Development and characterization of 20 polymorphic microsatellite markers for Livistona chinensis R. Br. var. subglobosa Becc., a fan palm in the Ryukyu Archipelago,Japan

Livistona chinensis R. Br. var. subglobosa Becc. is a subtropical fan palm distributed mainly in the islands of the Ryukyu Archipelago, Japan. Some small populations, separated by a few 100 km from the main species range, are also established on islands around northern Kyushu and Cape Ashizuri, Shikoku. Most of those are designated as national or prefectural natural treasures. We developed 20 microsatellite markers for this species and characterized them for polymorphisms within six populations. The number of alleles per locus ranged from two to nine, with an average of 4.2; the expected heterozygosity ranged from 0.16 to 0.61, with an average of 0.46. These markers will be useful for investigating disjunct range formation within L. chinensis var. subglobosa and the origins of the remote populations, providing crucial information for conservation of the populations.     

Genetic diversity and structure among natural populations of Sindora glabra in Hainan Island,China as revealed by ISSR markers

Sindora glabra, one of the second-class national protective plants in China, has important ecological and commercial values. To understand the genetic diversity and structure of S. glabra, eleven natural populations from four areas of Hainan Island in China were analyzed by inter-simple sequence repeat (ISSR) markers. Thirteen primers were screened out and used to amplify 157 DNA samples. A total of 122 bands were obtained, among which 114 (93.4%) bands were polymorphic. Genetic parameters including average number of effective alleles (1.547), Nei's gene diversity (0.321), Shannon's information index (0.482), and gene differentiation coefficient (0.1944) revealed a high level of genetic diversity maintained in S. glabra populations. The variation within populations accounted for 85.6% of total variation based on analysis of molecular variance. Genetic distance analysis showed that 11 populations could be divided into three groups and populations from the same areas were classified as one group, suggesting that high genetic diversity of S. glabra was attributable to geographic isolation. Together, introduction of germplasm from distant natural distribution areas would be a sound strategy for germplasm resource conservation of S. glabra and selection of elite individuals from populations of far relationship for hybridization is of great importance for breeding improvement programs in future.     

Intraspecific variation in Licuala glabra Griff. (Palmae) in Peninsular Malaysia– a morphometric analysis

We studied vegetative and reproductive characters of 74 herbarium specimens of Licuala glabra Griff. (Palmae) collected from 22 localities throughout Peninsular Malaysia. To test the null hypothesis that there are no distinct groups within the species, cluster analysis (nearest neighbour), principal coordinates and principal components analyses were performed on the set of 43 qualitative and quantitative characters. The results obtained suggested that: (1) there are three infraspeciflc groups within the species, viz., the two published varieties glabra and selangorensis as well as a possible third novel taxon; (2) there is clustering of individuals corresponding to their geographic localities; (3) clusters generated using only reproductive characters indicate a biogeographic distribution of populations correlated with the mountain ranges in the peninsula.     

Conservation Genetics of Mexican Beech, Fagus grandifoliavar. mexicana

Fagus grandifolia var. mexicana(Fagaceae) is a Mexican endemic tree, currently threatened with extinction. In order to assess the level and structure of genetic variation in four remaining populations, leaf samples were analysed using random amplified polymorphic DNA (RAPD) and cpDNA PCR-RFLP markers. A sample of the more widespread congener, F. grandifoliavar. grandifolia from the USA was also analysed for comparison. Thirty-three polymorphic RAPD bands were produced using 18 10-mer primers. AMOVA of RAPD data indicated significant (P < 0.002) population differentiation, with 15.6% of variation recorded between Mexican populations. PCR-RFLP analysis enabled three cpDNA haplotypes to be identified, denoted types A, B, and C. Types A and B were each restricted to an individual Mexican population, whereas Type C was fixed for two Mexican populations, and the population from the USA. Within-population genetic variation, quantified as percentage polymorphic bands, Shannon's Diversity Index and Nei's gene diversity measure, was found to be lower in Mexican populations than in that from the USA, and was positively related to population size. These results suggest that an unexpectedly high degree of genetic variation exists within Mexican beech, and this variation should be considered in developing the conservation strategy that is urgently required if extinction of this taxon is to be prevented.     

Random Amplified Polymorphic DNA Variation among and within Selected Ixora (Rubiaceae) Populations and Mutants

Random amplified polymorphic DNA (RAPD) analysis was used tostudy variation among and within selectedIxora (Rubiaceae) populationsand mutants. Six populations of I. congesta yielded identicalbanding patterns suggesting genetic uniformity of this species.However, six populations of I. coccinea varieties (three red-flowered,two yellow-flowered and one red-flowered wild-type) exhibitedinfraspecific differences in RAPD profiles. Small and largeleaves of an atavistic mutant cultivar of I. coccinea were alsosubjected to RAPD analysis. An extra band was amplified in thelarge leaves that was absent in small leaves, suggesting thatthe phenotypic alteration in this taxon is due to genetic mutationrather than epigenetic changes. Similarly, an extra band wasdetected in the white sectors of I. Variegated compared to thegreen sectors, suggesting that the shoot apical meristems ofthis cultivar exist as a genetic chimera. DNA gel blot hybridizationwas performed to confirm the specificities of selected bands.Our study indicates that differences among individuals of variouspopulations and mutants may be detected using RAPD markers.Copyright 1999 Annals of Botany Company Ixora L., variegated variety, RAPD fingerprinting, DNA gel blot, intraspecific genetic similarity, atavistic mutant.     

Population genetics in the conservation of the Azorean shrub Viburnum treleasei Gand.

We assess population genetic structure and variability in the endangered Azorean endemic Viburnum treleasei Gand., an evergreen shrub or small tree, occurring in eight out of nine islands of the archipelago. We combine RAPD and ISSR markers in eight populations and four islands covering the three subgroups of islands that compose the archipelago, and one population of V. tinus from the Portuguese mainland. Most of the genetic variability was found within populations, which is in accordance with the bi-parental reproductive strategy favored by the taxon. Gene flow estimations for the combined RAPD and ISSR markers suggest that the main cause for population variability between the studied populations is genetic drift. In accordance with the genetic structure indicators obtained, conservation measures should consider that translocation of individuals between islands must be avoided. In specific cases, the analysed populations may require the implementation of augmentation strategies due to their depleted state. With this study, a genetic background is now available to better define conservation measures for the taxon.     

Genetic Diversity inBrassica oleraceaL. (Cruciferae) and Wild Relatives (2n=18) using RAPD Markers

This paper presents the evaluation of genetic diversity in 29populations of wild taxa of theBrassica oleraceaL. group (2n=18)and two cultivars, using RAPDs as molecular markers. In a previouspaper (Lázaro and Aguinagalde,Annals of Botany82: 000–000,1998), 11 isozymes were used for the same purpose. Results obtainedwith the two molecular markers (isozymes and RAPDs) are compared.DNA from ten individuals per population was analysed using sixdifferent primers; the 151 detected bands were polymorphic,11 were common to all species, six to all taxa, only one toevery population; and no bands were shared by every individual.The dendrogram obtained using genetic distances clustersB. oleraceapopulationswithB. bourgeaui, B. alboglabra, B. montanaandB. incana. B.insularis, B. macrocarpa, B. villosaandB. rupestrispopulationsform another cluster. Populations ofB. creticaandB. hilarionisformthe third cluster. Genetic diversity inB. oleraceapopulations,theB. rupestriscomplex andB. creticasubspecies was estimatedusing the AMOVA programme; the latter was the most diverse.Copyright1998 Annals of Botany Company. Brassica oleraceaL., wild relatives, genetic diversity, genetic resources, RAPD markers, AMOVA.     

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.     

Random amplified polymorphic DNA (RAPD) markers reveal less genetic variation in the endangered plant Cerastiumfischerianum var. molle than in the widespread conspecific C. fischerianum var. fischerianum (Caryophyllaceae)

Random amplified polymorphic DNA (RAPD) analysis was used to assess the level of genetic diversity in an endangered plant Cerastiumfischerianum var. molle and the widespread conspecific C. fischerianum var. fischerianum. At both the variety and the population level, C. fischerianum var. molle had a lower level of RAPD variation than var. fischerianum. Analysis of molecular variance ( AMOVA ) showed that most of the genetic diversity exists among populations for both varieties. Genetic differentiation among populations probably resulted from the highly selfing habits of both varieties. To conserve C. fischerianum var. molle genetically, preservation of only a part of the population is insufficient.     

Random Amplified Polymorphic DNA (RAPD) Variation among Populations of the Insular Endemic Plant Campanula microdonta(Campanulaceae)

RAPD variation was examined in nine populations of Campanulamicrodonta Koidz., endemic to the Izu Islands, Japan. Ninety-eightbands were obtained for all populations, 94% of which were polymorphicat least within a population. Shannon's H values were calculated;these have frequently been used in RAPD studies to estimategenetic diversity. The values within populations did not correlatewith the allozyme gene diversity estimated by a previous studyor with distance from the Japanese mainland. The possible reasonsfor this discrepancy are different selection regimes betweenthe two markers, higher RAPD mutation rates, and each marker'sdifferent coverage of genomes. Cluster analysis of genetic similaritiessuggested that colonization of each island probably occurredonce, except for Miyake Island, where immigration has occurredat least twice. Copyright 2001 Annals of Botany Company AMOVA, Campanula microdonta Koidz., insular endemic plant, genetic diversity, population genetic structure, RAPD     

Genetic diversity among populations and size classes of buckeyes (Aesculus: Hippocastanaceae) examined with multilocus VNTR probes

 Little is known about genetic variation in members of the genus Aesculus (Hippocastanaceae), in particular A. flava (yellow buckeye) and A. glabra (Ohio buckeye). Here, three synthetic DNA probes (composed of tandemly repeated, core sequences) that reveal alleles at multiple variable-number tandem-repeat (VNTR) loci in these two species were used to investigate: 1) levels of genetic variation in one stand of A. flava and three isolated stands of A. glabra; 2) whether the stands of A. glabra are genetically differentiated from one another; 3) whether there has been selection for more heterozygous individuals through time in one stand each of A. flava and A. glabra; and 4) whether a possible genetic bottleneck had occurred during the formation of either species of Aesculus. First, variation of VNTR genetic markers within and among three populations of A. glabra separated by 60–180 km was examined. In each one hectare (ha) population, 22 individuals were randomly sampled. Among the three populations, the mean number of bands scored per individual was 80.35 and the average number of estimated loci surveyed was 54.17. Mean similarity and estimated heterozygosity within populations ranged from 0.634 to 0.743 and from 0.342 to 0.486, respectively. The mean similarity across populations was 0.657, while the mean estimated heterozygosity across populations was 0.484 for A. glabra. The most isolated site was the most genetically differentiated as indicated by differences in levels of similarity, heterozygosity, and Fst value comparisons. In a separate experiment, genetic variation in 22 large (reproductively mature; dbh > 8 cm) individuals was compared with that in 22 small (not yet reproductive; dbh < 1 cm) individuals collected within one ha stands for both A. flava and A. glabra. Mean similarity values among large versus small individuals of A. flava were 0.665 versus 0.662, while for A. glabra the corresponding values were 0.686 versus 0.691, respectively. Permutation tests of these similarity data detected no evidence for size class genetic differentiation in either species (both p-values > 0.050). Further, permutation tests for the number of bands per individual (average band number should be higher in more heterozygous individuals) detected no significant differences between size classes for either species. Thus, evidence of pronounced inbreeding and/or selection altering population genetics within small relative to large individuals was not detected. In addition, comparable similarity and heterozygosity values between these two closely related species (which still maintain an active zone of hybridization) suggests that either: 1) no extreme genetic bottleneck has accompanied the formation of these species from a common ancestor; or 2) signs of such a bottleneck have largely been eliminated. These studies demonstrate the utility of multilocus VNTR DNA probes for investigating genetic variation within and among plant populations, between size classes within a population, and between closely related species. Received May 15, 1998 Accepted September 11, 2001     

Allium ampeloprasum var. babingtonii (Alliaceae): an isoclonal plant found across a range of habitats in S.W. England

Despite the widespread distribution and common habitat dominance of clonal plant species the mechanisms for their ecological success often remain uncertain. Allium ampeloprasum var. babingtonii is a sterile plant that reproduces with physiologically independent asexual propagules. The variety is found mainly in the S.W. of England over a wide habitat range including sea cliffs and open woodlands and displays an autumn-spring phenology in common with its close Mediterranean relatives. The role of population genetic structure in the remarkable habitat distribution of the variety is not known. Genetic variation in A. ampeloprasum var. babingtonii was analysed using RAPD markers in single individuals of var. babingtonii from 16 distinct populations and five further individuals from a single population. This analysis revealed no polymorphism suggesting that all sampled individuals are part of a single clone. We offer the conjecture that in the absence of genetic variation an ecological release, due to temporal separation from competing species, may contribute to the ability of var. babingtonii to exploit heterogeneous environmental conditions.     

Genetic diversity of an endangered aquatic plant, Potamogeton lucens subspecies sinicus

The genetic diversity of the species, Potamogeton lucens subsp. sinicus var. teganumensis, which is critically endangered in Japan, was investigated. This species now occurs in only two known localities in Japan. One is a native population (Oitoike population), but the other (Teganuma-Okahotto population) is found in a small artificial pond that was dug in 1998. It is considered that the Teganuma-Okahotto population grew from a soil seed bank. Based on RAPD variation, we compared the genetic diversity of the two populations of P. lucens var. teganumensis in Japan and one population of P. lucens subsp. sinicus var. sinicus in China. The Teganuma-Okahotto population showed RAPD variation, suggesting that it may be derived from more than one seed buried in old sediments. This population also had the highest value of Shannon's Information Index among the three study populations. This finding suggests that seeds buried in sediments can contain genetic variability, and may be used to conserve the genetic diversity of rare and endangered plants.     

Genetic diversity of an endangered aquatic plant,Potamogeton lucens subspecies sinicus

The genetic diversity of the species, Potamogeton lucens subsp. sinicus var. teganumensis, which is critically endangered in Japan, was investigated. This species now occurs in only two known localities in Japan. One is a native population (Oitoike population), but the other (Teganuma-Okahotto population) is found in a small artificial pond that was dug in 1998. It is considered that the Teganuma-Okahotto population grew from a soil seed bank. Based on RAPD variation, we compared the genetic diversity of the two populations of P. lucens var. teganumensis in Japan and one population of P. lucens subsp. sinicus var. sinicus in China. The Teganuma-Okahotto population showed RAPD variation, suggesting that it may be derived from more than one seed buried in old sediments. This population also had the highest value of Shannon's Information Index among the three study populations. This finding suggests that seeds buried in sediments can contain genetic variability, and may be used to conserve the genetic diversity of rare and endangered plants.     

Further silphinene derivatives from Cineraria geifolia var. glabra

A reinvestigation of the sesquiterpenes from the aerial parts of Cineraria geifolia var. glabra afforded eight new silphinene derivatives. One of these compounds had been isolated previously but its structure had not been elucidated with certainty. The stereochemistry of a corresponding isovalerate from Callilepis salicifolia has to be revised.     

Relationship of Hypochaeris salzmanniana (Asteraceae, Lactuceae), an endangered species of the Iberian Peninsula, to H. radicata and H. glabra and biogeographical implications

Hypochaeris salzmanniana DC. (Asteraceae, Lactuceae) is an endangered species on the Iberian Peninsula, known from only eight coastal populations. Most authors have treated it as a variety, subspecies or simply as a synonym of H. glabra L. On the basis of morphological and cytological characters, Talavera recently separated H. salzmanniana (2n = 8) from H. glabra (2n = 10). Material of H. salzmanniana, H. glabra and H. radicata was collected from Spain, Italy, Sicily and Tunisia in order to assess taxonomic status and population relationships. Amplified Fragment Length Polymorphism (AFLP) analysis revealed three well-differentiated species. A close relationship between H. salzmanniana and H. radicata is also confirmed by AFLP analysis and chromosome number (2n = 8), morphology, and rDNA localization (FISH, fluorescence in situ hybridization). Hypochaeris salzmanniana and H. radicata share three fixed diagnostic AFLP fragments out of 348 fragments scored. The population structure of H. salzmanniana reveals distinct groups in southern Spain that are separated geographically. High differentiation among a western (Conil to Zahara), an intermediate (Punta Paloma and Los Algarbes) and an eastern (Algeciras and La Línea) group may reflect ancient separation. Population sizes and genetic compatibility differ greatly among populations and can be used to explain levels of within-population genetic diversity, together with recent documented loss of habitats resulting from tourist developments. Population structures of H. radicata and H. glabra show a similar geographical patterning: strongly differentiated populations from the Betic Cordillera and from the Iberian Massif, which are separated at present by the Guadalquivir river. Geological events at the end of the Tertiary (Tortonian–Messinian Miocene) might help explain patterns of differentiation in these three species of sect. Hypochaeris. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146 , 79–95.     

Patterns of Genetic Variation in in and ex situ Populations of the Threatened Chilean VineBerberidopsis corallina , Detected Using RAPD Markers

Berberidopsis corallina Hook. f. (Berberidopsidaceae) is a threatenedvine, endemic to the temperate rainforests of southern Chile.A RAPD analysis was carried out to assess the extent of geneticvariation in remaining wild populations and in British cultivatedplants, to assess the value of the latter for ex situ conservation.A total of 90 individuals (54 wild, 35 cultivated) were analysed,and a total of 54 polymorphic bands produced. A pairwise distancemeasure calculated from the RAPD data was used as input forprincipal coordinate analysis (PCO) and analysis of molecularvariance (AMOVA). AMOVA indicated that an exceptionally highproportion of total genetic variation (54.8%) was recorded amongpopulations; pairwise     

Population genetic structure of Titanotrichum oldhamii (Gesneriaceae), a subtropical bulbiliferous plant with mixed sexual and asexual reproduction

• Background and Aims Titanotrichum oldhamii is a monotypicgenus distributed in Taiwan, adjacent regions of China and theRyukyu Isands of Japan. Its conservation status is vulnerableas most populations are small and widely scattered. Titanotrichumhas a mixed system of reproduction with vegetative bulbils andseeds. The aim of this study was to understand the populationgenetic structure of Titanotrichum in relation to its specificreproductive behaviour and to determine possible implicationsfor conservation strategies. • Methods After an extensive inventory of most wild populationsof Titanotrichum in East Asia, samples from 25 populations withinits major distribution were carried out utilizing RAPD and inter-SSRmolecular fingerprinting analysis. • Key Results The findings support the conclusion thatmany populations reproduce predominantly asexually but thatsome genetic variation still exists within populations. However,significant amounts of variation exist between populations,perhaps reflecting population differentiation by drift. Thispartitioning of genetic diversity indicates that the level ofinter-population gene exchange is extremely low. These findingsare consistent with field observations of very limited seedproduction. The Chinese populations are similar to those ofNorthern Taiwan, while the Ryukyu populations fall within therange of variation of the north-central Taiwan populations.The Taiwanese populations are relatively variable and differentiationbetween north, east and south Taiwan is evident. • Conclusions The distribution of Titanotrichum seems tobe consistent with a former land connection between China, Taiwanand the Ryukyu Islands at a glacial maximum during the Quaternary,followed by progressive fragmentation of the populations. North-centralTaiwan is the centre of genetic diversity, possibly due to theproximity of the former land bridge between the regions, togetherwith the variety of suitable habitats in north Taiwan. The significanceof these findings for conservation is discussed.     

Genetic diversity of Mexican brook lamprey <Emphasis Type="Italic">Lampetra (Tetrapleurodon) geminis</Emphasis> (Alvarez del Villar, 1966)

Lampreys are the only surviving representatives of the oldest known vertebrates. The Mexican lamprey L. geminis (nonparasitic), is particularly interesting, because it is an endemic, biogeographical relict, and a threatened species. RAPD markers were used to describe genetic diversity in L. geminis A total of 77 specimens were collected from five populations, three in the R'o Grande de Morelia-Cuitzeo basin and two in the R'o Duero-Lerma-Chapala basin, Mexico. Eighty-eight RAPD markers were obtained from eight primers. Genetic diversity within each population was estimated using Shannon's index (S), heterozygosity (H) and gene diversity (h). These estimates revealed significant variation within populations, although a variance homogeneity test (HOMOVA) showed no significant differences among populations or between basins. Nei genetic distance values indicate a low genetic differentiation among populations. Analysis of molecular variance (AMOVA) indicates that most of the genetic diversity occurs within populations (91.4%), but that a statistically significant amount is found among populations (P0.001). Principal coordinates and cluster analyses of RAPD phenotypes show that specimens are not grouped by geographical origin. The genetic diversity found within L. geminispopulations may be explained by its breeding system and an overlapping of generations. The scarce genetic differentiation among populations is likely to the low rate of DNA change that characterizes the lamprey group.     

Low genetic diversity and recovery implications of the vulnerable Bankoualé Palm Livistona carinensis (Arecaceae), from North-eastern Africa and the Southern Arabian Peninsula

The Bankoualé Palm, Livistona carinensis is the only known species of Livistona occurring in Africa and is currently classified as vulnerable (IUCN 2004). This extreme outlier species of the genus is restricted to Yemen, Somalia and Djibouti, where all populations are in rapid decline. In Djibouti the palm is confined to three valley systems within the upland plateau of the Goda Massif. This study used microsatellite markers to investigate the genetic diversity and relationships within the species. At the species level L. carinensis contained very low genetic diversity. Most variation was due to the variation between the samples from Yemen and Somalia compared with those in Djibouti. The Djibouti populations were almost monomorphic across the nine loci tested. Interestingly, and despite the small sample sizes, the individuals from botanic gardens collections of the Yemen and Somalia populations were more genetically diverse than the Djibouti populations. This study indicates that the populations in Yemen and Somalia are highly significant for the conservation of the species genetic diversity. Given the lack of genetic diversity both within and among L. carinensis populations in Djibouti, plants could be cultivated for in-situ population enhancement from any seed that is available from within Djibouti with no significant genetic impacts of provenance mismatch. Clearly the populations from Djibouti, Somalia and Yemen are different genetic provenances raising some issues for the conservation and recovery of L. carinensis.