 为高效传粉者。因为长吻昆虫的阶段性出现所形成的不稳定传粉系统, 使低效的短吻昆虫可能会成为种群中花型变异的驱动力, 并使S型花受到更大的选择压力。H型花克服了柱头缩入的弊端, 可能会成为不稳定传粉系统下的一个替代花型而持续存在。     

lntrageneric Variation of Pollen Types in the Genus Phyllanthus L.

Basing on the pollen morphology of 22 Asian species, 17 from China, of PhylIanthus, 10 pollen types are recognized and described. Transformations of pollen type in this genus from a tricolporate one to a peripor(or) ste one are in succession and through 3 different pathways. It is interesting enough that the “successiformy” and “spiralizaton” pattern are all occurred.     

Phylogenetic relationship among related genera of Plumbaginaceae and preliminary genetic diversity of Limonium sinense in China

Phylogenetic relationship of Limonium and other genera of Plumbaginaceae in China were studied using the cp rbcL, matK and the intergene spacer of trnL-trnF. The analysis showed that Plumbaginaceae was strongly supported monophyletic group sister to Polygonacea, and two tribes were comfirmed by phylogenetic analysis in Plumbaginaceae. Preliminary genetic diversity of Limonium sinense in China was also analyzed in this study by nrDNA (ITS) and cp DNA (two regions of intergenic spacers, trnL-trnF and psbA-trnH). The results showed that the population genetic diversity was low perhaps for human activities and breeding system of this species. These results have been used to understand the evolutionary and demographic history of L. sinense, which is a requisite to establish efficient conservation measures for this species.     

国产省藤属植物的花粉形态学

对国产棕榈科省藤属(Calamius L.)15种植物的花粉进行了光学和扫描电镜观察,其中12种为首次报道。省藤属的花粉均为两沟型花粉,外壁覆盖层多为网状纹饰(大喙省藤C．macrorrhynchus)或具穿孔(华南省藤C.rhabdocladus)。首次发现省藤属花粉的外壁纹饰存在穿孔和外壁疣状突起的类型(阔叶鸡藤C.pulchellus)以及皱波状突起的类型(长鞭省藤C.flagellum)。花粉的大小、形状、外壁纹饰、外壁是否有突起,外壁厚度和网状纹饰网眼的大小,对于省藤属的种级分类有较大的意义。     

Study of the evolutionary relationships among Limonium species (Plumbaginaceae) using nuclear and cytoplasmic molecular markers

The genus Limonium, due to the patchiness of the natural habitats of its species as well as the high frequency of hybridization and polyploidy and the possibility of reproduction by apomixis, provides an example of all the principal mechanisms of rapid speciation of plants. As an initial study of evolution in this genus, we have analyzed intra- and interspecific variability in 17 species from section Limonium, the largest in the genus, based on RFLPs of cpDNA and nuclear rDNA ITS sequences. In the cpDNA analysis, 21 restriction enzymes were used, resulting in 779 fragments, 490 of which were variable and 339 parsimony informative. L. furfuraceum exhibited two relatively divergent cpDNA haplotypes. The relationships found among the species based on cpDNA restriction fragments were coincident using different methods of phylogenetic analysis. Due to the presumed reticulate evolution in the genus Limonium, the comparison of these results with data from the nuclear DNA was necessary; ITS sequences were analyzed. The final alignment contained 488 characters, of which 198 were variable and 156 parsimony informative. Two relatively divergent ITS types were present at the intraindividual level in L. delicatulum, a triploid species. Each type was related to ITS from different groups of diploid Limonium species, one with a base haploid chromosome number n = 8 (represented by L. cossonianum) and the other with n = 9 (represented by L. minutum). The different phylogenetic inference methods used for the analysis of ITS sequences rendered very similar topologies. In general, the relationships among the species studied were coincident with those obtained with the chloroplast genome. Both nuclear and cytoplasmic markers support the polyphyly of section Limonium, with at least two species, L. narbonense and L. vulgare, clearly divergent from the rest. Moreover, the remaining subsections into which section Limonium is currently divided seem to be artificial.     

Historical and biological determinants of genetic diversity in the highly endemic triploid sea lavender Limonium dufourii (Plumbaginaceae)

Microsatellite markers were used to evaluate the genetic diversity and population genetic structure in the critically endangered Limonium dufourii (Plumbaginaceae), a highly endemic triploid species from the coasts of eastern Spain. Sixty-five alleles from 13 microsatellite regions were amplified in a sample of 122 individuals collected from the six extant populations. Microsatellite patterns were consistent with the triploid nature of L. dufourii. Alleles were unambiguously assigned to two different parental subgenomes in this hybrid species and the greater contribution of the diploid parental subgenome was confirmed. Eleven, 25 and 26 multilocus genotypes were recorded from the haploid, diploid and from the combined information of both subgenomes, respectively. Genetic diversity was mostly distributed among populations (72.06% of the total genetic variation). Genotypes from Marjal del Moro populations grouped into two highly structured clusters (88.41% of the total variance). The observed patterns of distribution of genetic diversity are interpreted to result from multiple hybridization events and isolation between populations. Threats to this species are mainly anthropogenic (urbanization and tourism pressure), although stochastic risks cannot be ignored. Therefore, in order to preserve extant genetic variation of L. dufourii, in situ strategies such as the preservation of its habitat are a high priority. Several recommendations in order to assist ex situ measures to guarantee the success of conservation strategies and maintain the relationships between individuals and populations are proposed.     

Analysis of population genetic structure and variability using RAPD markers in the endemic and endangered Limonium dufourii (Plumbaginaceae)

Limonium dufourii (Plumbaginaceae) is a triploid species, with apomictic reproduction, endemic to the east mediterranean coast of Spain, where it is present in only six populations with a few individuals in most of them. L. dufourii is included in the Red List of Endangered Species by the IUCN. Genetic variation and population structure in this species has been studied using RAPDs. Twelve different primers provided 124 reliable bands of which 33 were polymorphic among the 165 individuals analysed. Those polymorphic bands were able to define 44 different patterns, of which all but six were present in only one population. Several methods for statistical evaluation have been used for intra- and interpopulation analysis of genetic variability. Relationships among patterns have led to the identification of four main clusters. Two of them show a perfect correspondence to the population of origin of those individuals that present them (Cullera and Torreblanca), and the other two (Groups A and B) include patterns found in individuals coexisting in the same populations (Marjal del Moro populations) and in El Saler. Most of the variation found in this species is due to differences among populations as shown by the analysis of molecular variance. This agrees with the expectation for an apomictic species such as L. dufourii . The analysis of homogeneity of variance shows that substantial differences in the amount of genetic variability present in the six populations exist. These results have been used to understand the evolutionary and demographic history of L. dufourii , which is a requisite in order to establish efficient conservation measures for this species.