On the general failure of coppice conversion into high forest in <Emphasis Type="Italic">Quercus pyrenaica</Emphasis> stands: a genetic and physiological approach

Genetic, dendrochronological and physiological studies are reviewed to investigate causes of tree stagnation of abandoned Quercus pyrenaica coppices. Tree decline – displayed by slow stem growth, branch dieback and scarce acorn production – has led to advice on the conversion of abandoned oak coppices into high forests by thinning. However, this practice has often failed. The shortage of genetic diversity and disequilibrium between above- and below-ground organs in large, over-aged stools (clones) are adduced consequences of centenary coppicing driving stand stagnation in Q. pyrenaica. Our results evidence that historical coppicing has not enhanced genetic diversity losses; on the contrary, it has allowed for the recruitment of new genotypes and the existence of uneven-aged stands of heterogenic clonal structure. Tree excavations have revealed widely spread root systems. High carbon expenditures in root respiration in large and centennial clones may constrain their aboveground development, and thinning practices aiming at converting Q. pyrenaica coppices into high forests might not succeed or even enhance a physiological root-to-shoot imbalance.     

Assessing the genetic legacy of a rare,clonal Australian shrub <Emphasis Type="Italic">Grevillea infecunda</Emphasis> (Proteaceae)

Reliance on clonal reproduction is associated with an increased risk of extinction. Grevillea infecunda is a rare, putatively sterile shrub restricted to 11 populations in a localized coastal region of south-eastern Australia. We assessed the genetic diversity, clonal diversity and spatial distribution of clones in all populations of G. infecunda to guide conservation management. Eight chloroplast haplotypes were identified from the trnL – trnF and trnQ – rps16 intergenic spacer regions. All individuals belonged to a single maternal lineage dominated by one haplotype (95/111 samples). Minor haplotypes differed from the common haplotype only by single mutational steps. However, microsatellite markers revealed 89 multilocus genotypes (MLGs) in 38 multilocus lineages (MLLs) of variable size. MLLs were not shared among populations and ramets from different MLLs rarely intermingled physically. New shoots arising after fire were confirmed to belong to previously-existing MLLs, indicating that this species exhibits adaptation to fire. Genetically similar MLGs were more likely to be found in close proximity than less similar MLGs, resulting in significant spatial autocorrelation to ca 350 m. Genetic diversity was moderate but genotypic diversity was low once likely clonality was taken into account. Clonality appears to have arisen several times within the holly-leafed grevilleas and examining G. infecunda is a step towards understanding why and how often clonality occurs, and the long-term evolutionary outcomes of this life history.     

Genetic diversity loss and homogenization in urban trees: the case of <Emphasis Type="Italic">Tilia </Emphasis>× <Emphasis Type="Italic">europaea</Emphasis> in Belgium and the Netherlands

Urban trees form a vital component of sustainable cities but the use of a restricted range of species and genotypes may pose a risk to global biodiversity. Despite several studies investigating tree species diversity, intraspecific genetic diversity of urban trees remains largely unexplored. Here, we characterized the genetic diversity of Tilia?×?europaea, one of the most widely planted urban tree species in Northwest Europe. We compared the genotypic diversity of historical plantings of Tilia spp. from the 17th century with the genotypic diversity of currently available planting stock in Belgium and the Netherlands. In total, 129 trees were sampled and genotyped with 14 microsatellite loci and 150 polymorphic Amplified Fragment Length Polymorphism markers. In Northwest Europe, homogenization of urban T.?×?europaea plantings already started at the 17th century. Genetic diversity within contemporary commercial planting stocks was extremely narrow and consisted mainly of two clones, sold under the name ‘Pallida’ and ‘Zwarte linde’. The genetic diversity found within the historical plantings was about four times higher than in the current commercial planting stocks. We recommend that tree nurseries should enlarge the genetic diversity of T.?×?europaea commercial planting stocks. The old clones have shown long-term disease resistance and could provide tree breeders with the valuable new genetic material. The range of available Tilia species and genotypes needs to be explored in future urban tree planning to optimize desired ecosystem services.     

Occurrence and Genetic Structure of the Systemic Grass Endophyte <Emphasis Type="Italic">Epichloë festucae</Emphasis> in Fine Fescue Populations

Epichloë species are systemic fungal endophytes that usually specialize in a certain group of related grass species. We examined the infection frequency of Epichloë festucae in populations of two fine fescue species (Festuca rubra and F. ovina) in natural and seminatural habitats at 86 study sites (total = 2514 plants) across Finland and northern Norway. Infection incidence varied significantly among grass species and populations. A substantial number of the F. rubra and F. ovina populations (53 out of 77 and 25 out of 30, respectively) were either endophyte-free or had very low (<20%) infection frequencies. The highest infection frequencies were found in subarctic areas. Moreover, infection incidence differed between habitats. In the area with the highest infection frequencies, we used microsatellite markers to study genetic diversity and the rates of gene flow of E. festucae among 12 F. rubra populations. Twenty out of the 25 fungal genotypes detected with four microsatellite markers were carrying multiple alleles in at least one locus, indicating multiple infections or vegetative hybridization of the fungus. One dominant genotype occurred in all 12 populations, representing 63.5% of all isolates. We found a moderate level of average genotypic variation and a low level of genetic differentiation (F _st = 0.0814). There was no correlation between infection frequency and genotypic diversity. Although the existence of a dominant genotype and the detected linkage disequilibrium suggest that the fungus is mainly asexual and vertically transmitted, the multiallelic loci and variation of genetic diversity among populations indicate occasional contagious spread and sexual or parasexual recombination of the fungus in some populations. Furthermore, the genotypes carrying multiallelic loci suggest the possibility of multiple infections or hybridization of the endophyte.     

High genetic diversity and differentiation of an extremely narrowly distributed and critically endangered decaploid rose (<Emphasis Type="Italic">Rosa praelucens</Emphasis>): implications for its conservation

Rosa praelucens is a critically endangered decaploid alpine rose with an extremely narrow geographic distribution in Northwestern Yunnan, China. We sampled almost all the extant individuals (527 individuals in 31 natural locations and 56 individuals preserved in three local living collections) to assess the genetic variation and to probe the genetic connectivity among the individuals and populations based on three cpDNA intergenic spacers and six fluorescent amplified fragment length polymorphism (AFLP) markers. The morphological traits from seven populations were also measured. R. praelucens exhibited high levels of morphological variation, genetic diversity, and differentiation. The extant individuals were clustered into eight groups in neighbor-net networks, and subsequent Bayesian analysis assigned them into three larger gene pools, both in accordance with their morphological traits, especially flower color. The living collections embraced two private cpDNA haplotypes and included three out of the species’ total eight AFLP genotypes. Rhizome clonal growth, decaploid, and mixed breeding system may largely contribute to high genetic diversity and differentiation in R. praelucens. We concluded that the endangered status of R. praelucens may mainly be due to habitat fragmentation and loss and inherent reproductive difficulties, rather than low genetic diversity. The populations contributing higher cpDNA genetic diversity, representing more AFLP genotypes, and encompassing private cpDNA haplotypes should be given conservation priority by creating plant-micro reserves. The living collections should also be targeted for further ex situ conservation, population recovery, and reintroduction of R. praelucens plants.     

Genetic diversity of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> recovered from Massachusetts between 1997 and 2014

Phytophthora blight caused by Phytophthora capsici limits the production of cucurbits and peppers in the United States and is a growing threat to sustainable vegetable production in New England. Little is known about the genetic diversity of P. capsici in New England, and a total of 210 P. capsici isolates from 18 sites were genotyped using 46 single nucleotide polymorphism markers, revealing 85 unique and 34 repeated multi-locus genotypes. Both mating types were recovered from 7 of the 18 locations. Isolates with identical genotypes (clonal lineages) ranged from 2 to 16. Three clonal lineages were recovered from multiple sites within the same year, although none were recovered across multiple years. Bayesian clustering revealed individuals with a complex genetic cluster composition. This, coupled with a high outcrossing rate (mean t = 0.87) and no clear clustering in principal coordinates analysis, suggests outcrossing among the populations. Phylogenetic and genetic distance analysis indicate differentiation based on farm location and movement among farms may be infrequent. There was no obvious differentiation based on cucurbit, tomato or pepper hosts.     

Microsatellite DNA analysis of spatial and temporal population structuring of <Emphasis Type="Italic">Phragmites australis</Emphasis> along the Hudson River Estuary

Phragmites australis is a perennial grass that has invaded wetlands of the northeastern United States over the past century. The Hudson River Estuary and surrounding watersheds are no exception in that populations of P. australis have spread dramatically along its shores and tributaries in the past 40 years. Recent studies have shown that genetically variable populations of P. australis can spread by seed dispersal in addition to clonal mechanisms. It is important to characterize the genetic variation of Hudson River populations as part of a management strategy for this species to determine the mechanisms by which its spreads and colonizes new habitats, particularly those with frequent anthropogenic disturbances. The goals of this study were to quantify levels of genetic variation and structuring in Hudson River populations of P. australis using microsatellite DNA analysis. A total of 354 culms of P. australis were collected from nine locations ranging from Albany, New York to Staten Island, New York in the summers of 2004 (N = 174) and 2011 (N = 180). Microsatellite data from eight loci indicated that the Hudson River Estuary has some of the highest levels of genetic variation of all U. S. Atlantic Coast regions containing P. australis. Gene diversity (Hs) across all loci in the 2004 collection was 0.45 (±0.02) and that of the 2011 collection was 0.47 (±0.07). Patches within sample sites were rarely monoclonal and had multiple genetic phenotypes. Moran’s Identity tests indicated that individuals within a patch were closely related, whereas little genetic relatedness was evident among individuals from sample sites >1 km apart. Spatial structuring was also not evident in autospatial correlation and principle coordinate analyses. These findings suggest that genetic diversity is maintained within stands by sexual reproduction and that seeds are important in dispersal of P. australis across the Hudson River Estuary. Ample habitats are available for establishment of new Phragmites stands due to high levels of anthropogenic disturbance from populations living along the Estuary. Wildlife managers should focus on monitoring habitats that provide seedbed for Phragmites and promote land use practices that prevent soil disturbance and establishment of new stands.     

Feed-backs between genetic structure and perturbation-driven decline in seagrass (<Emphasis Type="Italic">Posidonia oceanica</Emphasis>) meadows

We explored the relationships between perturbation-driven population decline and genetic/genotypic structure in the clonal seagrass Posidonia oceanica, subject to intensive meadow regression around four Mediterranean fish-farms, using seven specific microsatellites. Two meadows were randomly sampled (40 shoots) within 1,600 m² at each site: the “impacted” station, 5–200 m from fish cages, and the “control” station, around 1,000 m downstream further away (considered a proxy of the pre-impact genetic structure at the site). Clonal richness (R), Simpson genotypic diversity (D*) and clonal sub-range (CR) were highly variable among sites. Nevertheless, the maximum distance at which clonal dispersal was detected, indicated by CR, was higher at impacted stations than at the respective control station (paired t-test: P < 0.05, N = 4). The mean number of alleles (Â) and the presence of rare alleles ( _r) decreased at impacted stations (paired t-test: P < 0.05, and P < 0.02, respectively, N = 4). At a given perturbation level (quantified by the organic and nutrient loads), shoot mortality at the impacted stations significantly decreased with CR at control stations (R ² = 0.86, P < 0.05). Seagrass mortality also increased with  (R ² = 0.81, P < 0.10), R (R ² = 0.96, P < 0.05) and D* (R ² = 0.99, P < 0.01) at the control stations, probably because of the negative correlation between those parameters and CR. Therefore, the effects of clonal size structure on meadow resistance could play an important role on meadow survival. Large genotypes of P. oceanica meadows thus seem to resist better to fish farm-derived impacts than little ones. Clonal integration, foraging advantage or other size-related fitness traits could account for this effect.     

Mesoscale investigations based on microsatellite analysis of the freshwater sponge <Emphasis Type="Italic">Ephydatia fluviatilis</Emphasis> in the River-Sieg system (Germany) reveal a genetic divergence

Freshwater sponges play a major role in freshwater ecological system as important filter-feeding organisms and bioindicators. There are only few data about their ecological diversity and population genetic structure available, though a deeper knowledge is needed to propose proper conservation and effective management. The aim of this study was to assess data on distribution patterns of freshwater sponges to study the connectivity of genotypes of Ephydatia fluviatilis in a river system. We sampled specimens from River-Sieg system (River Agger and River Sieg, Germany). We hypothesized that strong anthropogenic influence would cause a uniform distribution of population structures. The genetic structure of E. fluviatilis populations was analysed with a set of eleven microsatellite loci from seven locations in River-Sieg system. Besides of E. fluviatilis, three other species co-occurred (Ephydatia mülleri, Spongilla lacustris, Eunapius fragilis). In contrast to our hypothesis, we observed an overall correlation between genetic and geographic distances among populations of this sessile species, which follows a clear isolation-by-distance pattern. A significant microsatellite polymorphism and high levels of genetic divergence between populations (F_ST) in upstream reaches were present. These results will provide important information for conservation management of populations with limited dispersal ability in connected river systems.     

Assessing genetic structure in a rare clonal eucalypt as a basis for augmentation and introduction translocations

Genetics can provide information that is critical for planning translocations for conservation, such as levels of diversity and divergence of target and source populations. For clonal plants, assessing population characteristics (size, diversity, mortality, gene flow) that influence conservation values also requires identification of different genetic individuals. We used 12 microsatellite markers to guide germplasm source recommendations for augmentation and introduction translocations to conserve the critically endangered Eucalyptus cuprea that occurs in fragmented populations in the semi-arid shrublands of Western Australia. Ramet clumps with identical multilocus genotypes were identified in all populations but clonal richness (R = 0 ? 0.86) and heterogeneity (D = 0 ? 0.98) varied among populations. Genetic diversity was low to moderate in all populations (mean H _o = 0.61, mean A = 3.78) and did not differ significantly between localities. There was evidence of inbreeding in some populations but outcrossing (t _m = 0.495) in the small number of families available for study (N = 4) and genotypic diversity of the larger extant populations suggest the generation of novel genotypes is a component of the reproductive strategy. Most diversity was within populations and differentiation among populations was moderate (F _ST = 0.100) suggesting mixing of source population for translocation is unlikely to lead to outbreeding depression. Principal Co-ordinate and Bayesian analyses indicated the Northern population is distinct from Central/Southern populations. We recommend use of mixed germplasm to conserve the moderate diversity characterising larger remnant populations and to enable the production of recombinants through sexual reproduction. But given seed availability and the distinction of the Northern population, an initial precautionary approach to a translocation proposed for south of the geographical range may be to source germplasm from the Central/Southern locality.     

Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.     

Evidence of the genetic diversity and clonal population structure of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strains isolated from different wine-making regions of China

Studies of the genetic diversity and population structure of Oenococcus oeni (O. oeni) strains from China are lacking compared to other countries and regions. In this study, amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST) methods were used to investigate the genetic diversity and regional evolutionary patterns of 38 O. oeni strains isolated from different wine-making regions in China. The results indicated that AFLP was markedly more efficient than MLST for typing O. oeni strains. AFLP distinguished 37 DNA patterns compared to 7 sequence types identified using MLST, corresponding to discriminatory indices of 0.999 and 0.602, respectively. The AFLP results revealed a high level of genetic diversity among the O. oeni strains from different regions of China, since two subpopulations and an intraspecific homology higher than 60% were observed. Phylogenetic analysis of the O. oeni strains using the MLST method also identified two major phylogroups, which were differentiated into two distinct clonal complexes by minimum spanning tree analysis. Neither intragenic nor intergenic recombination verified the existence of the clonal population structure of the O. oeni strains.     

Persistence of the gypsophile <Emphasis Type="Italic">Lepidospartum burgessii</Emphasis> (Asteraceae) through clonal growth and limited gene flow

Lepidospartum burgessii is a rare gypsophilic shrub with limited distribution in New Mexico and Texas. Most of the known plants are restricted to two large populations, with a few small, isolated populations scattered in the surrounding area. The low recruitment observed in the two largest populations may be due to low seed set resulting from high inbreeding and/or self-incompatibility. We used eight microsatellite loci to quantify diversity, relatedness, inbreeding, population structure, and frequency of clonal reproduction. Seed collections were made to quantify seed set and germination rates. Overall, there was a moderate level of clonal diversity within patches of L. burgessii indicating asexual growth is important for population persistence. Inbreeding coefficients were high both between and within populations. Most patches showed a significant level of relatedness between individuals. At a fine scale, patches within populations were significantly different from each other, however when all patches were combined, the two populations of L. burgessii were not genetically distinct. Compared to a population of its common congener, Lepidospartum latisquamum, L. burgessii populations had similar measures of diversity, more clonal reproduction, and lower germination rates. High relatedness and inbreeding may explain the low seed set and recruitment in L. burgessii, however factors such as insect herbivory and precipitation changes may further depress recruitment.     

Comparison of ISSR,IRAP and REMAP markers for assessing genetic diversity in different species of <Emphasis Type="Italic">Brassica</Emphasis> sp.

Molecular markers provide facilities in order to study genetic diversity and relationship among genotypes. In this study, genetic diversity among 35 genotype of Brassica sp. (belonging B. napus, B. juncea, B. rapa, B. nigra) were determined using 13 ISSR, 3 IRAP markers and 18 REMAP (primer combinations of ISSR and retrotransposon primer). The percentage of polymorphism for ISSR, IRAP and REMAP was 96.38, 94 and 96%, respectively. By comparison between markers, ISSRs indicated the highest expected heterozygosity (He) and Shannon’s information index (I) with value of 0.34 and 0.51, respectively, while REMAP marker had by far the highest number of polymorphic bands (340) and marker index (7.1) among all fragments scored over all markers. In pattern of clustering based on Bayesian methods, K = 8 was resulted for combined data clustering that was more organized clustering for genotypes compared to others. This research suggests the combined data of ISSR, IRAP and REMAP markers are most reliable than each solely marker whilst have been clustered genotypes in their taxonomic classification of Brassica without any mixture. Principle coordinate analysis (PCoA) separated 35 genotypes in four groups which all of genotypes were clustered correctly based on their taxonomic classification. The findings of this study provide the valuable insight into the Brassica species relationships in terms of similarity among genotypes which can be helpful in breeding programs, and also demonstrate that retrotransposon markers are legible for genetic diversity and next genetic analysis in Brassica genus.     

Diversity and enzymatic potentialities of <Emphasis Type="Italic">Bacillus</Emphasis> sp. strains isolated from a polluted freshwater ecosystem in Cuba

Genotypic and phenotypic characterization of Bacillus spp. from polluted freshwater has been poorly addressed. The objective of this research was to determine the diversity and enzymatic potentialities of Bacillus spp. strains isolated from the Almendares River. Bacilli strains from a polluted river were characterized by considering the production of extracellular enzymes using API ZYM. 14 strains were selected and identified using 16S rRNA, gyrB and aroE genes. Genotypic diversity of the Bacillus spp. strains was evaluated using pulsed field gel electrophoresis. Furthermore, the presence of genetic determinants of potential virulence toxins of the Bacillus cereus group and proteinaceous crystal inclusions of Bacillus thuringiensis was determined. 10 strains were identified as B. thuringiensis, two as Bacillus megaterium, one as Bacillus pumilus and one as Bacillus subtilis. Most strains produced proteases, amylases, phosphatases, esterases, aminopeptidases and glucanases, which reflect the abundance of biopolymeric matter in Almendares River. Comparison of the typing results revealed a spatio-temporal distribution among B. thuringiensis strains along the river. The results of the present study highlight the genotypic and phenotypic diversity of Bacillus spp. strains from a polluted river, which contributes to the knowledge of genetic diversity of Bacilli from tropical polluted freshwater ecosystems.     

Analysis of genetic diversity among medicinal therapist <Emphasis Type="Italic">Trigonella foenum</Emphasis>-<Emphasis Type="Italic">graecum</Emphasis> L. genotypes through RAPD and SSR markers

Trigonella is recognized as a medicinal therapist throughout the globe due to its multifaceted rare medicinal properties. It is indigenous from Iran to Northern India but has gained global acceptance towards cultivation and consumption for its yellow-to-amber colored seed which substantially contributes to food, pharmaceutical, nutraceutical and cosmetic industry. Genetic diversity serves as an excellent tool for developing improved crop varieties with breeder preferred traits. Unfortunately, very little information available on variability existing in commercial Trigonella genotypes considerably impedes the crop improvement. In this study, ninety Trigonella genotypes belonging to most productive North Indian states were subjected to multilocus genotyping using RAPD (49) and SSR (13) primers and detected an average of 55.60 and 50.16% polymorphism, respectively. The percentage polymorphism range (RAPD, 16.7–90.90; SSR, 33.30–66.66) average band informativeness (RAPD, 0.182–0.85; SSR, 0.21–0.91) and resolving power (RAPD, 0.95–9.984; SSR, 1.68–7.28) obtained revealed the wide range of diversity prevailing among these genotypes. Hierarchical clustering of genotypes in nine different clusters showed Trigonella’s genetic variability has wide genetic distribution across different agro-climatic zones. No consistency was observed while grouping Trigonella varieties based on eco-geographical region. Eventually, knowledge of these genetic differences significantly contributes in designing intra-specific crosses with potential interest to spice breeding programs. To the best of our knowledge, this is the first report of genetic diversity using SSR molecular markers in Trigonella foenum-graecum L.     

Genetic structure of diploid-polyploid complex of spined loach genus <Emphasis Type="Italic">Cobitis</Emphasis> (<Emphasis Type="Italic">Cypriniformes,Cobitidae</Emphasis>) in the Lower Danube

The extremely high diversity of spined loach biotypes in the Lower Danube has been detected by biochemical genetic investigation and cytometric analysis of 358 specimens collected in the riverbed and shallow channels. Along with two diploid species (C. elongatoides and C. “tanaitica”), six hybrid forms were revealed, namely, diploid C. elongatoides-“tanaitica”; triploid C. 2 elongatoides-“tanaitica,” C. elongatoides-2 “tanaitica,” and C. 2 elongatoides-species-1; and tetraploid C. 3 elongatoides-“tanaitica” and C. elongatoides-species-2-2 “tanaitica.” In addition, specimens with recombinant genotypes were also found. In spite of the apomictic mode of reproduction, the polyploids did not possess clonal structure, but according to the level of polymorphism and the genotype distribution, they were isomorphous to parental diploid species. Thus, in contrast to the polyploidy in Cobitids of the Dnieper, which have appeared in the basin due to the expansion, the polyploids of the Lower Danube are autochthonous and were derived by crossing with local diploid species. The process is apparently proceeds without any limitations.     

Hybridization rate and genotypic diversity of apomictic hybrids between native (<Emphasis Type="Italic">Taraxacum japonicum</Emphasis>) and introduced (<Emphasis Type="Italic">T. officinale</Emphasis>) dandelions in western Japan

Hybridization between the introduced and native plants may enhance invasiveness, especially in asexually reproducing species. Hybrid apomictic dandelions between native (Taraxacum platycarpum and T. japonicum) and exotic (T. officinale) species are distributed widely throughout Japan. To estimate the origin(s) and dispersal of the hybrids, we investigated the hybridization rate and genotypic diversity in mixed populations of T. japonicum, T. officinale and their hybrids at two green parks in western Japan. Among the plants identified as exotics from flower morphology, 86–96% were hybrids by genetic analysis. Genetic data with simple sequence repeat markers revealed a high clonal diversity of the hybrid both within and between populations, indicating multiple origins. A hybrid seed was found from among the 1891 seeds collected from T. japonicum in the parks, indicating ongoing hybridization in the field. T. officinale and hybrids were genetically differentiated between the two parks independent of the ploidy level; the allele frequency of T. officinale and tri- and tetraploid hybrids were similar within each park but different between the two parks. This suggests that the origins of hybrids were similar within the park but different between the parks. Overall, our results suggest that hybridization, including backcross, is an ongoing process, and that genetically diverse hybrids with various origins have been spreading in western Japan, probably because hybridization enhanced invasiveness at native habitat.     

Influences of the genetic neighborhood on ramet reproductive success in a clonal desert cactus

Clonal structure in clonal plants can affect sexual reproduction. Individual ramets can decrease reproduction if their neighbors are ramets of the same genet due to inbreeding depression or self-incompatibility. We assessed ramet reproductive success in the partial self-incompatible Ferocactus robustus (Cactaceae) as a function of floral display size in focal ramets and floral display size and clonal structure of their reproductive neighborhoods. Ramets were labeled, sized in number of stems, mapped and genetically identified through RAPD markers in one population. A pollen dispersal area of 15-m radius was established for each ramet to determine the clonal diversity in the neighborhoods. Flower production and fruit set were counted on a monthly basis during one reproductive season as a surrogate of ramet fitness. We expected a decrease in individual ramet reproductive success as a function of the number of reproductive ramets of the same genet in the neighborhood. A total of 272 sampled ramets revealed 116 multilocus genotypes, showing high clonal diversity in the population (G/N = 0.43, D = 0.98). Clonal diversity of neighborhoods ranged from 0.06 to 1 and fruit set varied from 0 to 76.9%. Individual ramet reproductive success was influenced by (1) mate availability, (2) floral display size of a genet within the reproductive neighborhood, and (3) the proportion of distinguishable genotypes. Floral display size of genets and ramets coupled with the genetic diversity within the reproductive neighborhood determines the low sexual reproduction in F. robustus.     

Molecular diversity and phylogeny of indigenous <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> strains associated with <Emphasis Type="Italic">Trifolium repens</Emphasis> plants in Romania

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.