Genetic diversity and structure of Atta robusta (Hymenoptera,Formicidae, Attini), an endangered species endemic to the restinga ecoregion

The genetic diversity and structure of the ant Atta robusta were assessed by ISSR (inter-simple sequence repeats) in 72 colonies collected from 10 localities in the Brazilian states of Espírito Santo (48 colonies) and Rio de Janeiro (24 colonies). The ISSR pattern included 67 bands, 51 of them (76.1%) polymorphic. Analysis of molecular variance (AMOVA) revealed a high level (57.4%) of inter-population variation, which suggested a high degree of genetic structure that was confirmed by UPGMA (unweighted pair-group method using an arithmetic average) cluster analysis. The significant correlation between genetic and geographic distances (r = 0.64, p < 0.05) indicated isolation that reflected the distance between locations. Overall, the populations were found to be genetically divergent. This finding indicates the need for management plans to preserve and reduce the risk of extinction of A. robusta.     

Small effect of fragmentation on the genetic diversity of Dalbergia monticola, an endangered tree species of the eastern forest of Madagascar, detected by chloroplast and nuclear microsatellites

Background and Aims

The oriental forest ecosystem in Madagascar has been seriously impacted by fragmentation. The pattern of genetic diversity was analysed on a tree species, Dalbergia monticola, which plays an important economic role in Madagascar and is one of the many endangered tree species in the eastern forest.

Methods

Leaves from 546 individuals belonging to 18 small populations affected by different levels of fragmentation were genotyped using eight nuclear (nuc) and three chloroplast (cp) microsatellite markers.

Key Results

For nuclear microsatellites, allelic richness (R) and heterozygosity (H_e,nuc) differed between types of forest: R = 7·36 and R = 9·55, H_e,nuc = 0·64 and H_e,nuc = 0·80 in fragmented and non-fragmented forest, respectively, but the differences were not significant. Only the mean number of alleles (N_a,nuc) and the fixation index F_IS differed significantly: N_a,nuc = 9·41 and N_a,nuc = 13·18, F_IS = 0·06 and F_IS = 0·15 in fragmented and non-fragmented forests, respectively. For chloroplast microsatellites, estimated genetic diversity was higher in non-fragmented forest, but the difference was not significant. No recent bottleneck effect was detected for either population. Overall differentiation was low for nuclear microsatellites (F_ST,nuc = 0·08) and moderate for chloroplast microsatellites (F_ST,cp = 0·49). A clear relationship was observed between genetic and geographic distance (r = 0·42 P < 0·01 and r = 0·42 P = 0·03 for nuclear and chloroplast microsatellites, respectively), suggesting a pattern of isolation by distance. Analysis of population structure using the neighbor-joining method or Bayesian models separated southern populations from central and northern populations with nuclear microsatellites, and grouped the population according to regions with chloroplast microsatellites, but did not separate the fragmented populations.

Conclusions

Residual diversity and genetic structure of populations of D. monticola in Madagascar suggest a limited impact of fragmentation on molecular genetic parameters.     

Genetic variation in cultivated Rheum tanguticum populations

To examine whether cultivation reduced genetic variation in the important Chinese medicinal plant Rheum tanguticum, the levels and distribution of genetic variation were investigated using ISSR markers. Fifty-eight R. tanguticum individuals from five cultivated populations were studied. Thirteen primers were used and a total of 320 DNA bands were scored. High levels of genetic diversity were detected in cultivated R. tanguticum (PPB = 82.19, H = 0.2498, H_B = 0.3231, I = 0.3812) and could be explained by the outcrossing system, as well as long-lived and human-mediated seed exchanges. Analysis of molecular variance (AMOVA) showed that more genetic variation was found within populations (76.1%) than among them (23.9%). This was supported by the coefficient of gene differentiation (G_st = 0.2742) and Bayesian analysis (θ_B = 0.1963). The Mantel test revealed no significant correlation between genetic and geographic distances among populations (r = 0.1176, p = 0.3686). UPGMA showed that the five cultivated populations were separated into three clusters, which was in good accordance with the results provided by the Bayesian software STRUCTURE (K = 3). A short domestication history and no artificial selection may be an effective way of maintaining and conserving the gene pools of wild R. tanguticum.     

Tests for inbreeding and outbreeding depression and estimation of population differentiation in the bird-pollinated shrub Grevillea mucronulata

Background and Aims

Plants show patterns of spatial genetic differentiation reflecting gene flow mediated by pollen and seed dispersal and genotype × environment interactions. If patterns of genetic structure are determined largely by gene flow then they may be useful in predicting the likelihood of inbreeding or outbreeding depression but should be less useful if there is strong site-specific selection. For many Australian plants little is known about either their population genetics or the effects on mating systems of variation in pollen transfer distances. Experimental pollinations were used to compare the reproductive success of bird-adapted Grevillea mucronulata plants mated with individuals from a range of spatial scales. A hierarchical survey of microsatellite DNA variation was also conducted to describe the scale of population differentiation for neutral markers.

Methods

The effects of four pollen treatments on reproductive performance were compared. These treatments were characterized by transfer of pollen from (a) neighbouring adults; (b) an adjacent cluster of adults (30–50 m distant); (c) a distant cluster (>5 km distant); and (d) open pollination. Sets of 17·9 ± 3·3 leaves from each of 15 clusters of plants were genotyped and spatial autocorrelation and F statistics were used to describe patterns of genetic structure.

Key Results

Grevillea mucronulata displayed evidence of both inbreeding and outbreeding depression, with ‘intermediate’ pollen producing consistently superior outcomes for most aspects of fitness including seed set, seed size, germination and seedling growth. Significant genotypic structuring was detected within clusters (spatial autocorrelation) and among adjacent clusters and clusters separated by >5 km distance (F_ST = 0·07 and 0·10).

Conclusions

The superior outcome of intermediate pollen transfer and genetic differentiation of adjacent clusters suggests that G. mucronulata selection disfavours matings among closely and distantly related neighbours. Moreover, the performance of open-pollinated seedlings was poor, implying that current mating patterns are suboptimal.     

Genetic divergence between Melipona quadrifasciata Lepeletier (Hymenoptera,Apidae) populations

Melipona quadrifasciata is a stingless bee widely found throughout the Brazilian territory, with two recognized subspecies, M. quadrifasciata anthidioides, that exhibits interrupted metasomal stripes, and M. quadrifasciata quadrifasciata, with continuous metasomal stripes. This study aimed to estimate the genetic variability of these subspecies. For this purpose, 127 colonies from 15 Brazilian localities were analyzed, using nine species-specific microsatellite primers. At these loci, the number of alleles ranged from three to 15 (mean: 7.2), and the observed heterozygosity (H_o) ranged from 0.03–0.21, while the expected heterozygosity (H_e) ranged from 0.23–0.47. The genetic distances among populations ranged from 0.03–0.45. The F_ST multilocus value (0.23) indicated that the populations sampled were structured, and the clustering analysis showed the formation of two subgroups and two more distant populations. The first group contained the subspecies M. quadrifasciata quadrifasciata, and the other, the subspecies M. quadrifasciata anthidioides and the two M. quadrifasciata populations with continuous metasomal stripes from northern Minas Gerais. These results confirmed that the yellow metasomal stripes alone are not a good means for correctly identifying the different subspecies of M. quadrifasciata.     

Evolution under domestication: ongoing artificial selection and divergence of wild and managed Stenocereus pruinosus (Cactaceae) populations in the Tehuac��n Valley, Mexico

Background and Aims

The Tehuacán Valley in Mexico is a principal area of plant domestication in Mesoamerica. There, artificial selection is currently practised on nearly 120 native plant species with coexisting wild, silvicultural and cultivated populations, providing an excellent setting for studying ongoing mechanisms of evolution under domestication. One of these species is the columnar cactus Stenocereus pruinosus, in which we studied how artificial selection is operating through traditional management and whether it has determined morphological and genetic divergence between wild and managed populations.

Methods

Semi-structured interviews were conducted with 83 households of three villages to investigate motives and mechanisms of artificial selection. Management effects were studied by comparing variation patterns of 14 morphological characters and population genetics (four microsatellite loci) of 264 plants from nine wild, silvicultural and cultivated populations.

Key Results

Variation in fruit characters was recognized by most people, and was the principal target of artificial selection directed to favour larger and sweeter fruits with thinner or thicker peel, fewer spines and pulp colours others than red. Artificial selection operates in agroforestry systems favouring abundance (through not felling plants and planting branches) of the preferred phenotypes, and acts more intensely in household gardens. Significant morphological divergence between wild and managed populations was observed in fruit characters and plant vigour. On average, genetic diversity in silvicultural populations (H_E = 0·743) was higher than in wild (H_E = 0·726) and cultivated (H_E = 0·700) populations. Most of the genetic variation (90·58 %) occurred within populations. High gene flow (Nm_FST > 2) was identified among almost all populations studied, but was slightly limited by mountains among wild populations, and by artificial selection among wild and managed populations.

Conclusions

Traditional management of S. pruinosus involves artificial selection, which, despite the high levels of gene flow, has promoted morphological divergence and moderate genetic structure between wild and managed populations, while conserving genetic diversity.     

ISSR and DAMD markers revealed high genetic variability within Flavoparmelia caperata in Western Himalaya (India)

Flavoparmelia caperata (L.) Hale is medicinally very important and possesses antifungal and antibacterial activities. F. caperata is the only species found in India. Inter simple sequence repeat (ISSR) and Directed amplification of minisatellite DNA (DAMD) methods were used to analyze the genetic variability within F. caperata from the Western Himalayan region of India. Eleven ISSR and 10 DAMD primers produced 139 and 117 polymorphic bands, and detected 91.44 and 82.34 % polymorphisms, respectively. Cumulative band data generated for ISSR and DAMD markers resulted in 86.86 % polymorphism across all the accessions of F. caperata. The average Polymorphic information content (PIC) value obtained with ISSR, DAMD, and cumulative band data were 0.28, 0.27, and 0.27, respectively. The clustering of the F. caperata accessions in the UPGMA dendrogram showed that these accessions are intermingled with each other in different subclusters irrespective of their geographical affiliations. The pattern of genetic variations within F. caperata accessions could be due to free exchange of spores that might have taken place among these accessions in the wild. ISSR and DAMD markers efficiently and reliably resulted in discrete banding patterns and polymorphic profiles. These markers despite targeting different regions of genome, revealed almost similar levels of polymorphism across all the accessions. The wide range of genetic distance and high level of polymorphism detected by ISSR and DAMD reflected a high genetic variability among the different accessions of F. caperata.     

Studies on genetic diversity among populations of Persicaria barbata (L.) H. Hara from India based on internal transcribed spacer sequences of nuclear ribosomal DNA

Internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 16 populations of Persicaria barbata (L.) H. Hara (Polygonaceae) belonging to five geographical locations of India (Arunachal Pradesh, Himachal Pradesh, Bihar, Karnataka and Andaman Island) was sequenced. Analysis of nucleotide sequences reveals polymorphism among the populations. UPGMA analysis conducted on the ITS datasets shows that the sampled populations of P. barbata are grouped according to their geographic locations and are supposed to be evolved under reproductive isolation which most probably are due to the long distance distribution and population fragmentation.     

Microsatellites reveal substantial among-population genetic differentiation and strong inbreeding in the relict fern Dryopteris aemula

Background and Aims

A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations.

Methods

Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores.

Key Results

Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (H_T = 0·447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (F_ST = 0·520). All loci strongly departed from Hardy–Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class.

Conclusions

The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy–Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.     

Highly differentiated populations of the narrow endemic and endangered species Primula cicutariifolia in China,revealed by ISSR and SSR

The perennial herb Primula cicutariifolia Pax is an endangered and endemic species with narrow distribution in eastern Anhui and Zhejiang provinces of China. In this study, the levels of genetic variation and the pattern of genetic structure in five natural populations of P. cicutariifolia were assessed by inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers. Both markers revealed that there was remarkably low genetic variation within populations (e.g., H_e = 0.19 and 0.18, for ISSR and SSR respectively) and high differentiation among populations (G_ST = 0.714 and 0.611; Φ_ST = 0.698 and 0.599, for ISSR and SSR respectively). The level of population genetic diversity was correlated to population size only detected by ISSR markers. The genetic structure of P. cicutariifolia may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self breeding system and biennial life form. To protect and avoid disappearance of P. cicutariifolia, much more attentions should be paid to protect all the remnant populations and their habitats, and three management units, i.e. Tianmushan, Damodao, and Panan units, were proposed.     

Conservation study of an endangered stingless bee (Melipona capixaba—Hymenoptera: Apidae) with restricted distribution in Brazil

Melipona capixaba, popularly known as “uruçu preta”, is a stingless bee restricted to the mountainous Atlantic Rainforest areas of Espírito Santo State, Brazil. Due to the endemism and small population size, this species discovered in 1994 is now considered “vulnerable to extinction”. Using ISSR, PCR–RFLP and microsatellites markers, we studied the genetic variability and structure of M. capixaba from 88 colonies collected throughout the distribution area of the species within Espírito Santo State. The microsatellite, ISSR and mitochondrial haplotype analyses showed that M. capixaba has low genetic variability compared to other insect species. The molecular analyses also indicated a high genetic similarity among the M. capixaba samples, with no clear pattern of structuring. The analyses of molecular variance results indicated that most of the total genetic variation in M. capixaba was explained by the genetic diversity within local populations. Results suggest that the analyzed samples could be treated as a single population for preservation purposes. Thus, given its endemism, local adaptation and low number of natural colonies, efforts for the conservation of M. capixaba should focus on preservation and increasing the number of colonies in the wild, so that M. capixaba can support constant captures and the effects of habitat deforestation in Espírito Santo State.     

Biased morph ratios and skewed mating success contribute to loss of genetic diversity in the distylous Pulmonaria officinalis

Background and Aims

In heterostylous plant species, skewed morph ratios are not uncommon and may arise from a range of factors. Despite the recognized importance of skewed morph ratios on overall reproductive success within populations, little is known about the impact of skewed morph ratios on population genetic diversity and differentiation in heterostylous species. This study specifically aimed to clarify the effect of population size and morph bias on population genetic diversity and differentiation in the temperate forest herb Pulmonaria officinalis. This species is characterized by a distylous breeding system and shows morph-specific differences in reproductive success.

Methods

Genetic diversity was determined for 27 P. officinalis populations in northern Belgium by using eight recently developed microsatellite markers. Multiple regressions were used to assess the relationship between genetic diversity, morph bias and population size, and F_ST-values were calculated for short- and long-styled morphs separately to study genetic differentiation as a function of morph type.

Key Results

For all genetic measures used, morph bias was more important in explaining patterns of genetic diversity than population size, and in all cases patterns of population genetic diversity followed a quadratic function, which showed a symmetrical decrease in genetic diversity with increasing morph bias. However, probably due to the reproductive advantage of L-morphs relative to S-morphs, maximum genetic diversity was found in populations showing an excess of L-morphs (60·7 % L-morph). On the other hand, no significant difference in pairwise genetic distances between populations was observed between L- (0·107) and S-morphs (0·106).

Conclusions

Our results indicate that significant deviations from equal morph ratios not only affect plant reproductive success but also population genetic diversity of heterostylous plant species. Hence, when defining conservation measures for populations of heterostylous plant species, morph ratios should be considered as an important trait affecting their long-term population viability.     

Differentiation between populations of Japanese grenadier anchovy (Coilia nasus) in Northwestern Pacific based on ISSR markers: Implications for biogeography

Northwestern Pacific provides unique scenarios for studying the roles of geography and ecology in driving population divergence and speciation. To elucidate the phylogeographic pattern of Coilia nasus in Northwestern Pacific, we examined ninety individuals from five localities along the coastal regions of China and the Ariake Bay of Japan by using seven ISSR markers. Analyses of molecular variance (AMOVA) showed that genetic differentiation among groups is relatively high (F_CT = 0.1904; P = 0.000). Bayesian analysis of ISSR data also revealed significant population structuring between Chinese and Japanese locations. Phylogenetic reconstructions show reciprocal monophyly in populations between China and the Ariake Bay of Japan. We conclude that the present-day phylogeographic pattern is the result of genetic isolation between Japanese and Chinese populations in the Northwestern Pacific following the glacial retreat, and that life-history traits and ecology may play a pivotal role in shaping the realized geographical distribution pattern of this species.     

Panax ginseng extract modulates oxidative stress,DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B1

Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on DNA and gene expression in rat. Female Sprague–Dawley rats were divided into eight groups (ten rats/group) and treated for 12 weeks including the control group, the group having received AFB₁ (80 µg/kg bw), the group having received FB₁ (100 µg/kg bw), the group having received AFB₁ plus FB₁ and the groups having received PGE (20 mg/kg bw) alone or with AFB₁ and/or FB₁. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB₁ and/or FB₁ plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB₁ and FB₁ have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties.     

Genetic,Epigenetic, and HPLC Fingerprint Differentiation between Natural and Ex Situ Populations of Rhodiola sachalinensis from Changbai Mountain,China

Rhodiola sachalinensis is an endangered species with important medicinal value. We used inter-simple sequence repeat (ISSR) and methylation-sensitive amplified polymorphism (MSAP) markers to analyze genetic and epigenetic differentiation in different populations of R. sachalinensis, including three natural populations and an ex situ population. Chromatographic fingerprint was used to reveal HPLC fingerprint differentiation. According to our results, the ex situ population of R. sachalinensis has higher level genetic diversity and greater HPLC fingerprint variation than natural populations, but shows lower epigenetic diversity. Most genetic variation (54.88%) was found to be distributed within populations, and epigenetic variation was primarily distributed among populations (63.87%). UPGMA cluster analysis of ISSR and MSAP data showed identical results, with individuals from each given population grouping together. The results of UPGMA cluster analysis of HPLC fingerprint patterns was significantly different from results obtained from ISSR and MSAP data. Correlation analysis revealed close relationships among altitude, genetic structure, epigenetic structure, and HPLC fingerprint patterns (R² = 0.98 for genetic and epigenetic distance; R² = 0.90 for DNA methylation level and altitude; R² = –0.95 for HPLC fingerprint and altitude). Taken together, our results indicate that ex situ population of R. sachalinensis show significantly different genetic and epigenetic population structures and HPLC fingerprint patterns. Along with other potential explanations, these findings suggest that the ex situ environmental factors caused by different altitude play an important role in keeping hereditary characteristic of R. sachalinensis.     

Genetic diversity analysis among and within populations of Pogostemon cablin from China with ISSR and SRAP markers

The diversity and genetic relationship among and within 16 populations of Pogostemon cablin from China were analyzed using ISSR and SRAP markers. High percentage of polymorphism observed by these two markers indicated a high level of genetic diversity existed among P. cablin populations. The percentage of polymorphic bands revealed by these two markers showed genetic variations were much higher between populations of P. cablin than within. The UPGMA clustering results using ISSR, SRAP or ISSR + SRAP combination showed most accessions from the same or adjacent regions were classified together. However, cluster results based on the absolute contents of patchouli alcohol and pogostone did not keep in good accordance with their geographic distributions, which revealed that the forming of patchouli alcohol and pogostone were relevant to the cultivation conditions except for genetic factors and environmental conditions. Finally, an appropriate strategy for conserving the patchouli germplasm was proposed.     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.     

Thermostable Xanthine Oxidase Activity from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t_1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg²⁺, Ag⁺ and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.     

Allozyme variation of populations of Castanopsis carlesii (Fagaceae) revealing the diversity centres and areas of the greatest divergence in Taiwan

• Background and Aims The genetic variation and divergence estimated by allozyme analysis were used to reveal the evolutionary history of Castanopsis carlesii in Taiwan. Two major questions were discussed concerning evolutionary issues: where are the diversity centres, and where are the most genetically divergent sites in Taiwan?• Methods Twenty-two populations of C. carlesii were sampled throughout Taiwan. Starch gel electrophoresis was used to assay allozyme variation. Genetic parameters and mean F_ST values of each population were analysed using the BIOSYS-2 program. Mean F_ST values of each population against the remaining populations, considered as genetic divergence, were estimated using the FSTAT program.• Key Results Average values of genetic parameters describing the within-population variation, the average number of alleles per locus (A = 2·5), the effective number of alleles per locus (A_e = 1·38), the allelic richness (A_r = 2·38), the percentage of polymorphic loci (P = 69 %), and the expected heterozygosity (H_e = 0·270) were estimated. High levels of genetic diversity were found for C. carlesii compared with other local plant species. Genetic differentiation between populations was generally low.• Conclusions From the data of expected heterozygosity, one major diversity centre was situated in central Taiwan corroborating previous reports for other plant species. According to the mean F_ST value of each population, the most divergent populations were situated in two places. One includes populations located in north central Taiwan between 24·80°N and 24·20°N. The other is located in south-eastern Taiwan between 22·40°N and 23·10°N. These two regions are approximately convergent with the most divergent locations determined for several other plant species using chloroplast DNA markers published previously. An important finding obtained from this study is that unordered markers like allozymes can be used to infer past population histories as well as chloroplast DNA markers do.     

Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, K_D (equilibrium constant) and B_max (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb’s free energy (∆G = −78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.