Seed survival and seasonal emergence in some species of Geranium, Ranunculus and Rumex

Seeds of 14 species were collected, usually in each of 2 or 3 yr, and mixed with the top 7.5 cm of sterilised soil confined in cylinders sunk in the ground outdoors and cultivated three times yearly. Seedling emergence was recorded for at least 3 and usually 5 yr. Most seedlings of Ranunculus bulbosus and Rumex acetosa appeared during the autumn of sowing, while the main emergence of Geranium pratense. Ranunculus acris, Rumex conglomeratus and R. maritimus was in the following spring. Emergence of G. pyrenaicum and G. robertianum took place throughout much of the year after sowing. Seeds of all these species were relatively short-lived in cultivated soil, and few seedlings appeared after the second year. Seed survival was greater in G. dissectum. G. molle, Rumex acetosella. Ranunculus sceleratus and especially R. repens, of which emergence in the fifth year after sowing amounted to 4% of the seeds sown. Emergence from the persistent seed bank was mainly from May to September for G. dissectum and G. molle and during autumn for Ranunculus flammula and R. sceleratus, while seedlings of Ranunculus repens and Rumex acetosella appeared sporadically over most of the year.     

The genetic structure of populations of the vulnerable aquatic macrophyte <Emphasis Type="Italic">Ranunculus nipponicus</Emphasis> (Ranunculaceae)

Ranunculus nipponicus (Makino) Nakai is a vulnerable aquatic macrophyte in the Kinki district, which is the southernmost distribution of this species in Japan. The genetic diversity and structure within and among eleven extant populations were assessed using the inter-simple sequence repeats (ISSR) polymerase chain reaction in association with combinations of propagation pattern (clonal and/or seeds) and genotypic geographical structure. In total, 53 bands were amplified, of which 18 (34%) were polymorphic. Analysis of the ISSR bands identified 46 genotypes among 81 individuals from one stream population and 72 distinct genotypes among 147 individuals in the Kinki district. An unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed some unity among upstream and downstream subpopulations within one stream and eleven populations. The Shannon index of genetic diversity was 0.109 for one stream population and 0.313 for total genetic diversity, suggesting relatively high genetic diversity. Analysis of molecular variance (AMOVA) revealed that 84.1% of the total genetic diversity occurred among populations and the remaining diversity (15.9%) occurred within populations. Significant genetic differentiation occurred among populations in the Kinki district. These results suggest that conservation of each population is important for maintaining genetic diversity of R. nipponicus in this district. An erratum to this article can be found at     

Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (H _E = 0.294 by ISSR and H _E = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (G _ST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.     

Historical divergence vs. contemporary gene flow: evolutionary history of the calcicole Ranunculus alpestris group (Ranunculaceae) in the European Alps and the Carpathians

Although many species have similar total distributional ranges, they might be restricted to very different habitats and might have different phylogeographical histories. In the European Alps, our excellent knowledge of the evolutionary history of silicate‐dwelling (silicicole) plants is contrasted by a virtual lack of data from limestone‐dwelling (calcicole) plants. These two categories exhibit fundamentally different distribution patterns within the Alps and are expected to differ strongly with respect to their glacial history. The calcicole Ranunculus alpestris group comprises three diploid species of alpine habitats. Ranunculus alpestris s. str. is distributed over the southern European mountain system, while R. bilobus and R. traunfellneri are southern Alpine narrow endemics. To explore their phylogenetic relationships and phylogeographical history, we investigated the correlation between information given by nuclear and chloroplast DNA data. Analyses of amplified fragment length polymorphism fingerprints and matK sequences gave incongruent results, indicative for reticulate evolution. Our data highlight historical episodes of range fragmentation and expansion, occasional long‐distance dispersal and on‐going gene flow as important processes shaping the genetic structure of the group. Genetic divergence, expressed as a rarity index (‘frequency‐down‐weighted marker values’) seems a better indicator of historical processes than patterns of genetic diversity, which rather mirror contemporary processes as connectivity of populations and population sizes. Three phylogeographical subgroups have been found within the R. alpestris group, neither following taxonomy nor geography. Genetic heterogeneity in the Southern Alps contrasts with Northern Alpine uniformity. The Carpathians have been stepwise‐colonised from the Eastern Alpine lineage, resulting in a marked diversity loss in the Southern Carpathians. The main divergence within the group, separating the ancestor of the two endemic species from R. alpestris s. str., predates the Quaternary. Therefore, range shifts produced by palaeoclimatic oscillations seem to have acted on the genetic structure of R. alpestris group on a more regional level, e.g. triggering an allopatric separation of R. traunfellneri from R. bilobus.     

Evaluating the relationship of autumn buttercup (Ranunculus acriformis var. aestivalis) to some close congeners using random amplified polymorphic DNA

Autumn buttercup, an endangered endemic of wet, saline meadows in Garfield County, Utah, is treated differently in various taxonomic references now in use in the region. The taxon is currently listed as endangered by the United States Fish and Wildlife Service under the name Ranunculus acriformis var. aestivalis L. Benson. Others treat it as R. acris var. aestivalis (L. Benson) Welsh. In an attempt to resolve the taxonomical disagreements and to quantitatively describe the genetic uniqueness of this taxon, putative close relatives of the autumn buttercup, namely R. occidentalis (Nevada County, CA), R. acris (introduced from Europe and now growing wild in Rich and Cache counties, UT), R. acriformis var. montanensis (Lincoln County, WY), R. acriformis var. acriformis (Emery and Sanpete counties, UT), and R. acriformis var. aestivalis were collected and analyzed using random amplified polymorphic DNA (RAPD) genetic markers amplified from total cellular DNA extracts. Twenty-three decanucleotide primers of arbitrary sequence were used for DNA amplification; over 350 markers were generated. The genetic marker data were analyzed using similarity coefficients and parsimony. Based on the results of those analyses and on a review of ecological and morphological characteristics, we have elevated the autumn buttercup to species rank as Ranunculus aestivalis (Benson) Van Buren & Harper.     

Isolation and characterization of thirteen microsatellite loci in four diploid species of Ranunculus cantonensis polyploid complex

Ranunculus is a cosmopolitan genus, and the evolution of the genus has been influenced by both polyploidy and hybridization. The interactions between some species of Ranunculus are regarded as a natural model system for studying hybridization. Four diploid species of the R. cantoniensis polyploid complex have relatively little reproductive isolation from each other, and they form a complicated hybrid pool in a multispecies hybrid zone. To clarify the genetic structure and spatiotemporal dynamics of gene flow in this multispecies hybrid zone, 13 polymorphic microsatellite loci were isolated from R. silerifolius var. dolicanthus. The loci's characteristics were observed in 30 individuals. The number of alleles per locus ranged from 6 to 20, and the PIC values ranged from 0.350 to 0.914. Eleven loci were significantly biased from the Hardy–Weinberg equilibrium. The 13 microsatellite loci were shown to be successfully amplified in three other related species. These polymorphic microsatellites will be powerful molecular tools for further studies of the Ranunculus genetic structure and gene flow.     

Characterization of novel microsatellite loci in rare minnow (<Emphasis Type="Italic">Gobiocypris rarus</Emphasis>) and amplification in closely related species in Gobioninae

Rare minnow (Gobiocypris rarus) is an endangered small fish endemic to upper reach of the Yangtze River. From a (GT)n enriched genomic library, 32 microsatellites were isolated and characterized. Nineteen of these loci were polymorphic in a test population with alleles ranging from 2–7, and observed and expected heterozygosities from zero to 0.8438, and 0.2679 to 0.8264, respectively. In the cross-species amplifications, 13 out of 19 polymorphic loci were found to be also polymorphic in at least one of the 7 closely related species of the subfamily Gobioninae. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the fine-scale population structure in rare minnow and its closely related species for the conservation purpose.     

RAPD variation among and within small and large populations of the rare clonal plant Ranunculus reptans (Ranunculaceae)

In the pre-alpine region of Europe numbers and sizes of populations of the clonal lake shore plant Ranunculus reptans have declined because of the regulation of lake water levels. We investigated genetic variation among and within 17 populations of different size (cover 1–10 000 m²) in R. reptans with RAPD (random amplified polymorphic DNA) profiles. We sampled 127 rosettes in 14 populations at Lake Constance and three populations at or near Lake Como. There was significant genetic variation between plants from the two lake regions (5.9%, analysis of molecular variance [AMOVA], P < 0.001), among populations within lake regions (20.4%, P < 0.001), and within populations (73.7%, P < 0.001). Under the assumptions of Wright's island model the variation among populations corresponds to a gene flow of N_em = 0.70. Within the 14 Lake Constance populations we detected significant genetic variation among subpopulations separated by only a few metres (4.0% of the within-population variation; P < 0.05). Molecular variance was 24% smaller in small populations covering <100 m² area than in larger ones (P < 0.03), indicating that samples from large populations were genetically more variable than samples representing comparable areas of smaller populations. We conclude that gene flow among populations is very limited and that genetic drift has caused reduced genetic variability of smaller populations. Conservation of genetic variability in R. reptans requires persistence of large and also of small populations (because of population differentiation), and it could be enhanced by increasing the size of small populations (to counter genetic drift).     

Eight new polymorphic microsatellite loci for genetic analyses in the endangered common hamster (Cricetus cricetus L.)

We developed primers for eight polymorphic microsatellite loci in the endangered common hamster (Cricetus cricetus L.). Population genetic parameters were analysed on the basis of 70 adult individuals captured from a single population. Allele numbers per locus ranged from eight to 17. Expected and observed heterozygosities per locus ranged from 0.67 to 0.91 and from 0.41 to 0.8, respectively.     

Ranunculus uttaranchalensis (Ranunculaceae): a new species from Western Himalaya,India

A new species of the genus Ranunculus, R. uttaranchalensis, is described from Gangotri National Park, Uttaranchal in Western Himalaya, India. It is distinguished from the closely allied R. lobatus in having radical leaves sub‐cordate with hairy lamina base and petiole hairy above; cauline leaves sessile, 3–7 lobed with linear to narrowly lanceolate lobes; sepals usually reddish brown, externally hairy; petals rounded‐obovate and oblong, receptacle glabrous except 1–3 hairs at the top.     

POPULATION STRUCTURE ALONG A STEEP ENVIRONMENTAL GRADIENT: CONSEQUENCES OF FLOWERING TIME AND HABITAT VARIATION IN THE SNOW BUTTERCUP,RANUNCULUS ADONEUS

Few studies have determined how gene flow and selection interact to generate population genetic structure in heterogeneous environments. One way to identify the potential role played by natural selection is to compare patterns of spatial genetic structure between different life cycle stages and among microenvironments. We examined patterns of spatial structure in a population of the snow buttercup (Ranunculus adoneus), using both adult plants and newly emerged seedlings. The study population spans a steep environmental gradient caused by gradual melting of snow within a permanent snowbed. Early-melting sites are characterized by denser vegetation, more fertile soils, and a longer growing season than late-melting sites tens of meters away. The flowering time of R. adoneus is controlled entirely by time of snowmelt, so the contiguous population is phenologically substructured into a series of successively flowering cohorts, reducing the opportunity for direct pollen transfer between early- and late-melting sites. For four highly polymorphic enzyme loci in this tetraploid species, there was subtle, but statistically significant, genetic differentiation between early, middle, and late-melting cohorts; adults usually showed greater differentiation among snowmelt zones than did seedlings. At two loci in adults and one locus in seedlings, homozygotes were more common than predicted at Hardy-Weinberg equilibrium, even when assuming maximum levels of double reduction during meiosis. This pattern suggests the occurrence of self-fertilization and/or population substructure. To determine how spatial isolation and phenological separation each contribute to genetic substructure, we used bivariate regression models to predict the numbers of allele differences between randomly paired individuals as a function of meters separation in space and days separation in flowering time. For newly emerged seedlings, we found that spatial separation was positively associated with genetic difference, but that the additional contribution of phenological separation to genetic difference was not significant. This implies that seeds and/or pollen move effectively across the snowmelt gradient, despite differences in flowering time. As was true for seedlings, spatial separation between paired adults contributed to greater genetic difference, but for a given spatial separation, the genetic difference between adult plants was reduced by phenological separation. This result implies that postemergence selection is favoring at least some seeds that migrate across the snowmelt gradient. Directional gene flow across the snowmelt gradient probably results from a genetic source-sink interaction, that is, the colonization of ecologically marginal late-melting sites by high quality seeds produced by the larger subpopulation in early-melting sites. Effective gene flow from high to low quality microenvironments is likely to impede adaptation to late-melting locations.     

Microsatellite DNA primers for the endangered vermilion darter,Etheostoma chermocki,and cross‐species amplification in other darters (Percidae: Etheostoma)

The endangered vermilion darter (Etheostoma chermocki) is endemic to the Black Warrior River system in the Mobile Basin in Alabama. Restoration and conservation of this species require an understanding of its population genetic structure, which can be characterized using microsatellite DNA. Nine microsatellite loci were developed; eight loci were polymorphic. Although observed heterozygosity was lower than expected heterozygosity in most polymorphic loci, only one locus showed significant deviation from Hardy–Weinberg equilibrium. These nine markers were tested in an additional 24 species of Etheostoma and appear to have sufficient allelic variation to be useful in studies of population genetic structure.     

中国云南毛茛科毛茛属一新种——哀牢山毛茛

描述了中国云南毛莨科Ranunculaceae一新种--哀牢山毛莨Ranunculus ailaoshanicus W.T.wang。此种在体态上与圆裂毛莨R．dongrergensis Hand．-Mazz．极为相似,但以茎较低矮,无叶,花较小,花瓣较小,长圆状椭圆形,脉不明显而相区别。     

Genetic diversity and genetic structure of populations of Ranunculus japonicus Thunb. (Ranunculaceae)

Abstract In order to clarify the genetic diversity and population structure of Ranunculus japonicus , allozymic analysis was conducted on 60 populations in southwestern Japan. Considerable genetic variati ons were detected among the populations of R. japonicus . The genetic diversities within species ( H _es = 0.215) and within populations ( H _ep = 0.172) were slightly higher than those of other perennial herbs with widespread distribution and outcrossing plants. Significantly higher values of fixation index were detected in some populations, which might have arisen from restricted mating partners. The majority of genetic variation (approx. 80%) resided within a population and a moderate level of genetic differentiation ( G _ST = 0.203) was observed among populations. The F _ST value (0.203) suggests the existence of a substantial population structure in this species. The highly significant correlation between geographic distance and F _ST values indicates that isolation by distance has played an important role in the construction of the genetic structure of this species.     

Aspects of the Comparative Physiology of Ranunculus bulbosus L. and Ranunculus repens L.: I. Response to Nitrogen

Ranunculus bulbosus and R. repens are closely related taxonomicallyand coexist in the same general habitat but differ in that R.bulbosus only exceptionally shows vegetative multiplicationcontrasting with R. repens which reproduces extensively by meansof long branched stolons which root and form new daughter plantsat the nodes. The response of these two species to nitrogen was similar inthat an increase from 10 to 50 ppm nitrogen gave rise to greatlyincreased total dry weight and higher shoot to root ratios,whereas further nitrogen increase from 50 to 200 ppm had littleeffect. However, they differed in that at low nitrogen levelsthe perennating organ (corm) took priority in R. bulbosus withsexual reproduction assuming greater importance at the highernitrogen levels, whereas in R. repens the main storage organ(the stock) was not especially favoured at low nitrogen levelsreflecting perhaps the differences between this structure andthe corm in R. bulbosus. In R. repens the greatest responseto nitrogen was shown by the stolons although this was due toincreased branching and a higher dry weight per unit lengthat the higher levels of nitrogen (50 and 200 ppm). Primary stolonlength was not greatly affected by nitrogen level. Dry weightsof flowers and fruits in R. repens were very much lower thanin R. bulbosus. It is suggested that in R. bulbosus the emphasis lies firstin replacement of the parent at approximately the same siteby a daughter from the new corm and secondly in sexual reproductivespread. In R. repens lateral spread by vegetative propagationtakes precedence over all else and under low nitrogen conditionsbranching is sacrificed and energy is diverted to maintaininglong primary stolons. These, in turn, have the potential forproducing daughter plants at some distance from the parent andthis may be of considerable importance in field situations sincethe daughter may come to occupy more favourable sites than theparent.     

Isolation and characterization of microsatellites from a marine foodfish species ribbonfish Trichiurus haumela

The ribbonfish, Trichiurus haumela, is an important marine foodfish species. Supply of this fish relies on capture from the wild. No information about genetic diversity and population structure of wild stocks is available. Therefore, we developed the first set of 10 polymorphic microsatellites. The average allele number was 13.2 per locus ranging from six to 20. The expected heterozygosity was 0.84, whereas the observed heterozygosity was 0.71. This set of microsatellites provides a tool for assessing genetic diversity and population structure of this species.     

A New Species of Ranunculus L. from Xinjiang

A new species of the genus Ranunculus L., R. hamiensis J . G. Liu, isdescribed from Xinjiang.     

Isolation and characterization of polymorphic microsatellite markers in <Emphasis Type="Italic">Cupressus </Emphasis><Emphasis Type="Italic">chenggiana</Emphasis> S. Y. Hu (Cupressaceae)

Cupressus chenggiana S. Y. Hu (Cupressaceae) is an endemic and endangered conifer species in southwest China. In order to study the population genetics and design the effective conservation methods, we aimed to develop microsatellite primers for this species in the present study. We developed eight new microsatellite loci for this species through biotin capture method. Polymorphism of each locus was further assessed in 18 individuals from three geographically distant populations. The number of alleles per locus ranged from 6 to 11 with an average of 8.13. The observed and expected heterozygosities ranged from 0.219 to 0.296 and from 0.374 to 0.470, with averages of 0.254 and 0.417, respectively. We further found that three of nine microsatellite loci developed previously for another congeneric species showed polymorphic banding patters. We performed primer-crossing tests of these loci in the other two congeneric species which are closely related to C. chenggiana (C. gigantea and C. duclouxiana). These microsatellite markers would be effective for analyzing genetic diversity and population genetic structure of this species and its morphological differentiation with the close relatives.     

Diploid and autotetraploid sexuals and their relationships to apomicts in the Ranunculus cassubicus group: insights from DNA content and isozyme variation

 From the predominantly aposporous Ranunculus cassubicus group, a subgroup of the R. auricomus complex, two species with both diploid and tetraploid cytodemes (R. cassubicifolius W. Koch and R. carpaticola Soó) were known. Nine population samples of both species have been analyzed for variation of ploidy levels and isozymes. DNA image analysis showed that three populations of R. cassubicifolius from Bavaria and Salzburg are diploid, two from Lower Austria tetraploid. In R. carpaticola, two populations from Central Slovakia and one from Romania are diploid, one from northern Slovakia is hexaploid. The polyploid populations had somewhat smaller C-values than expected from diploids. Ploidy levels are consistent within populations, contradicting previous hypotheses that cycles of diploid-tetra-ploid-dihaploid apomictic individuals occur in natural populations of goldilocks. Isozyme-allozyme analysis of nine polymorphic loci showed that individual variation and genetic measures of di-ploid and tetraploid populations are equivalent to those of sexual taxa. Multiple allelism and unbalanced gene dosages in tetraploid R. cassubicifolius give evidence for autopolyploidy that is most probably of multiple origin. The observed excess of homozygotes and the high diversity between populations in the diploid populations of R. cassubicifolius are most probably due to geographical isolation of population groups, that might have happened during the Würm glaciation and might have promoted the separation of autotetraploids in the easternmost part of the distribution range. Genetic distance values analyzed by UPGMA of all sexual populations separated the two taxa and the cytodemes within R. cassubicifolius. Multidimensional scaling of individuals (including the apomicts) based on a presence/absence matrix of alleles confirmed this differentiation with an overlapping zone between R. cassubicifolius and R. carpaticola. The hexaploid R. carpaticola population showed reduced genotypic diversity, an increased number of heterozygotes and fixed heterozygosity as typical for apomictic mode of reproduction. This population shared alleles of both species without any specific ones, multidimensional scaling placed genotypes among R. cassubicifolius. Thus, the hexa-ploid apomicts might have originated from hybrids of R. cassubicifolius and R. carpaticola. In general, evolution of agamic lineages in the R. cassubicus complex might have been facilitated by autopoly-ploids, because they can provide a bridge between the reproductive systems that are otherwise isolated by ploidy levels, and also a starting point for spontaneous origin of apomixis. Received October 8, 2001; accepted May 10, 2002 Published online: November 7, 2002 Addresses of the authors: Elvia H?randl (E-mail: elvira.hoerandl@univie.ac.at), Department of Systematics and Evolution of Higher Plants; Johann Greilhuber (E-mail: Johann.Greilhuber@univie. ac.at), Department of Systematic Karyology and Embryology of Plants; both: Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.