Impact of the flower stalk-boring moth Neoschoenobia testacealis (Lepidoptera: Crambidae) and water-level fluctuations on the flower and fruit production of the yellow water lily Nuphar subintegerrima (Nymphaeaceae) in irrigation ponds of western Japan

The flower and fruit production of the yellow water lily Nuphar subintegerrima and the seasonal occurrence of the flower stalk-boring moth Neoschoenobia testacealis were surveyed in two irrigation ponds in western Japan. Water-level fluctuations at each pond were measured. Flowering was observed from late May to September, and fruiting from late May to October. The yellow water lily produced cumulative numbers of 189 and 181 flowers and 121 and 129 fruits in the two ponds’ quadrats. The fruit setting rates at the two ponds were 0.64 and 0.71, and decreased in the summer. The percentage of bored flower stalks was 16 and 5%. Mature larvae bored flower stalks severely during the summer. Whenever the boring larvae attacked the flower stalks, the flower buds and flowers were aborted. There was a significant negative correlation between the fruit setting rate and the boring rate per month (number of bored flower stalks/number of all flower stalks). Whenever abruptly rising water levels (about 10 cm) submerged the flowers, the flowers were aborted too and could not set fruits. The percentages of non-setting fruit due to the rising water levels at the two ponds were 6 and 8%. Overall, our results indicated that flower stalk boring was a more important factor than water-level fluctuation in the sexual reproduction of N. subintegerrima and that N. subintegerrima was well suited to small irrigation ponds.     

Genetic diversity and geographical differentiation of Dipteronia Oliv. (Aceraceae) endemic to China as revealed by AFLP analysis

The genus Dipteronia Oliv. endemic to central and southern China consists of two species, Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry, both of them are rare and endangered. AFLP markers were used to characterize the genetic diversity and geographical differentiation of the genus. Eight out of 32 PstI + 3/MseI + 3 selective primer combinations screened were applied to the analysis on 142 individuals of 17 D. sinensis and 4 D. dyeriana populations, respectively. A total of 324 fragments with 316 polymorphic were amplified. The proportion of polymorphic loci (PPB) was 97.53%. The Nei's gene diversity in D. sinensis and D. dyeriana was 0.3319 and 0.3047, respectively. About 43.6% (G_ST = 0.4356) of the genetic variation occurred among the populations, indicating a relatively high genetic differentiation among the populations. Cluster analysis grouped the 21 populations into two groups according to their species delimitation. The populations of D. sinensis were further divided into three subgroups corresponding to their geographical distributions. Correlation analysis revealed a significant correlation (p < 0.05) between geographical distance and genetic distance of these populations, suggesting that the relatively high genetic differentiation among the populations of D. sinensis might be caused by geographical isolation.     

Genetic structure of Pyrenophora teres net and spot populations as revealed by microsatellite analysis

The population structure of the fungal pathogen Pyrenophora teres, collected mainly from different regions of the Czech and Slovak Republics, was examined using a microsatellite analyses (SSR). Among 305 P. teres f. teres (PTT) and 82 P. teres f. maculata (PTM) isolates that were collected, the overall gene diversity was similar (? = 0.12 and ? = 0.13, respectively). A high level of genetic differentiation (F_ST = 0.46; P < 0.001) indicated the existence of population structure. Nine clusters that were found using a Bayesian approach represent the genetic structure of the studied P. teres populations. Two clusters consisted of PTM populations; PTT populations formed another seven clusters. An exact test of population differentiation confirmed the results that were generated by Structure. There was no difference between naturally infected populations over time, and genetic distance did not correlate with geographical distance. The facts that all individuals had unique multilocus genotypes and that the hypothesis of random mating could not be rejected in several populations or subpopulations serve as evidence that a mixed mating system plays a role in the P. teres life cycle. Despite the fact that the genetic differentiation value between PTT and PTM (F_ST = 0.30; P < 0.001) is lower than it is between the populations within each form (F_ST = 0.40 (PTT); F_ST = 0.35 (PTM); P < 0.001) and that individuals with mixed PTT and PTM genomes were found, the two forms of P. teres form genetically separate populations. Therefore, it can be assumed that these populations have most likely undergone speciation.     

Genetic diversity of wild populations of Rheum tanguticum endemic to China as revealed by ISSR analysis

Rheum tanguticum is an important but endangered traditional Chinese medicine endemic to China. The wild resources have been declining. Establishing the genetic diversity of the species would assist in its conservation and breeding program. Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population genetic structure in 13 wild populations of R. tanguticum from Qinghai Province. Thirteen selected primers produced 329 discernible bands, with 326 (92.94%) being polymorphic, indicating high genetic diversity at the species level. The Nei's gene diversity (He) was estimated to be 0.1724 within populations (range 0.1026–0.2104), and 0.2689 at the species level. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly within populations (71.02%), but variance among populations was only 28.98%. In addition, Nei's differentiation coefficients (G_st) was found to be high (0.3585), confirming the relatively high level of genetic differentiation among populations. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.573, P = 0.002), and the unweighted pair-group method using arithmetic average (UPGMA) clustering and Principal coordinates analysis (PCoA) demonstrated similar results. Meanwhile, the genetic diversity of R. tanguticum positively correlated with altitude and annual mean precipitation, but negatively correlated with latitude and annual mean temperature. This result might be an explanation that the natural distribution of R. tanguticum is limited to alpine cold areas. We propose conservation strategy and breeding program for this plant.     

Genetic diversity and inter-specific relations of western Mediterranean relic Abies taxa as compared to the Iberian A. alba

Several Abies species are currently present in the Mediterranean region and most of them are endemic taxa and tertiary relicts. Using six nuclear microsatellites, we studied the genetic structure and inter-specific relationships among West Mediterranean firs, A. pinsapo (Spain), A. maroccana and A. tazaotana (Morocco). Based on the hypothesis that A. pinsapo could historically exchange genes with A. alba growing in the Pyrenees via secondary contact, we investigated the level of genetic admixture between these species using a Bayesian approach. The studied populations showed moderate genetic diversity (mean H_E = 0.598) and a high level of genetic differentiation (F_ST = 0.225) that was especially pronounced between A. alba and the African firs. All populations experienced a strong bottleneck effect that was likely induced by climatic changes occurring in the West Mediterranean during the last glacial cycle and the Holocene. According to Bayesian clustering, both African taxa grouped together in a single cluster, the two A. pinsapo populations formed a second cluster, and two additional clusters were detected within A. alba. Our results indicate that A. tazaotana is genetically very close to A. maroccana, and hence these two taxa should probably not be considered as separate species. We found no genetic admixture between A. pinsapo and A. alba and only minor between A. pinsapo and the African fir populations suggesting an isolation effect of the Gibraltar Strait. Current limited distributions of firs in the Mediterranean region together with changing climate may lead to further deterioration of the genetic diversity levels. Hence, future efforts should focus on monitoring the demography and genetic threats to existing populations.     

Population genetic differentiation of Schisandra chinensis and Schisandra sphenanthera as revealed by ISSR analysis

Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils. are well-known Chinese medicinal plants. The population genetic variation of the two species was studied using inter simple sequence repeat (ISSR) molecular markers. High levels of genetic diversity are revealed in both S. chinensis (P = 88.36%, h = 0.2894, I = 0.4396) and S. sphenanthera (P = 84.09%, H = 0.2782, I = 0.4280). However, the population genetic differentiation is significantly different between the two species. The S. sphenanthera harbors as high as 27% of the genetic variation among populations but 73% within populations, whereas in S. chinensis 17% of the genetic variation occurs among populations and 83% within populations. Both significant (P < 0.05) heterozygosity excess and shifted mode of allele frequency distribution are detected in four out of six populations of S. chinensis and one out of five populations of S. sphenanthera, suggesting the occurrence of recent population bottlenecks in the two species. The different patterns of genetic variation in S. chinensis and S. sphenanthera are discussed in relation to their differences in pollination mechanism, geographic distribution and historical events, and the level of gene flow and genetic drift.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Genetic structure of Phlebotomus (Larroussius) ariasi populations, the vector of Leishmania infantum in the western Mediterranean: Epidemiological implications

In recent years there has been growing interest in analyzing the geographical variations between populations of different Phlebotomus spp. by comparing the sequences of various genes. However, little is known about the genetic structure of Phlebotomus ariasi. In this study, we were able to sequence a fragment of the mitochondrial Cyt b gene in 133 sandflies morphologically identified as P. ariasi and proceeding from a wide geographical range covering 35 locations in 11 different regions from five countries. The intra-specific diversity of P. ariasi is high, with 45 haplotypes differing from each other by one to 26 bases and they are distributed in two mitochondrial lineages, one limited geographically to Algeria and the other widely dispersed across Mediterranean countries. The Algerian lineage is characterized by having 13 fixed polymorphisms and is made up of one sole haplotype. The European/Moroccan P. ariasi lineage is characterized by being made up of a great diversity of haplotypes (44) which display some geographical structuring. This could be one of the multiple factors involved in the epidemiological heterogeneity of the foci of leishmaniasis. Phlebotomus chadlii is the sister group of European/Moroccan P. ariasi. The separation of the Algerian haplotype, H45, from the rest of the specimens, European/Moroccan P. ariasi and P. chadlii, is well supported by the bootstrap analysis.     

Genetic variation and relationships in Laetiporus sulphureus s. lat., as determined by ITS rDNA sequences and in vitro growth rate

The aim of this study was to characterise the genetic variation and molecular relationships of the brown rot polypore, Laetiporus sulphureus s. lat., from Europe, South America, Africa, and Asia, using ITS sequences of the nu-rDNA and by comparing the growth rate in vitro. In a NJ analysis of the sequences of 130 individuals of L. sulphureus s. lat., eight distinct clusters emerged, supported by BS values of 70-100 %. Within each cluster, the ITS rDNA sequence variation was below 3 %. The sequences were also analysed together with Laetiporus sequences available from GenBank. Results demonstrated the possible presence of L. huroniensis in Europe (invalidly named L. montanus) and L. gilbertsonii in South America, and provided more information on the Pan-American and European distribution of one of the clades, currently known in North America as L. sulphureus. L. conifericola formed a separate distinct clade. Moreover, the analysis revealed two unknown Laetiporus taxa in Korea, one in South Africa, and one in Europe. As L. sulphureus is described from Europe (France), and we show that more than one taxon exist here, it is presently not possible to define L. sulphureus s. str. Certain biological differences between some of the clades (in vitro growth rates, chemical composition, and pigmentation) were demonstrated and discussed.     

Genetic differentiation in endangered Gynostemma pentaphyllum (Thunb.) Makino based on ISSR polymorphism and its implications for conservation

Studies were performed to investigate the genetic variation of 14 natural populations of Gynostemma pentaphyllum (Thunb.) Makino, an outcrossing clonal plant species in China, using inter-simple sequence repeat (ISSR) markers. Fourteen selected primers were used to amplify DNA samples from 140 individuals, and totally 194 loci were detected. The percentage of polymorphic bands (PPBs) showed that the genetic diversity was pretty high at the species level (PPB = 96.39%) but quite low at the population level (PPB = 1.03–25.26%). Shannon's information index (I) and Nei's gene diversity (h) displayed a similar trend to PPB. According to the hierarchical analysis of molecular variance (AMOVA) and Nei's analysis of gene diversity, the percentages of genetic variation among populations were 88.66 and 88.94%, respectively, indicating a high level of inter-population genetic differentiation. The low levels of genetic diversity within populations and high genetic differentiation among populations were assumed to result from the limited gene flow, the clonal nature and genetic drift. Based on the genetic data, effective conservation strategies were proposed for conserving this traditional Chinese medicinal herb. Concerning the management of G. pentaphyllum, we suggested that in situ conservation be an important and practical measure for maintaining the genetic diversity and that a possibly maximum number of populations be conserved. Populations EMS and HLT, in which particularly low levels of genetic variation were characterized, should be given the priority for ex situ conservation.     

Four new hypocretenolides (guaian-12,5-olides) from Leontodon rosani (Asteraceae,Cichorieae)

Subaerial parts of Leontodon rosani (Ten.) DC. collected in the South Italian Basilicata region yielded four new hypocretenolides, 11β,13-dihydro-15-hydroxyhypocretenolide (2), 15-hydroxyhypocretenolide (3), 11β,13-dihydro-15-hydroxyhypocretenolide-β-glucopyranoside (4), and 15-hydroxyhypocretenolide-β-glucopyranoside (5). Structure elucidations were based on MS experiments and extensive one- and two-dimensional NMR experiments. None of the isolated compounds showed any activity in an in vitro acetylcholinesterase inhibition assay. The chemosystematic impact of the occurrence of hypocretenolides in L. rosani is discussed with regards to the supposed allotetraploid origin of the title species. Moreover, HPLC retention times and online mass signals for all currently known hypocretenolide derivatives are described to ease future studies screening for this rare group of natural products.     

Hybridizations and genetic relationships among Lindernia species (Scrophulariaceae): L. procumbens and two subspecies of L. dubia

Lindernia procumbens and L. dubia are common annual weeds in flooded rice fields of Japan. Two subspecies of L. dubia, subsp. major and subsp. dubia, are usually recognized in Japan but they are both regarded as synonyms of L. dubia elsewhere. In a cluster analysis based on AFLP, most L. dubia subsp. major formed a separate cluster from L. dubia subsp. dubia although 11% of haplotypes classified using AFLP were not coincident with classification using the shape of leaf bases, which is the commonly used identification trait. Artificial F₁ plants between L. procumbens and L. dubia subsp. major, and between L. procumbens and L. dubia subsp. dubia did not produce seed. Forty percent of capsules produced by F₁ plants from these two subspecies were slimmer and 80% pollen were sterile in slimmer capsules. However, seed number of most F₁ capsules was not different from that of self-fertilized plants, suggesting that there was no complete reproductive isolation between the subspecies. Natural hybridization of these subspecies may have occurred but we are not aware of it because F₁ plants are rare and F₂ plants are indistinguishable from these subspecies.     

Re-evaluation of morphological and chloroplast DNA variation in Juniperus osteosperma Hook and Juniperus occidentalis Torr. Little (Cupressaceae) and their putative hybrids

Relationships between variation in cpDNA and morphology were examined to test the hypothesis of hybridization between Juniperus osteosperma and Juniperus occidentalis. Principal components analysis of 11 taxonomically-important characters distinguished individuals collected from eastern Nevada and Utah from those of southern Oregon. In contrast, many individuals collected from sympatric populations in western Nevada were morphologically intermediate to these two groups. Comparative sequencing of the trnS-trnG intergenic spacer and restriction site analysis of a trnL-trnF PCR product revealed nine haplotypes, and examination of haplotype-morphology associations allowed identification of species-specific genetic markers as well as those that transcend species limits. The confinement of morphological intermediacy and transcendent haplotypes to zones of sympatry, the discovery of haplotypes characteristic of one species in the morphological background of the other, and determination that the character intermediacy encountered is of the type expected under interspecific gene flow are marshaled in support of introgressive hybridization between J. osteosperma and J. occidentalis.     

Genotypic characterization and species identification of Fasciola spp. with implications regarding the isolates infecting goats in Vietnam

Ribosomal RNA sequences (361 or 362 bp) of the second internal transcribed spacer 2 (ITS-2) and a portion of mitochondrial cox1 (423 bp) for Fasciola spp. obtained from specimens collected in indigenous and hybrid goats and sheep in Vietnam were characterized for genotypic status and hybridization/introgression. Alignment of 48 ITS-2 sequences (also those from goats and sheep in this study) indicates that F. gigantica and F. hepatica differ typically from each other at seven sites whereas one of these is a distinguishing deletion (T) at the 327th position in F. gigantica relative to F. hepatica. The isolates from the mountainous goats in the North of Vietnam (Yen Bai province) showed the ITS-2 composition relatively identical to that of F. hepatica. The ITS-2 sequences from populations of Fasciola isolates in goats had probably experienced introgression/hybridization as reported previously in other ruminants and humans. All Vietnamese goat-of-origin specimens had high pairwise percentage of mitochondrial cox1 sequences to F. gigantica (97-100%), and very low identity to F. hepatica (91-93%), suggesting their maternal linkage to be traced to F. gigantica. The presence of hybrid and/or introgressed populations of liver flukes bearing genetic material from both F. hepatica and F. gigantica in the goats/sheep in Vietnam, regardless of indigenous or imported hosts, appears to be the first demonstration from a tropical country.     

利用微卫星遗传标记探讨达氏鳇的多倍体倍性

鲟形目(Acipenseriformes)鱼类是一类多倍体起源的鱼类,种问易于杂交,且由于存在大量微型染色体,其染色体数目和倍性也难于确定.到目前为止包括达氏鳇(Huso dauricus)在内的一些鲟鱼物种基因组大小和染色体倍性仍旧存在争议.本实验采用微卫星遗传标记技术,通过观察9个微卫星位点的等位基因数目,发现达氏鳇在大部分位点中显示的倍性大于二倍性,同时利用多倍体分析软件POLYSAT推断了26尾达氏鳇的个体倍性.结果表明,26尾达氏鳇中有20尾显示为六倍体,占总数的76.92％;4尾显示为八倍体,占总数的15.38％;2尾显示为四倍体,占总数的7.79％.因此,我们认为达氏鳇应该为八倍体物种,结果中出现的四倍体和六倍体个体,是由于这些个体在我们选取的微卫星位点中存在部分纯合等位基因,导致了多数个体显示为六倍体.这一结论与近年来支持达氏鳇为进化性八倍体物种的研究结果一致.     

Genetic diversity and population structure of the endangered species Psammosilene tunicoides revealed by DALP analysis

Psammosilene tunicoides W. C. Wu et C.Y. Wu (Caryophyllaceae) is a rare plant endemic to China. We used the direct amplification of length polymorphism (DALP) method to analyse the genetic diversity of 17 natural populations of this species from Yunnan, Sichuan and Guizhou provinces. The percentage of polymorphic bands (PPB) was 99.1% at the species level and 19.9% at the population level. The total genetic diversity (Ht) was 0.2827; the genetic diversity within populations (H_s) was 0.0594 and the coefficient of gene differentiation (G_st) was 0.7899. The results indicated low levels of genetic diversity within populations and extremely high genetic differentiation among populations. This pattern might be caused by genetic drift resulting from intensive harvesting and habitat fragmentation. It might also be a result of local adaptation. We further performed experiments on its breeding system and found a high rate of selfing in P. tunicoides, which may be partially responsible for its population structure.     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.