Molecular phylogeny and divergence times of Onosma (Boraginaceae s.s.) based on nrDNA ITS and plastid rpl32‐trnL(UAG) and trnH–psbA sequences

We analysed 87 species of Onosma (Boraginaceae) from throughout its distribution range to investigate its evolutionary history. Using nrDNA ITS and two plastid (rpl32‐trnL_(UAG) and trnH–psbA) markers, we reconstructed phylogenetic relationships within Onosma by conducting maximum parsimony, maximum likelihood, Bayesian, and BEAST analyses. The analyses revealed that Onosma as currently circumscribed is not monophyletic. However, the vast majority of Onosma species appear to belong to a single clade, the so‐called Onosma s.s. Outside of this core clade is a clade containing O. rostellata, a subclade of Sino‐Indian species and Maharanga emodii. Podonosma orientalis (as O. orientalis) appear only distantly related to Onosma but is more closely related to Alkanna, as also suggested in previous molecular studies. The Onosma s.s. clade includes all representatives of O. sect. Onosma, and encompasses three subsections, i.e. Onosma, Haplotricha and Heterotricha, corresponding to asterotrichous, haplotrichous and heterotrichous groups, respectively, but none of these subsections was retrieved as monophyletic. We observed significant incongruence between nuclear and chloroplast phylogenies regarding the phylogenetic status of the heterotrichous group. A dozen of the Iranian haplotrichous species formed a lineage which may not hybridize with asterotrichous species. Divergence time estimates suggested that the early radiation of Onosma s.l. took place at the Oligocene‐Miocene boundary and the diversification within Onosma s.s. occurred during middle to late Miocene and Pliocene.     

Evolutionary significance of hybridization in Onosma (Boraginaceae): analyses of stabilized hemisexual odd polyploids and recent sterile hybrids

Interspecific hybridization is an important evolutionary force promoting plant speciation. In the genus Onosma, one of three main evolutionary lineages presumably evolved by hybrid speciation. The assumed hybrid lineage (Heterotricha) consists of two species complexes with bimodal karyotypes containing different numbers of large (L) and small (S) chromosomes, the tetraploid Onosma pseudoarenaria (2n = 12 L + 14S) and the triploid Onosma arenaria (2n = 12 L + 8S). The latter represents a rare case of hemisexual, asymmetrically compensating allopolyploids. Representatives of the other two lineages of the genus, Haplotricha (2n = 12 L) and Asterotricha (2n = 14S), have been considered to be the ancestral taxa of O. pseudoarenaria and O. arenaria, although this has yet to be investigated critically. In the present study, we examined genetic [amplified fragment length polymorphism (AFLP), internal transcribed spacer (ITS) , and chloroplast (cp)DNA)], reproductive (pollen viability and seed production) and cytogenetic (chromosome counts, genome size assessment) patterns to resolve the hypothesized allopolyploid formations in the Heterotricha group, single or polytopic allopolyploid origins, as well as ongoing interspecific gene flow as one piece of evidence for understanding past hybrid speciation events in the genus. Discordant patterns in maternally inherited cpDNA (Heterotricha accessions bearing the haplotypes related to asterotrichous species) and the nuclear ITS and AFLP markers (Heterotricha clustering with haplotrichous Onosma fastigiata), as well as karyological features, support the hybrid origin of the stabilized Heterotricha lineage. Genetic variation that is both large and geographically correlated indicates multiple origins of Heterotricha allopolyploids or, less likely, a single origin with recurring introgression from the progenitor species. The nuclear markers and cytogenetic features also provide evidence for the ongoing hybridization between O. arenaria and Onosma echioides (2n = 14S), which gives rise to sterile triploids of 2n = 6 L + 15S. We contrast the two cases of triploids with LLS (hemisexual O. arenaria from the stabilized Heterotricha lineage) and LSS (recent sterile hybrids) karyotypes, which could help to understand the mechanisms ensuring the establishment and reproductive fitness of the odd allopolyploids in Onosma. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 89–107.     

Palynological analysis of five selected <Emphasis Type="Italic">Onosma</Emphasis> taxa

Pollen size and exine structure of the following five Onosma L. species from Slovakia and Italy were studied by light and scanning electron microscopy: Onosma echioides, O. tornensis, O. visianii, O. arenaria, and O. pseudoarenaria. Among the taxa studied, O. echioides has the smallest pollen grains in average (10.5–17.5 × 8.0–14.5 μm), and O. pseudoarenaria the largest grains in average (15.3–20.5 × 12.3–16.3 μm). Pollen grains of all 5 species are small sized, 3-syncolporate, subprolate, heteropolar, with ovate equatorial outlines and circular to rounded triangular polar outlines; the tectum is microechinate. A positive correlation is found between pollen size and chromosome number. The value of pollen characters for taxonomic purposes and the position of the taxa studied within the genus Onosma are discussed.     

Effects of nitrogen supplementation of the culture medium on the growth, total lipid content and fatty acid profiles of three microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis)

To improve the properties of microalgae as sources for biodiesel production, three microalgae (Tetraselmis subcordiformis SHOU-S05, Nannochloropsis oculata SHOU-S14 and Pavlova viridis SHOU-S16) were cultured in media supplemented with different amounts of nitrogen (0, 0.22, 0.44, 0.88 and 1.76 mmol N·L^?1). The growth, total lipid contents, lipid classes and fatty acid profiles of the three microalgae were assayed after 10 days of cultivation. The results indicated that the specific growth rates of T. subcordiformis, N. oculata and P. viridis were lowest (0.014, 0.033 and 0.018, respectively) in the treatments without nitrogen supplementation and increased significantly with increasing nitrogen supplementation. The microalgae treated with 0.22 mmol N·L^?1 had the highest total lipid contents, which were 29.77, 35.85 and 32.10 % in T. subcordiformis, N. oculata and P. viridis, respectively. The total lipid contents as well as the proportions of neutral lipid in the three microalgae decreased significantly with increasing nitrogen supplementation between 0.22 and 1.76 mmol N·L^?1. The fatty acid profiles of the three microalgae were significantly different. The obvious characteristic of the fatty acid profile of T. subcordiformis was a high amount (17.68–22.22 %) of 18:3n3. However, EPA (C20:5n3) and C16 fatty acids were significantly high in N. oculata and P. viridis, respectively. N. oculata and P. viridis accumulated more 16-carbon fatty acids and fewer polyunsaturated fatty acids in nitrogen-limited media. It is therefore suggested that a limited nitrogen treatment is effective for improving the properties of P. viridis and N. oculata as sources for biodiesel.     

Bipolaris euphorbiae as a Biological Control Agent for Wild Poinsettia (Euphorbia heterophylla): Host-Specificity and Variability in Pathogen and Host Populations

Bipolaris euphorbiae (anamorphic Ascomycota: Pleosporaceae) has been studied since the early 1980s as a potential biocontrol agent of wild poinsettia (Euphorbia heterophylla), a noxious invader of soybean fields in Brazil. A new isolate (KLN05) was arbitrarily selected after the aggressiveness of six isolates of the fungus obtained from different sites was evaluated on wild poinsettias grown from seeds obtained from six different geographic origins in Brazil: Viçosa-state of Minas Gerais; Itabuna-state of Bahia; Niterói-state of Rio de Janeiro; Nova Laranjeira, Londrina and Foz do Iguaçú-state of Paraná. Bipolaris euphorbiae grew between 10 and 30 °C, with the optimum for colony growth at 25 °C. The best temperature range for conidial germination was between 20 and 30 °C. Conidial germination was not inhibited by mixing conidia with imazethapyr (106 g of active ingredient [a.i.]/l), glyphosate (720 g of a.i./kg), fomesafen (250 g of a.i./l), carfentrazone (400 g of a.i./l), atrazine (500 g of a.i./l) and glyphosate+carfentrazone (720 g of a.i/kg+400 g of a.i/l). Bipolaris euphorbiae was inoculated on plants belonging to 32 species of 12 different plant families and was only capable of infecting E. heterophylla.     

Effect of high ferric ion concentrations on total lipids and lipid characteristics of Tetraselmis subcordiformis,Nannochloropsis oculata and Pavlova viridis

Microalgae as sources for biodiesel production have been widely investigated. Microalgae biomass, lipid content and fatty acid profiles of microalgae are limiting factors for the cost-effective production of biodiesel. In this paper, the effects of high ferric ion concentrations on three species of microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis) were studied. The microalgae were cultured in different concentrations (1.2?×?10^?2, 1.2?×?10^?1, 1.2 and 12 mmol L^?1) of ferric ion. The growth, lipid content and fatty acid profiles of the three microalgae were analysed. When algae were cultured in 1.2 mmol L^?1 ferric ion for 10 days, the final cell density and specific growth rates of T. subcordiformis, N. oculata and P. viridis decreased significantly (p?<?0.05), and the total lipid contents of the microalgae, 33.72, 37.34 and 29.48 % (dry mass) in T. subcordiformis, N. oculata and P. viridis, respectively, were higher than those at other concentrations. The neutral lipid/total lipid ratios of the three microalgae species increased with increasing ferric ion concentration. Neutral lipids accounted for 50.75, 48.37 and 46.59 % of the total lipid in T. subcordiformis, N. oculata and P. viridis, respectively, when cultured in 12 mmol L^?1 ferric ion. The proportions of saturated fatty acids in all three species cultured in 12 mmol L^?1 ferric ion were significantly higher than those cultured in lower ferric ion concentrations. An optimum ferric ion concentration can improve the properties of T. subcordiformis, N. oculata and P. viridis as sources for biodiesel.     

Protein content, amino acid composition and nitrogen-to-protein conversion factors of Ulva rigida and Ulva capensis from natural populations and Ulva lactuca from an aquaculture system, in South Africa

The South African abalone aquaculture industry is expanding and there is a lack of information on the nutritional profiles of the seaweeds that are used as feed. The current study quantified the protein contents of Ulva rigida and Ulva capensis from natural populations and Ulva lactuca from a commercial, integrated seaweed/abalone aquaculture system. Three methods of protein quantification were used: the Bradford method, crude protein, and specific nitrogen to protein (N-Prot) conversion factors, and results were compared to each other. The results showed that values obtained with the “traditional” conversion factor of 6.25 were on average higher than Bradford values by factors of 64.1 % in U. capensis, 77.1 % in U. rigida and 58.9 % in U. lactuca. This pattern is in line with other published work on seaweed and microalgae. Analyses of amino acid composition showed that aspartic acid was the most abundant amino acid and that these species were also rich in glycine and alanine but poor in histidine, methionine and cysteine. N-Prot factors were as follows: U. capensis, 5.58; U. rigida, 5.12 and U. lactuca, 5.65. An average N-Prot factor of 5.45 provides a more accurate estimate of the protein content of the Ulva species studied than the “traditional” factor of N?×?6.25.     

Patterns of genetic variation in Pilosella echioides and its selected relatives: results of variation in ploidy level,facultative apomixis and past and present hybridization

We used allozymes to elucidate the genetic variation of Pilosella echioides and P. rothiana in the Pannonian Basin and its relationship with morphology and modes of reproduction. The former species consists of sexual diploid, apomictic tetraploid, and very rare sexual tetraploid populations; the latter is exclusively tetraploid and apomictic. As expected, we detected the highest intra-population variation in diploid populations of P. echioides. Nonetheless, 73 % of populations of tetraploid P. echioides and 64 % of P. rothiana consisted of 2–7 multilocus allozyme genotypes, the means being 5.75 in P. echioides and 2.64 in P. rothiana. Both the proportion of distinguishable genotypes (G/N) per population and genotype diversity (D) per population significantly differed between diploid P. echioides (means 0.415 and 0.828, respectively) on the one hand and tetraploid P. echioides (means 0.252 and 0.387, respectively) and P. rothiana (means 0.213 and 0.347, respectively) on the other. Rather surprisingly, we found an excess of homozygotes (positive F _IS) in diploids, which indicates inbreeding. Tetraploids of P. echioides have most likely originated from only a few polyploidization events and have spread thanks to agamospermy—at least populations from the NW part of the area under study seem to be monophyletic. Genetic differences within the putatively hybridogeneous species P. rothiana are small. It seems plausible that it has a common origin and that it spreads independently of its parents (P. echioides and P. officinarum). A certain level of genetic diversity can be caused by residual sexuality or less likely by repeated polytopic hybridization between P. echioides and P. officinarum. Pilosella sterrochaetia is reported here from Hungary for the first time. It is an extremely rare primary diploid hybrid between diploid P. echioides and P. leucopsilon. Its intermediate nuclear genome size also confirms its hybrid origin.     

Geographical distribution of myxomycetes living on Cryptomeria japonica bark in Japan

There is little available data on the biogeography of myxomycetes at the regional scale within any given climate zone. To investigate the geographical distribution of these protozoans, we focused on corticolous myxomycetes associated with the bark of Cryptomeria japonica trees, which we sampled extensively throughout Japan. Myxomycete sporophores developed in 73% of 2244 moist-chamber cultures of 188 bark samples from 24 regions, including 31 species. The most abundant species were Paradiacheopsis rigida and Cribraria confusa, which accounted for over 20% of all myxomycetes sampled. Non-metric multidimensional scaling was used to analyze the distribution of myxomycetes in relation to geographical variables and bark pH. The distribution of myxomycetes was influenced by altitude, temperature and bark pH. Temperature gradients and relative abundance of species were negatively correlated in P. rigida and Comatricha laxa and positively correlated in C. confusa, Macbrideora cornea, and Diderma chondrioderma. Bark pH was also positively correlated with the relative abundance of C. confusa, D. chondrioderma, and Physarum nutans and negatively correlated with that of P. rigida, P. cribrata, Enerthnema papillatum, and E. melanospermum. Geographical distribution of corticolous myxomycete communities was determined based on temperature and bark pH, which acted as local barriers in our study area.     

First report of a green toad (Bufo viridis sensu lato) in the Early Pleistocene of Spain: Palaeobiogeographical and palaeoecological implications

For the first time unequivocal fossil remains of a green toad (Bufo viridis s.l.) are described in the Iberian Peninsula. The fossils come from the Cueva Victoria site, a late Early Pleistocene (ca. 1.1–1.2 Ma) karstic filling in semi-arid southeastern Spain (Murcia region). By extension, other remains from two other Early Pleistocene Spanish localities, Barranco León D (ca. 1.3 Ma) and Almenara-Casablanca 3 (ca. 1.1 Ma), are cautiously attributed to the group B. viridis. The B. viridis group was previously reported with some uncertainty to the west of its current distribution area in Western Europe (Spain and France) in the Pliocene (Bufo cf. viridis) and less probably in the Early Miocene (Bufo aff. viridis). Since no osteological differences have been established between the recently described extant species of B. viridis s.l. (e.g. Bufo balearicus, Bufo siculus, Bufo boulengeri, B. viridis sensu stricto and Bufo variabilis) no precise palaeobiogeographical relationships can be drawn for the Spanish fossils. However, the occurrence of a third species of bufonid toad during the Pleistocene in the South of the Iberian Peninsula raises some interesting ecological questions in relation to the local disappearance of the green toad, which can be hypothetically linked to the intensification of the Pleistocene glacial/interglacial climate dynamic or to probable competition with another toad, Bufo calamita.     

Seasonal variation in phenolic composition, antibacterial and antioxidant activities of Ulva rigida (Chlorophyta) and assessment of antiacetylcholinesterase potential

The phenolic composition and antibacterial and antioxidant activities of the green alga Ulva rigida collected monthly for 12 months were investigated. Significant differences in antibacterial activity were observed during the year with the highest inhibitory effect in samples collected during spring and summer. The highest free radical scavenging activity and phenolic content were detected in U. rigida extracts collected in late winter (February) and early spring (March). The investigation of the biological properties of U. rigida fractions collected in spring (April) revealed strong antimicrobial and antioxidant activities. Ethyl acetate and n-hexane fractions exhibited substantial acetylcholinesterase inhibitory capacity with EC₅₀ of 6.08 and 7.6 μg mL^?1, respectively. The total lipid, protein, ash, and individual fatty acid contents of U. rigida were investigated. The four most abundant fatty acids were palmitic, oleic, linolenic, and eicosenoic acids.     

Genetic diversity inferred from AFLP fingerprinting in populations of Onosma echioides (Boraginaceae) from serpentine and calcareous soils

Abstract

Onosma echioides is a non-obligate serpentinophytic borage occurring discontinuously on calcareous and serpentine outcrops at the northwest limit of its range. Mean concentrations of Ca, Mg and heavy metals in root and shoot samples of eight populations from the two soil types were first determined. Subsequently, the genetic polymorphism of the same accessions was estimated by means of Amplified Fragment Length Polymorphism (AFLP) fingerprinting technique. Root and shoot samples from serpentine outcrops showed higher levels of Ni, Cr and Mg, and lower Ca/Mg ratios compared with those from calcareous soils. Based on 353 polymorphic AFLP bands, the two edaphic groups of populations showed comparable levels of genetic diversity. A remarkable genetic differentiation between populations and a high level of within-population genetic variance were found. Results of Mantel's test supported a significant correlation between genetic and geographical distances, while no difference in relation to the edaphic factor was detected. Molecular data suggested isolation as the key factor shaping the infraspecific genetic structure of O. echioides, which may be in relation with the short-distance, zoochorous systems of seed dispersal and pollination of this species.     

Association of loganin contents with the genetic characterization of natural populations of Palicourea rigida Kunth determined by AFLP molecular markers

Extracts of the medicinal plant Palicourea rigida Kunth, popularly known as douradinha, are widely used for treating urinary tract disorders. Unfortunately, nowadays this is one of the species endemic to Brazilian Cerrado that is at greatest risk of extinction.The aim of the this work was to use AFLP molecular markers to determine the genetic structure and diversity of eight natural populations of P. rigida and to associate their genetic characteristics with loganin production in order to obtain provide relevant information to promote programs for the conservation of this valuable medicinal plant.A total of 120 polymorphic bands were scored and higher proportion of genetic diversity was found in inter-populations (64%) rather than in intra-populations (36%). F_st value was found to be significantly greater than zero (0.3601), demonstrating the complex genetic structure of P. rigida populations. Accessions collected from Cristalina, GO, showed higher percentage of polymorphic loci (65.5%) and the highest genetic diversity. Analysis of Molecular variance (AMOVA) demonstrated 63.9% of intra-population genetic variation. The lowest genetic variability was detected among accessions from the population found in Sacramento, MG. No spatial standard was observed for P. rigida population, suggesting a partially isolated island model. It was observed a minor but significant positive correlation (r = 0.22) between chemical and genetic matrices. The association between chemical and genetic data indicated that environmental factors promoted the loganin production in populations growing in Luziânia, GO, and therefore accessions from those populations should be considered as prime material for initiating the conservation process of P. rigida.     

Survival possibilities of the dragonfly <Emphasis Type="Italic">Aeshna viridis</Emphasis> (Insecta,Odonata) in southern Sweden predicted from dispersal possibilities

We use public records from 1980 to 2014 to analyse survival of the EU Annex IV species Aeshna viridis in Sweden, a dragonfly strongly associated with the plant Stratiotes aloides. We clustered localities with S. aloides based on assumed dispersal abilities of A. viridis, using a dispersing radius of 2–100 km, calculating the proportion of sites with S. aloides that A. viridis is able to reach. If mean dispersal capability is high (40 km or above) 92.6 % or more of the localities are connected. For a good disperser, the probability of long-time survival is good. We further analysed the species richness of other Odonata and aquatic plants at 98 localities from the dataset. A. viridis co-occurred with more Odonata in the presence of S. aloides and running water but not in lakes. S. aloides sites had a higher number of other aquatic plants. Area had no impact on the occurrence of the species. For the present situation we surveyed 32 localities with known occurrence of the species. Only half of the sites for S. aloides contained any specimens while A. viridis occurred in the same number of sites. The species co-occurred in only 8 of 32 sites. In four sites A. viridis larvae appeared among Menyanthes trifoliata, Phragmites australis, Potamogeton natans and Sphagnum spp., indicating that at high latitudes A. viridis breeds among other species. Indirect monitoring based only on S. aloides would underestimate the number of populations of the dragonfly.     

Use of response surface methodology to study the effect of media composition on aflatoxin production by Aspergillus flavus

Aflatoxins are one of the most important secondary metabolites. These extrolites are produced by a number of Aspergillus fungi. In this study, we demonstrate the effect of media components and enhanced aflatoxin yield shown by A. flavus using response surface methodology in response to different nutrients. Different components of a chemically defined media that influence the aflatoxin production were monitored using Plackett–Burman experimental design and further optimized by Box–Behnken factorial design of response surface methodology in liquid culture. Interactions were studied with five variables, namely sorbitol, fructose, ammonium sulfate, KH₂PO₄, and MgSO₄.7H₂O. Maximum aflatoxin production was envisaged in medium containing 4.94 g/l sorbitol, 5.56 g/l fructose, 0.62 g/l ammonium sulfate, 1.33 g/l KH₂PO₄, and 0.65 g/l MgSO₄·7H₂O using response surface plots and the point prediction tool of the DESIGN EXPERT 8.1.0 (Stat-Ease, USA) software. However, a production of 5.25 μg/ml aflatoxin production was obtained, which was in agreement with the prediction observed in verification experiment. The other component (MgSO₄.7H₂O) was found to be an insignificant variable.     

Comparison of molecular genetic utilities of TD,AFLP, and MSAP among the accessions of <Emphasis Type="Italic">japonica,indica</Emphasis>, and <Emphasis Type="Italic">Tongil</Emphasis> of <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is one of the most important food crops in the world. Genetic diversity is essential for cultivar improvement programs. We compared genetic diversity derived from insertion–deletion (in–del) or base substitutions by amplified fragment length polymorphism (AFLP), from transposon transposition mutations by transposon display (TD), and from cytosine methylation by methylation-sensitive amplified polymorphism (MSAP) in japonica, indica, and Tongil type varieties of Oryza sativa L. Polymorphic profiles from the three marker systems allowed us to clearly distinguish the three types of varieties. The indica type varieties showed the highest genetic diversity followed by the Tongil and japonica type varieties. Of the three marker systems, TD produced the highest marker indices, and AFLP and MSAP produced similar marker indices. Pair-wise comparisons of the three marker systems showed that the correlation between the two genetic markers systems (AFLP and TD, r = 0.959) was higher than the correlations between the genetic and epigenetic marker systems (AFLP and MSAP, r = 0.52; TD and MSAP, r = 0.505). Both genetic marker systems had similar levels of gene differentiation (G _ST ) and gene flow (N _m ), which differed in the epigenetic marker system. Although the G _ST of the epigenetic marker system was lower than the genetic marker systems, the N _m of the epigenetic marker system was higher than in the genetic marker systems, indicating that epigenetic variations have a greater influence than genetic variations among the O. sativa L. types.     

Assessment of genetic diversity and interspecific relationships among three species of Podophyllum using AFLP markers and podophyllotoxin content

The genus Podophyllum (common name: May Apple) has high medicinal value due to the presence of anticancer molecule, podophyllotoxin. A total of 35 individuals belonging to three species of Podophyllum viz. P. hexandrum Royle, P. sikkimensis R. Chatterjee and Mukherjee both Indian species, and their American counterpart, P. peltatum L. have been investigated with a view to ascertain variation in their (1) podophyllotoxin content, and (2) molecular profiles generated through AFLP markers. The active principle content varied within the representative individuals of different populations of a species and between species; the species-wise podophyllotoxin content (% of dry wt) ranged as follows: P. hexandrum-Munsyari populations: 0.39–1.20 %, P. hexandrum-Kullu populations: 0.58–1.50 % (highest), P. peltatum: 0.50–1.30 %, and P. sikkimensis: 0.06–0.73 % (lowest). Detection of podophyllotoxin in P. sikkimensis, although at low levels, would appear to be the first report of its occurrence in this species. The genetic diversity and relationship amongst 35 sampled individuals of three species have been analyzed using 20 AFLP markers, which resulted into 1,358 loci of which 595 were polymorphic revealing 44 % polymorphism. High level of genetic diversity was observed (percent of polymorphic bands, PPB = 88.01 %; PIC = 0.813) among the species, while it was low within the individual species (PIC = 0.57 %; Marker index, MI = 4.77). Genetic similarity among the species (calculated with Euclidean coefficient) showed two major clusters. Cluster one contained all the individuals of P. peltatum (American May Apple) whereas cluster two grouped together individuals representing various populations, belonging to both the species of Indian May Apple (P. hexandrum, and P. sikkimensis). The observed paired relationship (45–50 % similarity; calculated from AFLP data) of intercontinental species in the Podophyllum group (P. hexandrum, and P. sikkimensis vs. P. peltatum) would appear to be paraphyletic. The AFLP data of the analyzed representatives have been used to examine the sister relationships among these species, and would be beneficial to find ways to strengthen the gene flow among populations to maintain the natural genetic variation within the populations of Podophyllum species.     

Population genetic diversity and divergence of the halobiotic herb Limonium sinense estimated by AFLP and ISSR, and implications for conservation

Limonium sinense is a halobiotic herb endemic to China that has been traditionally used for hundreds of years for its good restorative function. Genetic variation and population structure of this species were investigated by using amplified fragment length polymorphisms (AFLPs) and inter simple sequence repeats (ISSRs). A high level of genetic diversity was detected [AFLP: H _E = 0.284, percentage of polymorphic loci (PPL) = 92.68 %; ISSR: H _E = 0.257, PPL = 85.71 %] at the species level with POPGENE. Based on analysis of molecular variation (AMOVA), the among-population component accounted for 29.03 % (AFLP) and 28.81 % (ISSR) of the genetic variation, indicating that most of the genetic variation was between individuals within populations. The Shannon diversity index (I) was higher for AFLP (0.432) than for ISSR (0.395). Five main clusters were shown in the unweighted pair-group method with arithmetic mean (UPGMA) dendrogram created using TFPGA, consistent with the result of principal coordinate analysis using NTSYS. In situ conservation is advocated first. Keeping a stable environment for this halobiotic herb is necessary. For ex situ conservation, it is important to establish a germplasm bank. AFLP and ISSR markers were proved to be efficient tools in assessing the genetic variation among populations of L. sinense. The patterns of variation appeared to be consistent for these two marker systems, and they can be used for management of genetic structure, protection of the halobiotic plant, and conservation of germplasm.     

Chloroplast microsatellite diversity of Opisthopappus Shih (Asteraceae) endemic to China

The genus Opisthopappus consists of two species, O. longilobus and O. taihangensis. Both of them are important economic and ornamental plants but endangered in China. In this study, genetic variation in four populations of O. longilobus and nine populations of O. taihangensis was estimated by chloroplast microsatellites (cpSSR). Low genetic diversity was detected in O. longilobus and O. taihangensis populations. The percentages of polymorphic loci (PPL), Nei’s gene diversities (h), and Shannon’s indices (I) was 53.56 %, 0.074, and 0.139, respectively, for O. longilobus, while PPL = 44.44 %, h = 0.018, I = 0.048 for O. taihangensis. Thirteen chloroplast haplotypes were identified from 153 individuals analyzed of two species. Ten haplotypes were found in O. longilobus, while seven in O. taihangensis. Haplotype 1 was the common and ancestral one. The level of haplotype diversity was also low (Hd _{O. longilobus}  = 0.4875, Hd _{O. taihangensis}  = 0.3452). AMOVA analysis revealed that the majority of the genetic differences was partitioned within populations of O. longilobus and O. taihangensis, as confirmed by PCoA and cluster analysis. Inter-population genetic distances among population of O. longilobus and O. taihangensis correlated insignificantly with geographic distances (r = 0.286, P = 0.128 for O. longilobus vs. r = 0.356, P = 0.058 for O. taihangensis). The self-compatibility, effective gene flow, narrow geographical distribution, and historical factors may be the main factors causing differentiation in the genetic structure of O. longilobus and O. taihangensis populations.