Phylogeny ofTyphonium (Araceae) inferred from restriction fragment analysis of chloroplast DNA

The interrelationship of the ten species of the genusTyphonium and related genera in subtribe Arinae of the Araceae was inferred by chloroplast DNA restriction fragment analysis. A total of 42 site mutations were observed and 26 site mutations were shared by two or more species. A majority rule consensus tree was made by performing 100 bootstrap replicates using Wagner Parsimony. Two groups ofTyphonium were recognized significantly as monophyletic groups, i.e. 1)Typhonium larsenii andT. kunmingense, and 2)T. trilobatum, T. blumei andT. flagelliforme.     

DIVERSITY OF SHOOT MORPHOLOGY IN TYPHONIUM (ARACEAE)

The details of shoot organization, including number of leaves per shoot, position of foliage leaves and cataphylls, position of the lateral continuation shoot, nature of axillary buds and phyllotaxis, and the pattern of shoot extension were observed and compared in five species of Typhonium, Dracunculus vulgaris, Sauromatum venosum, Arum italicum, and Helicodiceros muscivorus, resulting in the recognition of four types of stems. Three of the four types were found in the genus Typhonium. One type was found in Typhonium giganteum and Sauromatum venosum and is presumably the same in Biarum. Another type, found in T. trilobatum, T. blumei, and T. flagelliforme, is distinct in the position of the lateral continuation shoot, which arises from the axil of the n-1 leaf and is adnate to the axis to above the n leaf. Based on the results, two groups, one of which is further subdivided into two subgroups, are recognized in subtribe Arinae (subfam. Aroideae tribe Areae).     

Population genetic diversity in the polyploid complex of wheatgrasses using isoenzyme and RAPD data

Thirty five bands (alleles) from six enzyme systems and fifty seven random amplified polymorphic DNA (RAPD) fragments were selected to analyse the genetic diversity of 33 polyploid wheatgrasses (Triticeae) populations of species Thinopyrum junceiforme and Elytrigia pycnantha, and two hybrids, one pentaploid and one novel 9-ploid. Dice’s similarity coefficient, the UPGMA-derived phenograms from RAPD, and allozymes markers showed that the clustering of wheatgrass populations was based on ploidy level. These markers had similar levels of diversity between populations, with high genetic similarity within the same ploidy-level and within population’s individuals. The tetraploid Th. junceiforme populations are closely related, with a large similarity distances varied from 0.8 to 1. Based on the isozyme and RAPD analyses, diploid taxa are related to polyploids with similarity coefficients 0.4.     

Molecular characterization of five medicinally important species of Typhonium (Araceae) through random amplified polymorphic DNA (RAPD)

The interrelationship of five medicinally important species of Typhonium (Araceae) including T. venosum, which was previously placed under the genus Sauromatum, was inferred by analysis of random amplified polymorphic DNA (RAPD). DNA from pooled leaf samples was isolated and RAPD analysis was performed using 20 decamer oligonucleotide primers. Out of a total of 245 bands amplified, 12 were found to be monomorphic while 233 bands were polymorphic including 86 species-specific bands. The genetic similarities were analyzed from the dendrogram constructed by the pooled RAPD data using a similarity index. The dendrogram showed two distinct clades, one containing T. roxburghii, T. trilobatum and T. venosum and the other containing the remainder two species, i.e. T. diversifolium and T. flagelliforme. Both the clusters shared a common node approx. at 23.7% level of similarity. The maximum similarity of 31.2% was observed between T. venosum and T. trilobatum. In view of its close genetic similarity with other members of Typhonium, transfer of Sauromatum venosum to the genus Typhonium and merger of the two genera was supported.     

Population Genetic Structure of Astyanax scabripinnis (Teleostei, Characidae) from an Urban Stream

Despite the great anthropogenic interference on urban streams, information is still scarce about the genetic variability and structure of native fish populations inhabiting such streams. In the present study, random amplified polymorphic DNA (RAPD) markers were used to analyze genetic variability and structure of populations assigned to the Neotropical fish species Astyanax scabripinnis from an urban stream located in Londrina, Paraná State, southern Brazil. Thirty individuals of this species were collected from three sites throughout the upper Cambé stream. A total of 10 primers amplified 159 loci, of which 128 (80.5%) were polymorphic. Each of the three populations showed very similar proportions of polymorphic loci, which ranged from 63.5 to 64.8%. Unbiased genetic distances varied from 0.0612 to 0.0646. Theta_p-test values indicated moderate to high genetic differentiation among individuals from different localities. The number of migrants varied from 1.34 to 1.46, suggesting a low gene flow between populations. The genetic similarity among all individuals studied ranged from 0.424 to 0.848. The results suggest that populations of A. scabripinnis in Cambé stream are undergoing genetic differentiation.     

An investigation on the genetic background of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> by similarity analysis of its partial genomic DNA and phylogenetic comparison of deduced related species

Nostoc flagelliforme, which is distributed on arid and semi-arid steppes of northwestern parts of China, has attracted increasing interest for its stress tolerance. In order to gain more insight into the genetic background of N. flagelliforme, we sequenced its partial genomic DNA for similarity analyses against current public databases, followed by phylogenetic comparison of N. flagelliforme and the potentially related species deduced from the similarity analyses. Approximately 430 kb genomic sequence (~5% of genome as a rough estimate) was determined from 106 distinct genomic clones. Nucleotide BLAST showed that ~23.1% of the partial genomic sequence was similar to N. punctiforme genomic DNA and ~12.4% to its plasmid DNA. Similar protein search by online FASTA-protein program showed 46.2% of the similar proteins had their corresponding orthologs in N. punctiforme genome. Furthermore, phylogenetic comparison based on 16S rRNA sequences showed N. flagelliforme and N. punctiforme clustered closer among the deduced related species. These results indicated that N. punctiforme might also be potentially close neighbor species of N. flagelliforme, in addition to the formerly regarded close neighbor species N. commune and N. sphaeroids. In general, these data enriched our recognition of the evolutionary relationship between N. flagelliforme and other Nostoc species, especially N. punctiforme.     

Typhonium muaklekense sp. nov. (Araceae) from Thailand

Typhonium muaklekense D. Sookchaloem & S. Maneeanakekul is described and illustrated. This new species resembles T. roxburghii Schott and T. varians Hett. & Sookch. in the spathe. Drawings, photographs and a table comparing diagnostic features of T. muaklekense, T. roxburghii and T. varians are provided.     

Genetic Variation in Hippophae rhamnoides ssp. sinensis (Elaeagnaceae) Revealed by RAPD Markers

Hippophae rhamnoides ssp. sinensis is endemic to China, and it is a dioecious, outcrossing plant. Although many studies have been undertaken mainly on its agricultural, nutritional, medical, and ornamental value, little is known about its population genetics. This study uses random amplified polymorphic DNA to investigate the genetic diversity and population genetic structure of 13 natural populations of the subspecies sinensis. Fifteen primers amplified 107 reproducible bands, with 95 (88.79%) being polymorphic. The gene diversity within population was 0.168, considerably lower than that of tree species and most perennial, outcrossing species, but higher than that of annual or short-lived, selfing species. The Gst value showed that 18.3% of the total genetic variation resided among populations, a little lower than that of outcrossing species. The present results are quite similar to those previously reported in another subspecies, H ssp. . rhamnoides rhamnoides. The low genetic differentiation among populations in ssp. sinensis may be attributed to the long-distance dispersal of seeds facilitated by birds, in addition to its characteristics of outcrossing, wind pollination, and widespread distribution. No association between genetic distance and geographical distribution was found. The population relationships revealed by the UPGMA dendrogram parallel this result, in that genetic distance did not increase with geographic separation. This pattern of population differentiation may imply the adaptation of ssp. s populations to the local environment, given that its habitats vary greatly across its distribution.     

A comparison of the genetic diversity in Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry

Dipteronia is an endemic genus to China and includes only two species, Dipteronia sinensis and D. dyeriana. Based on random amplified polymorphic DNA (RAPD) markers, a comparative study of the genetic diversity and genetic structure of Dipteronia was performed. In total, 128 and 103 loci were detected in 17 D. sinensis populations and 4 D. dyeriana populations, respectively, using 18 random primers. These results showed that the proportions of polymorphic loci for the two species were 92.97% and 81.55%, respectively, indicating that the genetic diversity of D. sinensis was higher than that of D. dyeriana. Analysis, based on similarity coefficients, Shannon diversity index and Nei gene diversity index, also confirmed this result. AMOVA analysis demonstrated that the genetic variation of D. sinensis within and among populations accounted for 56.89% and 43.11% of the total variation, respectively, and that of D. dyeriana was 57.86% and 42.14%, respectively. The Shannon diversity index and Nei gene diversity index showed similar results. The abovementioned characteristics indicated that the genetic diversity levels of these two species were extremely similar and that the interpopulational genetic differentiation within both species was relatively high. Analysis of the genetic distance among populations also supported this conclusion. Low levels of interpopulational gene flow within both species were believed to be among the leading causes for the above-mentioned phenomenon. The correlation analysis between genetic and geographical distances showed the existence of a remarkably significant correlation between the genetic distance and the longitudinal difference among populations of D. sinensis (p < 0.01), while no significant correlation was found between genetic and geographical distances among populations of D. dyeriana. This indicated that genetic distance was correlated with geographical distances on a large scale rather than on a small scale. This result may be related to differences in the selection pressure on species by their habitats with different distribution ranges. We suggest that in situ conservation efforts should focus on establishing more sites to protect the natural populations and their habitats. Ex situ conservation efforts should focus on enhancing the exchange of seeds and seedlings among populations to facilitate gene exchange and recombination, and to help conserve genetic diversity. __________ Translated from Acta Phytoecologica Sinica, 2005, 29(5): 785–792 [译自: 植物生态学报, 2005, 29(5): 785–792]     

Random amplified polymorphic DNA (RAPD) markers reveal genetic homogeneity in the endangered Himalayan species Meconopsis paniculata and M. simplicifolia

Random amplified polymorphic DNA (RAPD) marker-based analysis was carried out to study the extent of genetic polymorphism between populations of the two endangered Himalayan poppy species, Meconopsis paniculata and M. Simplicifolia. Of the 90 primers tested, 38 revealed marked inter-species genetic polymorphism between individuals of the two species from geographically isolated populations. However, intra-species genetic homogeneity was also evident with respect to a number of primers both within and between populations. A comprehensive analysis incorporating data from RAPDs, DNA fingerprinting and isozyme pattern was carried out and, based on the presence or absence of bands, three matrices of similarity indices were estimated. These matrices were subsequently utilized in cluster analysis. In order to compare the three clusters generated using these three different marker systems, a Mantel matrix-correspondence test was carried out on the basis of comparisons of co-phenetic values. The overall representation of relationships by cluster analysis was similar for all three marker systems and this was substantiated by high correlations among the three analyses revealed by the Mantel matrix-correspondence test. Our results point to very low or absence of, genetic polymorphism in M. paniculata and M. simplicifolia, and are in broad agreement with our previous observations on genetic diversity of Meconopsis species which point to a genetic basis for the possible extinction of this economically important genus.     

Genotype relationships inMicroseris elegans (Asteraceae,Lactuceae) revealed by DNA amplification from arbitrary primers (RAPDs)

The genetic relationships among 10 inbred lines representing 10 populations of the autogamous annualMicroseris elegans from throughout California has been determined using random amplified polymorphic DNAs (RAPDs). Seventeen arbitrary 10 base pair primers produced 134 amplification products; 81 of these were shared by two or more strains. The 3 genotypes from Northern California are closely related as are 3 genotypes from Middle Californian populations which are not nearest neighbors. DNA fingerprinting with the oligonucleotide (GATA)₄ gave compatible results, but the comparison was limited to samples run on one gel. Isoenzyme patterns are compatible with the DNA results, but limited by the very low number of informative polymorphisms. The clustered relationship among genotypes within a species and their geographic distribution suggests very restricted genetic recombination and an origin of new populations from randomly dispersed achenes within the range of the species.     

Population variation and phylogeny in the endangered <Emphasis Type="BoldItalic">Chamaesyce skottsbergii</Emphasis> (Euphorbiaceae) based on RAPD and ITS analyses

Chamaesyce skottsbergii var. skottsbergii is federally listed as an endangered taxon, and is found in small and isolated populations restricted to calcareous soils in dry shrubland habitats on the Hawaiian islands of Oahu and Molokai. Concern over the genetic relationship among these disjunct populations arose as a result of threats to the habitat of the Oahu population. The populations were examined using random amplified polymorphic DNA (RAPD) markers and sequence analysis of the internal transcribed spacer (ITS) region of the rDNA cistron. Chamaesyce skottsbergii var. vaccinioides, a closely related variety found in several small populations on Molokai, was used for baseline comparison of the genetic divergence among populations. RAPD analysis demonstrated that variation within and among populations is the highest for any Hawaiian species examined. Polymorphism was greater than 95% within populations and was 99.4% at the species level. Similarly, measures of genetic similarity indicate that differentiation among these populations is higher than is known for some species. Both RAPD and ITS sequence analysis indicate that populations of C. skottsbergii var. skottsbergii on Oahu and Molokai are genetically distinct, and the extent of this genetic differentiation supports the recognition of these populations as distinct varieties. The Molokai population is in fact much more closely related to var. vaccinioides than to var. skottsbergii on Oahu, and thus should be recognized by the previously used variety name, C. skottsbergii var. audens. Further conservation measures for each of the varieties are addressed.     

Comparison of cytotoxic activities betweenin vitro and field grown plants ofTyphonium flagelliforme (Lodd.) blume

Anin vitro cytotoxicity screening of theTyphonium flagelliforme extracts indicated high cytotoxicity effect on human lung carcinoma NCl-H23 cells and human mammary gland carcinoma T-47D cells, but the extracts were not active on human liver carcinoma HepG2 cells. NCl-H23 cells were more susceptible toT. flagelliforme extracts than T-47D cells. EDP₅₀ values of the hexane fractions of the mature plant and thein vitro plantlet ofT. flagelliforme on NCl-H23 cells were less than 2 μg/mL Extract from the mature plant was relatively more cytotoxic than the one fromin vitro plantlet except for the hexane fraction. The chloroform and butanol fraction of the mature plant had higher cytotoxicity effect than the fraction fromin vitro plantlet on NCl-H23 cells. All the 3 fractions (hexane, chloroform, and butanol) of the mature plant exhibited higher cytotoxicity effects on human mammary gland carcinoma T-47D cells than the 3 fractions ofin vitro plantlet. However, the human liver carcinoma cells were resistant toT. flagelliforme extracts except for higher concentration of hexane fractions of both the mature and thein vitro plants and the chloroform fraction of the mature plant. Micropropagated plantlets ofT. flagelliforme could hence be used as herbal materials for the treatment of human lung and breast cancers.     

Population Structure of the Giant Garter Snake, Thamnophis gigas

The giant garter snake, Thamnophis gigas, is a threatened species endemic to California’s Central Valley. We tested the hypothesis that current watershed boundaries have caused genetic differentiation among populations of T. gigas. We sampled 14 populations throughout the current geographic range of T. gigas and amplified 859 bp from the mitochondrial gene ND4 and one nuclear microsatellite locus. DNA sequence variation from the mitochondrial gene indicates there is some genetic structuring of the populations, with high F_ST values and unique haplotypes occurring at high frequency in several populations. We found that clustering populations by watershed boundary results in significant between-region genetic variance for mtDNA. However, analysis of allele frequencies at the microsatellite locus NSU3 reveals very low F_ST values and little between-region variation in allele frequencies. The discordance found between mitochondrial and microsatellite data may be explained by aspects of molecular evolution and/or T. gigas life history characteristics. Differences in effective population size between mitochondrial and nuclear DNA, or male-biased gene flow, result in a lower migration rate of mitochondrial haplotypes relative to nuclear alleles. However, we cannot exclude homoplasy as one explanation for homogeneity found for the single microsatellite locus. The mitochondrial nucleotide sequence data supports conservation practices that identify separate management units for T. gigas.     

Evaluation of genetic relationships in Dalbergia species using RAPD markers

Conservation of identified germplasm is an important component forefficient and effective management of plant genetic resources. Traditionally,species identification has relied on morphological characters like growth habit,floral morphology like flower colour, and agronomic characteristics of the plant.Dalbergia species are important wind-dispersed tropicaltimber trees which exhibit high intrafruit seed abortion because of intensesibling competition for maternal resources. Studies were undertaken foridentification and genetic relationships in five species ofDalbergia and to evaluate genetic diversity withinpopulations of Dalbergia sisso, D.latifolia, D. paniculata, D.assamica and D. spinosa by using randomamplified polymorphic DNAs (RAPD) markers. Analysis was started by using 30decamer primers that allowed to distinguish five species and to select a reducedset of primers. The selected primers were used for identification and forestablishing a profiling system to estimate genetic relationships and toevaluate the genetic variability among the individuals in a population ofDalbergia species. A total of 120 distinct DNA fragments(bands), ranging from 0.3 to 4.0 kb, were amplified byusing nine selected random decamer primers. The genetic similarity was evaluated onthe basis of presence or absence of bands, which revealed a wide range ofvariability within the species. The cluster analysis indicated that five speciesof Dalbergia formed two major clusters. The first clusterconsisted of D. spinosa, D. latifolia and D.sisso. The second cluster was represented by two species, i.e.D. paniculata and D. assamica.A maximum similarity of 60% was observed in D. paniculata andD. assamica and they formed a minor cluster.Dalbergia latifolia and D. sissoformed another minor cluster with more than 50% similarity. Dalbergiaspinosa shared up to 40% similarity with D.latifolia and D. sisso. All the species sharemore than 20% similarity among themselves. The closest genetic distance existedwithin populations of different Dalbergia species. Thus,these RAPD markers have the potential for conservation of identified clones andcharacterization of genetic relatedness among the species. This is also helpful intree breeding programs and provides an important input into conservation biology.     

Inter- and intra-genetic variation of four wild populations of Prosopis using rapd-pcr fingerprints

The species of Prosopis (Leguminosae) are welladapted to grow in the arid regions of the world. In Mexico, nearly 70%of its territory accounts for arid lands and nine species of this genus arefound in the country which are currently utilized for multiple purposes. Giventhat a sustainable exploitation of this important natural resource is necessary,knowledge of the genetic variability of natural populations is required. Thegenetic variation among and within four wild populations of Prosopisglandulosa, P. juliflora, and two ofP. laevigata, located in the Mexican States of Chihuahua(1), Sinaloa (2), Guanajuato (3) and Hidalgo (4), respectively, was determinedusing random amplified polymorphic DNA (RAPDs). There was a genetic variation of72.48% among these four populations, and within them the estimated geneticvariation was 27.52%. Between the populations of P.laevigata the genetic variation obtained was 7.15%, and within them92.85% (AMOVA, P < 0.001). Based on the assumption ofWright's island model, the genetic variation found among populations correspondsto a gene flow (N_em) of 0.09, while the gene flow detected betweenthe populations of P. laevigata was 3.2. A total of 27 RAPDphenotypes were identified in the four populations; among them, 11 correspondedto P. laevigata. The phylogenetic analyses showed that thethree species are differentiated in three clades and that probably P.juliflora was the origin of the other two species. The protogynoustrait of their flowers and the outcrossing are the main factors that may explainthe genetic variation observed in these populations.     

Molecular data indicate multiple independent colonizations of former lignite mining areas in Eastern Germany by <Emphasis Type="Italic">Epipactis palustris</Emphasis> (Orchidaceae)

Former lignite mining areas in Eastern Germany are valuable secondary habitats for many plant and animal species endangered in the natural landscape. Here, we present a study on genetic structure and diversity of 16 populations of the threatened orchid Epipactis palustris (Orchidaceae) from five mining pits and 11 natural habitats, which we carried out in order to ascertain how many times this species immigrated into former lignite mining areas, and where the source populations are located. We used two different anonymous genetic marker methods, random amplified polymorphic DNA (RAPD) and microRNA-primed genomic fingerprinting (miRPF) to analyze patterns of genetic variation. Results of a multivariate analysis based on asymmetric Soerensen similarity, principal coordinate analysis and a neighbor-joining cluster analysis indicate high within population-variability and a moderate genetic differentiation among E. palustris populations. We found no differences between genetic diversity values of populations from former mining areas and those of natural habitats. Thus, we could not find evidences for genetic bottlenecks in the mining populations due to founder events. Source populations are predominantly close surrounding populations as geographic distance and genetic dissimilarity were correlated. However, exchanges may reach beyond 125 km and repeated independent colonization events are highly likely.     

Genetic Diversity and Genetic Structure of an Endangered Species, Trillium tschonoskii

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H_E) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.     

RAPD variation in wild populations of four species of the genus Hordeum (Poaceae)

 The genetic variation of 102 natural populations of wild barley growing in Spain was assessed using RAPDs (random amplified polymorphic DNA). The plant material included the annual species H. marinum subsp. marinum (22 populations) and subsp. gussoneanum (14), H. murinum subsp. murinum (7) and subsp. leporinum (35), and the perennial species H. bulbosum (17) and H. secalinum (7). Ten of the tested 64 arbitrary 10-mer primers amplified polymorphic DNA in all taxonomic units. Analyses was performed within and between populations, species and subspecies. The primers gave a total of 250 RAPD products. The level of polymorphism varied between taxonomic units depending on the primers employed and the plant reproductive system. In general, the most variable were the allogamous species H. secalinum and H. bulbosum and the autogamous H. marinum subsp. marinum. Among the amplified bands, 69 (27%) were shared by at least two different taxonomic units. The remaining bands were specific. The results demonstrate differences in the degree of similarity between taxonomic units. Jaccard’s similarity coefficients for interval measure within and between populations were used to produce a cluster diagram using the unweighted pair-group method (UPGMA). The different populations of the species and subspecies of Hordeum fell into three groups. The first group contained the populations belonging to both subspecies of H. marinum, plus those of H. secalinum. The populations of H. marinum subsp. gussoneanum were very closely associated. Those of H. marinum subsp. marinum were grouped in a broad cluster. The second group, occupying the innermost position of the tree, was very closely associated with the populations of both subspecies of H. murinum. The third branch segregated H. bulbosum. A series of RAPD markers were investigated by cleaving the amplified products of the same size with restriction endonucleases that recognize targets of 4- or 6-bp. The production of equivalent fragments following cleavage by the same enzyme would seem to demonstrate their homology in samples from different individuals, populations or taxonomic units. Received: 18 May 1997 / Accepted: 22 August 1997     

Genetic variation in the Italian crested newt, Triturus carnifex, and the origin of a non-native population north of the Alps

Genetic variation over 40 protein loci and 46 populations representing three taxa of crested newts revealed moderate genetic distances between Triturus carnifex carnifex, T. c. macedonicus and T. cristatus. Two populations from the Geneva Basin (presumed to be introduced) were genetically similar to T. c. carnifex and dissimilar to T. c. macedonicus and T. cristatus, showing that they belong to T. c. carnifex and not to native T. cristatus. A significant pattern of spatial genetic variation was found within T. c. carnifex along a north to south axis, from Croatia to Calabria. The Genevan populations showed highest genetic similarity with T. carnifex from Tuscany, suggesting that the propagule originated from that area. Effects of a population genetic bottleneck associated with the introduction could not be documented. The observed high allelic variation in Genevan T. c. carnifex could not be directly explained by introgression from T. cristatus. Comparisons across the range, including zones of hybridization within the T. cristatus superspecies, indicated that some alleles typical for the Genevan population may represent the so-called `hybrizymes'.