Anthoxanthum odoratum andA. alpinum: Life history parameters at two different altitudes

The closely related species, diploidAnthoxanthum alpinum Á. Löve etD. Löve and tetraploidAnthoxanthum odoratum L., have distinct distributions:A. alpinum is an arctic-alpine species, whileA. odoratum is a species of lower altitudes and more southerly latitudes. However, some authors suggest that both species are potentially able to maintain their populations in the distribution area of the other species. Reciprocal transplant experiments in two mountain localities (one withA. alpinum, the other withA. odoratum) were carried out to study the survival and vegetative and generative reproduction of tussocks of both species in the area of distribution of the other species. The mortality of tussocks ofA. alpinum was higher in the “alien” locality, the life span of tillers was greatly reduced and the size of tussocks decreased considerably during the experiment compared to its “native” locality. These results suggest that there was a lack of favourable microsites forA. alpinum in the “alien” locality probably caused by the presence of strong competitors.A. odoratum flowered far less in the “alien” locality (both on tussocks and tillers) compared to the “native” one. These results suggest that environmental conditions are not suitable for the flowering ofA. odoratum in the “alien” locality. We conclude thatA. alpinum could have been limited in extending its distribution into the area ofA. odoratum by the presence of strong competitors, whileA. odoratum could have been limited by its ability to reproduce by seed.     

Allozyme,chloroplast DNA and RAPD markers for determining genetic relationships between Abies alba and the relic population of Abies nebrodensis

Allozyme, chloroplast (cpDNA) and random amplified polymorphic DNA (RAPD) markers have been used to estimate genetic and taxonomic relationships among different populations of Abies alba and the relic population of A. nebrodensis. Twelve isozyme gene loci, as well as restriction fragment length polymorphism (RFLP) at cpDNA spacer regions between t-RNA genes were analysed. Moreover, a set of 60 random sequence 10-mer primers were tested. Over all isozyme loci, evident differences in allele frequencies among A. nebrodensis and A. alba populations were found, particularly at 2 loci, phosphoglucose isomerase (Pgi-a) and shikimate dehydrogenase (Skd-a). More than 10% of the total genetic diversity was due to differences among populations. High values of genetic distances among populations were also found. Out of the 60 primers tested, 12 resulted in a polymorphic banding pattern both within and among populations. A total of 84 RAPD fragments were produced by the 12 selected primers. A phenogram of relationships among populations was constructed based on RAPD band sharing: the differentiation of the A. nebrodensis population was evident. The analysis of molecular variance (AMOVA) was used to apportion the variation among individuals within populations and among populations. There was considerable variation within each population: even so, genetic divergence was found among populations. This pattern of genetic variation was very different from that reported for inbred species. Identical cpDNA amplification and restriction patterns were observed among all the individuals sampled from the populations. Taken together, the results of allozyme and RAPDs show a clear differentiation among A. nebrodensis and A. alba populations and provide support for their classification into two different taxonomic groups.     

Detection and analysis of genetic variation in Hordeum spontaneum populations from Israel using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to analyse genetic diversity within and between Hordeum spontaneum populations sampled from Israel. Nei's index of genetic differentiation was used to partition diversity into within and between population components. Fifty-seven per cent of the variation detected was partitioned within 10 H. spontaneum populations. Using principal component and multiple regression analysis, part of the variation detected between populations was seen to be associated with certain ecogeographical factors. Fifty-eight per cent of the distribution of the phenotypic frequencies of three RAPD phenotypes detected using a single primer in 20 H. spontaneum populations could be accounted for by four ecogeographical variables, suggesting adaptive variation at certain RAPD loci.     

Isozyme studies in the genusCheirolophus (Asteraceae:Cardueae-Centaureinae) in the Iberian Peninsula, North Africa and the Canary Islands

The phylogenetic and phenetic analysis of 109 RAPD polymorphisms inS. nebrodensis, a perennial and self-incompatible endemic of four mountain ranges in Spain, andS. viscosus, a self-compatible annual widespread in Europe, as well asS. lividus, S. sylvaticus andS. vulgaris revealed a sister group relationship between the first two species. This result contrasts sharply with the earlier hypothesis based on isozyme variation thatS. viscosus originated from within a paraphyleticS. nebrodensis and that the two species represent a progenitor-derivative pair. After considering possible reasons for the sister group relationship found, including the possibility of rooting artefacts, it is concluded that neither the RAPD data nor the isozyme data allow to draw safe conclusions about the mode of speciation and therefore the relative age of the two species. As a consequence, the limited genetic variation ofS. viscosus in comparison toS. nebrodensis as revealed by both the RAPD and the isozyme data may reflect its population history, geographical distribution, reproductive ecology, or mode of dispersal just as well as its recent origin from a paraphyleticS. nebrodensis. The result of this study calls for a critical reexamination of other taxon pairs postulated to have a progenitor-derivative relationship on the basis of isozyme evidence.     

The allotetraploid invasive weedBromus hordeaceus L. (Poaceae): Genetic diversity, origin and molecular evolution

Bromus hordeaceus (sectionBromus, Poaceae), a predominantly self-fertilizing tetraploid (2n=28), is an annual weed native to the Mediterranean Basin, which now has a world-wide distribution. High morphological variation led to the recognition of four subspecies, three of which correlated with habitat-type. We examined genetic diversity at enzyme loci in 15 populations from the Mediterranean and the Atlantic region. Although sampled over a larger range of ecological and geographical conditions, the North-African populations appeared less genetically differentiated than populations from Brittany, suggesting higher levels of gene flow among the first ones (Nm=3.756 and 1.066 respectively). No genetic differentiation was encountered among the four subspecies. The populations were homozygous at homologous loci, suggesting high rates of selfing, but they frequently exhibited fixed intergenomic heterozygosity. The meiotic chromosome behaviour and disomic inheritance encountered are in accordance with the previously proposed allopolyploid origin of the species. The diploidsB. arvensis andB. scoparius have been previously implicated in the parentage ofB. hordeaceus on the basis of morphology and serology. We comparedB. hordeaceus with related diploid species belonging to the same section (sectionBromus) using different sources of data (flow cytometry, karyotypes, RAPD and DNA sequences). Molecular phylogeny based on internal transcribed spacer sequences of nuclear ribosomal genes provided the first clear scheme of relationships among monogenomic species of the section. A new hypothesis is proposed concerning the origin ofB. hordeaceus: We found that it diverged earlier than all other species of sectionBromus excluding the diploidB. caroli-henrici which is basal in this group. The 13 autapomorphies accumulated byB. hordeaceus, and the absence of intra-individual sequence heterogeneity are also consistent with the relatively ancient origin of the species within the section.     

Taxonomy of theHieracium alpinum group in the sudeten Mts., the West and the Ukrainian East Carpathians

A taxonomic revision of theHieracium alpinum group (sensu Flora Europaea) in the Sudeten Mts., the West and the Ukrainian East Carpathians is provided. Six species are distinguished in the area studied, viz.Hieracium alpinum, H. halleri, H. augusti-bayeri, H. melanocephalum, H. tubulosum, andH. schustleri. H. alpinum occurs throughout the mountain ranges studied, three other taxa are endemic to the Sudeten Mts. (H. melanocephalum, H. tubulosum, H. schustleri), one is confined to the West Carpathians (H. halleri, in addition to the Alps) and one is endemic to the Ukrainian East Carpathians (H. augusti-bayeri). A diploid cytotype (2n=18) was ascertained inH. alpinum from the Ukrainian East Carpathians and the same chromosome number was found forH. augusti-bayeri. An agamospermous mode of reproduction was confirmed for the triploid cytotype ofH. alpinum, the triploidH. melanocephalum andH. halleri and the tetraploidsH. tubulosum andH. schustleri; the diploid taxa were found to be sexual. Pollen production in diploid taxa is high and pollen grains are homogeneous in size; triploid species, as well as tetraploidH. tubulosum, do not produce polllen; tetraploidH. schustleri has pollen grains of variable size. Data on the ecology and distribution of the species are also given.     

Consistency of population genetics parameters estimated from isozyme and RAPDs dataset in species of genus <Emphasis Type="Italic">Prosopis</Emphasis> (Leguminosae,Mimosoideae)

Genetic variability, population structure and differentiation among 17 populations of 5 species and 2 natural interspecific hybrids of section Algarobia of genus Prosopis were analyzed from data of 23 isozyme and 28 RAPD loci. Both markers indicated that the studied populations are highly variable. P. alba populations in average showed lower values of genetic variability estimates from isozyme data, but this trend was not observed for RAPD markers. The hierarchical analyses of the distribution of genetic variability showed that the highest proportion of variation occurred within populations, the differentiation among species was intermediate and the lowest component was observed among populations within species. The consistency between results from both dataset implies that they are not biased and reflect the actual genetic structure of the populations analyzed. The matrices of Euclidean distances obtained from the two sets of markers were highly correlated according to Mantel test. In both cases the corresponding phenogram and MDS plot tended to cluster conspecific populations while hybrid populations were not intermediate between putative parents. Some disagreements between isozyme and RAPD phenograms were observed mainly in the affinities of hybrid populations. Such inconsistencies might result from reticular rather than dichotomic evolutionary relationships. The phenetic associations retrieved gave no support to the division of the section Algarobia into series.     

Isozyme and PCR-based genotyping of epidemic Phytophthora colocasiae associated with taro leaf blight

The Oomycetous fungus Phytophthora colocasiae causing leaf blight of taro is widely distributed in India. Wide geographic range or sexual recombination provides genetic differentiation within this species. To determine how genetic variation is partitioned in P. colocasiae, 14 isolates were isolated from different regions of India, where the incidence of leaf blight is great. Molecular and biochemical techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae. Seven polymorphic enzyme systems revealed 23 isozyme patterns, each uniquely characterised by the presence or absence of electromorphs. Further, 10 oligodeoxynucleotide primers were selected for random amplified polymorphic DNA (RAPD) assays, which resulted in 123 polymorphic bands for 10 isolates of P. colocasiae. The data were entered into a binary matrix and a similarity matrix was constructed using a DICE similarity (SD) index. A UPGMA cluster based on SD values was generated using a NTSYS computer program. Shannon's index was used to partition genetic diversity. Similarly, isozymes and RAPDs yielded high estimates of genetic variability. Genetic diversity estimates via isozyme and RAPD pattern indicated 78.26% and 100%, respectively, total diversity among populations. This type of genetic variation in P. colocasiae indicates that variation due to asexual and/or possibly infrequent sexual mechanisms is possible and that genetic differentiation has taken place as a result of geographic isolation. The presence of larger than expected RAPD variation in isolates of P. colocasiae and the presence of distinct different zymotypes among these isolates suggests that genetic recombination (or less likely hybridisation) is at least possible in this fungus and that geographic differentiation has taken place. Even isolates obtained from the same habitat have different RAPD patterns, indicating that many populations of this fungus are made up of more than one genet and that few are derived clonally.     

Inter- and intraspecific genetic variation inHippophae (Elaeagnaceae) investigated by RAPD markers

Genetic diversity has been investigated by the application of molecular markers in, for the first time, all the taxa recognised in recent treatises of the genusHippophae. RAPD (random amplified polymorphic DNA) analyses were conducted with 9 decamer primers, which together yielded 219 polymorphic markers. We found 16 fixed RAPD markers, i.e. markers that either occurred in all plants of a population or were absent from all plants. Several of these markers were useful for analysis of interspecific relationships, whereas others can be considered as taxon-specific markers. Clustering of taxa and populations in our neighbour-joining based dendrogram was in good agreement with some recently suggested taxonomic treatises ofHippophae. Amount and distribution of genetic variability varied considerably between species. Partitioning of molecular variance withinH. rhamnoides supported earlier findings that a considerable part of the total variance resides among subspecies (59.6%) Within-population variability also differed considerably. Percentage polymorphic RAPD loci and Lynch and Milligan within-population gene diversity estimates showed relatively high values for some species close to the geographic centre of origin in Central Asia, e.g.H. tibetana and the putatively hybridogenousH. goniocarpa. Spatial autocorrelation analyses performed on 12 populations ofH. rhamnoides revealed positive autocorrelation of allele frequencies when geographic distances ranged from 0 to 700 km, and no or negative autocorrelation at higher distances. At distances between 700 and 1900 km, we observed deviations from the expected values with strongly negative autocorrelation of allele frequencies. A corresponding relationship between geographic and genetic distances could not be found when the analysis instead was based on one population from each of 8 species.     

Low levels of genetic variation inPhenakospermum guyannense (Strelitziaceae), a widespread bat-pollinated Amazonian herb

Phenakospermum guyannense is a monotypic, arborescent, long-lived monocot that is widespread in Amazonian South America. This outcrossing species is pollinated primarily by phyllostomid bats. Given these life-history characteristics,P. guyannense is expected to exhibit high levels of genetic variation and gene flow. We used isozyme electrophoresis and randomly amplified polymorphic DNA (RAPD) to characterize genetic variation in populations ofP. guyannense from French Guiana. Both measures detected a surprisingly low level of genetic variation, with only five out of twenty (25%) allozyme loci polymorphic (P), 1.35 alleles per locus (A), and an expected heterozygosity (H_e) of 0.090 at the species level. Isozymic genetic variation was even lower within populations (P = 17.5, A = 1.24, H_e = 0.074), and was corroborated by a RAPD assay that used 26 arbitrary primers (P = 3.61, A = 1.04, H_e = 0.014). Although overall levels of variation were low, the detectable variation was distributed as would be expected for an outcrossing species with extensive gene flow (mean G_ST = 0.230). We suspect thatP. guyannense is depauperate in genetic variation because of a series of bottlenecks that affected the species over this portion of its range.     

Phenetic investigation of non-nodulating African species ofAcacia (Leguminosae) using morphological and molecular markers

Morphological and RAPD markers were used to assess the relationships among nodulating and non-nodulating species of AfricanAcacia. Non-nodulating species of AfricanAcacia are only found within subg.Aculeiferum sect.Monacanthea. African species of sect.Monacanthea examined were found to form a group distinct from the other African species examined on a morphological and molecular basis. All lack the ability to nodulate, suggesting that non-nodulation may be used as a taxonomic tool. The species of sect.Aculeiferum were separated by RAPD and morphological analysis into two groups depending on whether they were armed with prickles in pairs and/or prickles in threes, or solitary. A third group of species was identified within sect.Acacia: further subdivision of this group was achieved into subsectt.Pluriseriae andUniseriae. The position ofA. albida relative to other AfricanAcacia species was found to be distinct but not totally independent of the genus. The partitioning and distribution of the genetic variability within the genus is further elucidated by the RAPD analysis of populations ofAcacia species. A population analysis ofA. polyacantha demonstrated geographical and site-specific variation.     

Iridoid glucosides of the genus Veronica s.l. and their systematic significance

 The mode of reproduction, pollen production, chromosome numbers, genetic variation (RAPD, allozymes) and overall similarity were studied in 6 species of Hieracium sect. Alpina in the Tatry Mts. (the Western Carpathians, Slovakia). All species were confirmed to be agamospermous and, except of H. krivanense and H. slovacum, lacking pollen grains. For the first time, a chromosome number is reported for H. krivanense (2n=4x=36). Considerable genetic variation was revealed in H. alpinum and a correlation between geographic and genetic distances was found in this species. Between-population variation in RAPD and allozyme phenotypes was found in H. pinetophilum and H. crassipedipilum. In all other species, allozyme and RAPD variation was low or absent. With few exceptions, the species differ in their allozyme as well as RAPD patterns. The relatedness of one population of endemic H. slovacum and H. halleri was confirmed. It is shown, that Carpathian species of the H. fritzei group are derived from at least two ancestors. Received July 3, 2000; accepted June 24, 2002 Published online: November 20, 2002 Addresses of the authors: H. Štorchová, (e-mail: storchova@ueb.cas.cz) Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 135, CZ-165 00 Praha 6, Czech Republic. I. V. Bartish, J. Chrtek Jr., J. Kirschner, M. Tetera, J. Štěpánek, Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pruhonice, Czech Republic.     

RAPD Analysis of the Genome in Species of the Genus Hordeum

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum.High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgaresensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between theHordeumspecies, between the H. spontaneumpopulations, and between regional H. vulgarecultivars and a dendrogram was constructed.     

RAPD analyses of hybridization events inCardamine (Brassicaceae)

Recent hybrid speciation events between the diploidCardamine rivularis and the diploidC. amara leading to the triploidC. xinsueta were investigated with random amplified polymorphic DNA (RAPD). The hybrid nature ofC. xinsueta was confirmed by the additivity of RAPD markers which discriminate the parent species. An unexpectedly high level of genetic variation withinC. xinsueta argues for several hybridization events and/or backcrossing withC. rivularis. A low number of nonparental bands inC. xinsueta indicates the younger age of this taxon. In contrast to the situation inC. xinsueta, low variability was observed in the hexaploidC. schulzii, a derivative species ofC. xinsueta: C. schulzii probably originated recently by a single autopolyploidization event. The higher number of non-parental bands inC. schulzii is explained by the changing genetic variability of the parental populations. Evidence for potential gene flow across the ploidy levels is also provided.     

Genetic diversity analysis by RAPD inCathaya argyrophylla Chun et Kuang

Genetic diversity level ofCathaya argyrophylla was confirmed by random amplified polymorphic DNA (RAPD) markers. Seventy five samples (individuals), collected from Hunan and Sichuan provinces of China were used in the study. 21 10-mer oligonucleotide primers detected 106 sites, and 34 (32%) of them were polymorphic. The level of genetic variation in C.argyrophylla was lower than those of other conifers, and was considered to be associated with the complexity of habitats. The percentages of polymorphic sites (PPS) in the Hunan and Sichuan populations were 18% and 25% respectively. 7.99% of genetic variation existed between the two populations; this value was higher than the mean value (6.8%) among populations in conifers displayed by allozyme. Some subpopulations ofC. argyrophylla were greatly differentiated because of site mutation and genetic drift. The highest value of genetic difference between subpopulations amounted to 16. 23%. In addition, a concept of diversity coefficient (DC), a value used to measure the genetic diversity level, and its calculation were proposed. The low genetic diversity level ofC. argyrophylla was thought to be one of the factors causing its endangered status.     

High RAPD but no cpDNA sequence variation in the endemic and endangered plant, Heptacodium miconioides Rehd. (Caprifoliaceae)

Heptacodium miconioides Rehd. is an endangered species endemic to China and has suffered rapid decrease of distribution range and population size. This species has been disappeared in central China where the modal specimen was collected. We analyzed the genetic variation of the remaining populations to reveal whether the genetic diversity also suffered decrease and to provide some suggestions for conservation. All the nine known remaining populations were sampled. Genetic variation was analyzed based on RAPD markers and two fragments of cpDNA sequence, intergenic spacers of petG-trnP and trnS-trnG. No variation was observed in the two fragments of cpDNA sequence. However, the species exhibited high level of RAPD variation compared to other threatened or rare plants. Measures of genetic diversity within populations were strongly related to the log of estimated population size, indicating that large populations usually have more genetic diversity than that of small ones. About 25% of the variation was partitioned among populations. Significant relationship was observed between differentiation and geographical distance, indicating a pattern of isolation-by-distance. Given for few populations remaining, all the populations should be protected and urgent efforts be paid on the small populations to avoid their local extinction.     

Analysis of genetic variability of South American wild rice populations (Oryza glumaepatula) with isozymes and RAPD markers

The knowledge of population structure and genetic diversity of wild relatives of rice is needed to investigate their evolutionary history and potential use in breeding programs. Very little is known about the wild rice species ( Oryza spp.), particularly those that are native to South America. A study using isozyme and RAPD markers was conducted to estimate the level of genetic diversity of four South American wild rice populations ( Oryza glumaepatula ) recently collected in the Amazon forest and western Brazil rivers. F -statistics and genetic diversity parameters calculated from isozyme and RAPD markers indicated high values for inbreeding coefficients and differentiation among the four populations. In agreement with this, a pattern of greater variation between than within populations was observed with both types of markers. These findings were corroborated by an AMOVA analysis, which indicated that a large portion of the total genetic variation was attributed to regional divergence. The partition of the AMOVA analysis among populations showed that most of the genetic diversity was due to differences among populations. This distribution pattern of genetic variation of O. glumaepatula populations is in agreement with the expectation for an autogamous species and provides important baseline data for conservation and collection strategies for this species.     

Interspecific hybridization in the genus Hieracium s. str.: evidence for bidirectional gene flow and spontaneous allopolyploidization

Although reticulation has indisputably played an important role in the evolutionary history of the genus Hieracium s. str. (Asteraceae), convincingly documented cases of recent interspecific hybridization are very rare. Here we report combined evidence on recent hybridization between two diploid species, Hieracium alpinum and H. transsilvanicum. The hybrid origin of the plants from the Romanian Eastern Carpathians was supported by additive patterns of nuclear ribosomal DNA polymorphism (ITS), an intermediate position of hybrid plants in principal coordinate analysis based on amplified fragment length polymorphism phenotypes (AFLP), and additivity at one allozyme locus. Flow cytometric analyses and chromosome counting showed that two hybrids were diploid (2n ~ 2x ~ 18) while one was surprisingly tetraploid (2n = 4x = 36). To our knowledge, this is the first record of spontaneous polyploidization following interspecific crossing in the genus. Allozyme data, especially the presence of unbalanced heterozygosity at one locus, suggest the origin of this tetraploid via a triploid bridge with subsequent backcrossing to H. alpinum. According to PCR-RFLP analyses of the trnT-trnL intergenic spacer, all H. ×krasani hybrids examined had the H. alpinum haplotype while H. transsilvanicum served as a pollen donor. The hybrids occurred at the locality with abundant H. alpinum plants where paternal H. transsilvanicum was missing. Previously reported instances of interspecific hybridization between the same parental taxa showed an opposite direction of crossing and relative abundance of parental taxa. This suggests that the direction of hybridization might be influenced by the frequency of parental taxa at the locality.     

Diversity of Rhizobia isolated from various Hedysarum species

Cultural and physiological properties, serology, plasmid profiles and infective traits were determined for 23 strains of rhizobia isolated from various Hedysarum species: H. coronarium (common name: sulla) (16), H. carnosum (1), H. alpinum (3), H. mackenzii (2) and H. pallens (1) from Portugal, Spain, Tunisia, Alaska and Israel. Strains isolated from H. alpinum, H. mackenzii and H. pallens have slow growth rates on yeast-extract mannitol medium and were unable to nodulate H. coronarium plants, whereas the latter were effectively nodulated by all sixteen fast growing strains from sulla. Regardless of the country of origin all H. coronarium strains fell into one serogroup and were not serologically related with strains of other Hedysarum species. The RAPD (random amplified polymorphic DNA) fingerprinting method which was carried out on five H. coronarium and three H. alpinum strains allowed distinction to be made among serologically related rhizobia. No particular plasmid profile pattern was observed in relation to the host or geographical origin of the strains.