Molecules and morphology in concert: tests of some hypotheses in arctic Potentilla (Rosaceae)

We developed a combined molecular and morphological approach to unravel complex variation at low taxonomic levels, exemplified by some arctic members of Potentilla. Twenty-one populations from Svalbard were analyzed for random amplified polymorphic DNAs (RAPDs) and 64 morphological characters to test the hypotheses that (1) the P. nivea complex (section Niveae) consists of three taxa (P. chamissonis, P. insularis, and P. nivea), (2) three "eco-morphotypes" in P. pulchella (section Multifidae) should be considered different taxa, and (3) P. insularis originated as an intersectional hybrid (Niveae × Multifidae). Twenty-two RAPD multilocus phenotypes were observed in the 136 plants analyzed based on 35 markers. Three fairly distinct groups of RAPD phenotypes were identified in the P. nivea complex based on multivariate analyses and an analysis of molecular variance (AMOVA; 77.6% among-group variation). The variation within the P. nivea complex was more or less continuous in multivariate analyses of the morphological data. We identified, however, several individual morphological characters that separated unambiguously among the three groups of RAPD phenotypes, revealing that these groups correspond to the previously hypothesized taxa. Many identical RAPD multilocus phenotypes were observed in the "eco-morphotypes" of P. pulchella, suggesting that its conspicuous morphological variation is caused by plasticity or by genetic variation at a small number of loci. The hypothesis of the hybrid origin of P. insularis was not supported by the RAPD data. Overall, very little RAPD variation was observed within populations of the four taxa (2.1-16.7% in AMOVA analyses; average genotypic diversity, D, was 0.10-0.30). We conclude that detailed, concerted analysis of molecules and morphology is a powerful tool in low-level taxonomy.     

Variation in the Festuca brachyphylla (Poaceae) complex in Svalbard, elucidated by chromosome numbers and isozymes

Contrasting with former taxonomic treatments, chromosome numbers and isozyme data support the delimitation of the seminiferous representatives of the Festuca brachyphylla complex in Svalbard into four species: F. baffinensis, F. brachyphylla, F. hyperborea and F. edlundiae. Unique enzyme markers were found for all species. Festuca brachyphylla proved hexaploid, and the others, tetraploid. The chromosome numbers of F. hyperborea and F brachyphylla (as circumscribed at present) are new to Svalbard. Festuca baffinensis is the most distinct species within the complex, probably representing a separate evolutionary lineage. The three other species seem closely related, showing mutually equidistant relationships. Some deviating plants found on disturbed ground might represent hybrid derivatives or an introduced foreign strain of the elsewhere variable F. brachyphylla. Materials of diploid F. ovina from northern Fennoscandia was enzymatically closely related to the F brachyphylla complex in Svalbard. Festuca brachyphylla, F. edlundiae , and F. hyperborea all had a stronger affinity to F ovina than to F baffinensis , indicating that the F brachyphylla complex is an artificial taxonomic group. There are reasons to believe that the origin of the polyploid taxa of the F brachyphylla complex can be traced to diploid species of the F. brachyphylla and F ovina complexes.     

Trans-Atlantic dispersal and phylogeography of Cerastium arcticum (Caryophyllaceae) inferred from RAPD and SCAR markers

Cerastium arcticum is an autogamous pioneer species with a distribution limited to the North Atlantic region. It has been suggested that such species must have survived in ice-free refugia on both sides of the Atlantic throughout the last, or even several, of the Pleistocene glaciations, because they lack special adaptations for long-distance dispersal. To address the possibility for recent trans-Atlantic dispersal of C. arcticum, we analyzed random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) differentiation among 26 populations of this high-polyploid species. Three SCAR markers were obtained that verified the main patterns identified in the RAPD analysis. Eighty-four multilocus RAPD phenotypes were observed in the 126 plants analyzed, based on 35 polymorphic markers. Multivariate analyses and analyses of molecular variance (AMOVAs) identified two highly divergent groups of populations: one arctic group (western and eastern Greenland, and the archipelagos of Svalbard and Franz Josef Land) and one nonarctic group (southern and northern Norway, and Iceland), indicating that C. arcticum is composed of two lineages with different evolutionary histories. However, there was little geographic structuring within each lineage, in spite of the fact that both lineages are disjunctly distributed across the Atlantic. Occurrence of very similar, in some cases even identical RAPD multilocus phenotypes on both sides of the Atlantic in this autogamous allopolyploid is most probably caused by postglacial dispersal. The present geographic distribution of C. arcticum may thus have been established after trans-Atlantic expansion from two Weichselian refugia, one for each evolutionary lineage. Unexpectedly, the level of intrapopulational variation increased towards the north. This may reflect that interpopulational migration is most extensive in the treeless arctic environment, where the species has a more continuous distribution than in the more southerly areas.     

Infraspecific variation and phylogeography of the high-polyploid Iberian endemic Anthoxanthum amarum Brot. (Poaceae; Pooideae) assessed by random amplified polymorphic DNA markers (RAPDs) and morphology

High morphological and ecological diversity has been observed in the north-west Iberian Peninsula endemic Anthoxanthum amarum Brot., for which two different morphotypes (northern and southern) have been described on the basis of qualitative traits and geographical origin. In the present study, a combined molecular and morphological method was applied to ten populations of this species with the following aims: (1) to assess whether the variation observed was taxonomically meaningful; (2) to assess the influence of the environment on the variation in the morphological characters; and (3) to track the potential phylogeographical information deduced from our random amplified polymorphic DNA marker (RAPD) data in order to draw inferences about the past history of this species in the north-west Iberian Peninsula. To achieve these aims, 26 macromorphological characters were recorded in 279 specimens, and 77 RAPD phenotypes were identified in the 79 plants studied. The association analyses performed using the morphological and molecular data showed that no clear separation existed between the morphs, and a strong correlation between qualitative characters and the environment was detected. Moreover, both the multivariate analyses and the assignment test based on RAPD data revealed that the genetic variation was hierarchically structured, and three genetically distinct groups could be identified. Two of these clusters might correspond to different expansion routes proposed in the literature for different plant species in the north-west Iberian Peninsula.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 179–192.     

Amplified fragment length polymorphism versus random amplified polymorphic DNA markers: clonal diversity in Saxifraga cernua

Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories.     

The species distinction of the narrow-leaved Festuca from East Africa based on AFLP fingerprinting and morphology

The distinctness of the species Festuca claytonii , F. elgonensis and F. pilgeri (endemic species in East Africa) was assessed by comparison to the widespread narrow-leaved species F. abyssinica , F. chodatiana and F. richardii . The six species were analysed using 56 morphological characters and 375 AFLPs. Principal components and discriminant analyses of the morphological data revealed that the endemic species F. claytonii and F. pilgeri are good species, but that F. abyssinica, F. elgonensis and F. richardii do intergrade. Relevant morphological characters were identified for an overall species distinction but also for pairwise comparisons. A principal coordinate analysis of the AFLPs confirmed the species distinction mentioned above. In conclusion, four narrow-leaved species of Festuca occur in Uganda, viz. F. abyssinica , F. chodatiana , F. claytonii and F. pilgeri . F. elgonensis and F. richardii should be included in F. abyssinica . A key to the species is given.     

Festuca brachyphylla, F. saximontana and related species in North America

American material of Festuca brachyphylla Schult. & Schult., F. saximontana Rydb. and related species was studied. Based on morphology and anatomy F. brachyphylla was separated into three subspecies, two of which are only found in very restricted areas of the southern part of North America, one in California (ssp. breviculmis Frederiksen ssp. nov.) and one in Colorado and neighbouring states (ssp. coloraden–sis Frederiksen ssp. nov.). F. jenseni Gjaerevoll & Ryvarden was found to be the same as F. brachyphylla i.flavida Polun. and the forma level is chosen. F. brachyphylla var. groenlandica Schol. is raised to F. groenlandica (Schol.) Frederiksen. Within F. saximontana a new combination (f. saximontana Rydb. var. purpusiana Frederiksen & Pavlick) is made. American distribution of the taxa studied is mapped. A key including all the taxa has been prepared.     

Isozyme analysis of theCerastium alpinum-C. arcticum complex (Caryophyllaceae) supports a splitting ofC. arcticum Lange

As part of a larger investigation of theC. alpinum-C. arcticum complex of arctic and North Atlantic areas, isozyme variation ofC. alpinum, C. arcticum, and related taxa was analysed. A total of 124 multilocus phenotypes was divided into more or less distinct groups by numerical analyses. Most groups correspond well to previously recognized taxa. However, what has traditionally been considered asC. arcticum was divided into two distinct groups, consisting of northern (Svalbard, Greenland) and more southern (Norway, Iceland) populations, respectively. The division ofC. arcticum into two taxa is also supported by other kinds of data and the two taxa probably deserve species rank. Serpentine plants from Shetland had multilocus phenotypes similar to those ofC. arcticum from Iceland and should be included in the southern taxon.     

Genetic similarity among male bees of Euglossa truncata Rebêlo & Moure (Hymenoptera: Apidae) revealed by RAPD markers

The genetic RAPD (random amplified polymorphic DNA) markers have been used successfully in taxonomical studies of several groups of organisms. In the present study these molecular markers were used to analyze the genetic similarity among eighteen males of Euglossa truncata Rebêlo & Moure exhibiting variations in two morphological characters (colour of the antennal scape and metaepisternal hairs) which are frequently used to identify this species of euglossine bee. The twelve primers used in the RAPD analysis amplified 127 loci, of which 40 (31.5%) were polymorphic, showing some variation among the individuals. The coefficients of genetic similarity among the individuals ranged from 0.79 to 0.95, indicating a rather high genetic similarity among the 18 male bees studied. No RAPD band was specific to any morphological character analyzed. The results indicate that all bees analyzed belong to the same species. The high genetic similarity among the eighteen euglossine males studied indicates that the variations observed in the morphological characters are not in disagreement with the identification of this species of Euglossina and these characters can vary among males of E. truncata.     

Genomic variation and relationships in aerial yam (Dioscorea bulbifera L.) detected by random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) markers were used to assess intraspecific variability and relationships in aerial yam (Dioscorea bulbifera L.). A total of 23 accessions from different geographic locations in Africa, Asia, and Polynesia were analyzed by 10 arbitrarily chosen GC-rich decamer primers. Using cesium chloride purified genomic template DNA, highly reproducible polymorphic fingerprints were generated by all 10 primers, resulting in a total of 375 informative characters. Only eight bands were monomorphic among all investigated accessions. A binary character matrix was generated by scoring for presence/absence of a band at a particular position, transformed into a matrix of pairwise distances using either the Jaccard or a simple matching coefficient, and analyzed by neighbour joining, UPGMA (unweighted pair group method with arithmetic averaging) cluster analysis, or split decomposition. All methods of data evaluation resulted in similar groupings that reflected the geographical origin of the samples. The African accessions formed a distinct isolated group, whereas Asian and Polynesian accessions proved to be more heterogeneous. With two exceptions (var. suavior and var. sativa), the RAPD data supported previous varietal classification based on morphological characters. Stepwise reduction of the number of evaluated characters did not affect branching patterns of the trees above a minimum threshold of 150. Key words : Dioscorea bulbifera, random amplified polymorphic DNA (RAPD), genetic variation, genetic relatedness.     

Molecular evidence for polyploid origins in Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies

The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis: (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua. Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis. The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin.     

Evidence for Hybridization by Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Between Leishmania braziliensis and Leishmania panamensis/guyanensis in Ecuador

ABSTRACT. The taxonomic attribution of four Leishmania stocks isolated from humans in Ecuador has been explored by both multilocus enzyme electrophoresis and random amplified polymorphic DNA. For three loci, MLEE results showed patterns suggesting a heterozygous state for a diploid organism, while the corresponding homozygous states are characteristic of the Leishmania panamensis/guyanensis complex and Leishmania braziliensis . RAPD profiles exhibited for several primers a combination of the Leishmania panmensis/guyanensis complex and L. braziliensis characters. These data hence suggest that the four stocks are the results of hybridization between L. panamensis/guyanensis and L. braziliensis . MLEE data show that the results cannot be attributed to either mixture of stocks, or an F1 in the framework of a simple Mendelian inheritance.     

Clonal diversity along a water depth gradient in a declining reed stand as detected by three different genetic methods

Isozyme, RAPD (random amplified polymorphic DNA) and microsatellite techniques were applied to determine the genetic diversity of a reed stand declining at the open water fringe for several decades and the accuracy of these methods was evaluated. Sampling was carried out along three transects parallel with and one transect at right angle to the lakeshore. In isoenzyme investigation, esterase and further 11 polymorphic loci of seven isozymes (with a total of 26 alleles) were analysed and 39 clones were distinguished. RAPD analysis involved 11 random decamer primers and, based on 141 repeatably amplifying and polymorphic RAPD fragments, 52 multilocus genotypes were differentiated. In microsatellite investigation, four primer pairs provided 47 polymorphic allelic variants that determined 37 multilocus phenotypes.Besides the differences, all the three methods revealed high number and intermingling (mixed) arrangement of clones at lower water depths, and low clone number and successive structure at the open water fringe of the stands. Only microsatellite analysis revealed different stages of colonization: old successful clone (colonizing the deeper water but almost completely disappearing at the lakeshore edge) and successful but young clone (with large extension at the lakeshore and just reaching the open water edge of the stand) were also distinguished. Finally, the present paper demonstrated that an appropriate data evaluation could make the result of RAPD comparable to that of microsatellite studies.     

云南金钱槭形态变异与遗传变异的相关性研究

对我国特有珍稀濒危保护植物云南金钱槭的形态变异水平、遗传变异模式以及两者之间的相关性进行了研究。形态学性状分析结果表明 :各居群形态性状变异系数的平均值从大到小排列为 :文山居群 (WSh)、屏边居群 (PB)、黑龙潭居群 (HL T)、蒙自居群 (MZ) ;文山居群与屏边、黑龙潭、蒙自居群间已产生显著或极显著水平的形态差异 ,而后三者间的差异未达显著水平。RAPD分析检测到 10 3个位点 ,其中多态位点 84个 ,云南金钱槭物种水平的多态位点比率为 81.5 5 % ,与其它珍稀濒危植物相比该种的遗传多样性水平不低。 AMOVA和 N ei基因多样性指数分析显示 ,尽管大部分遗传变异仍存在于居群内 (分别为5 7.86 %、5 7.33% ) ,但居群间的遗传变异已达较高水平 (分别为 4 2 .14 %、4 2 .6 7% )。相关分析结果显示 ,云南金钱槭的形态变异与海拔、土壤有机质等生态因子有着显著或极显著水平的相关性 ,但与遗传变异的相关性未达显著水平 ,说明云南金钱槭的形态变异虽然具有一定的遗传基础 ,但可塑性及环境压力对形态变异的产生作用更大一些。基于形态性状和 RAPD数据的聚类分析则进一步说明 ,云南金钱槭的形态变异受到环境因子的强烈影响而与遗传背景的关系不显著     

用RAPD分子标记探讨沙拐枣属的种间关系

利用随机扩增多态性DNA（RAPD）技术分析了14种沙拐枣属（Calligonum L.)植物,通过对16个Sangon公司十聚体随机引物进行PCR扩增,3个引物能产生多态性带。对3个引物扩增产生的45条扩增产物,计算单匹配系数,应用UPGMA方法构建亲缘关系树状图。分析结果表明：（1）物种间遗传差异明显,具有丰富的遗传多样性;（2）14种沙拐枣属植物明显聚为4类,与传统的形态学分类结果基本一致。     

Comparative studies of genetic diversity in kenaf (Hibiscus cannabinus L.) varieties based on analysis of agronomic and RAPD data

Kenaf (Hibiscus cannabinus L.) is a fiber crop classified in the genus Hibiscus (Malvaceae), and has a great potential for its multipurpose utilization, in addition to its traditional usage. Varietal identification of kenaf is always problematic and knowledge on genetic diversity of kenaf varieties is also limited, which significantly hindered our effective utilization and conservation of the valuable kenaf germplasm. In order to find a proper method for identifying kenaf varieties and studying their variation, morpho-agronomic characters and random amplified polymorphic DNA (RAPD) markers were analyzed among 14 kenaf varieties commonly used in Japan. Data from morphological analysis showed that the included kenaf varieties could be divided into three major groups. The characters, such as middle stem diameter, whole stalk weight, and days to 50% flowering, are highly responsible for the variation of the kenaf varieties, but it is difficult to identify individual varieties merely by the morpho-agronomic characters. On the other hand, clearly separation of the kenaf varieties was achieved based on the RAPD variation patterns. Genetic relationship of the kenaf varieties can also be traced through the analysis of RAPD and morph-agronomic variation. It is concluded from the present study that RAPD analysis is an effective tool in identifying of kenaf varieties and determining their genetic relationships, particularly when combined with the analysis of morpho-agronomic characters.     

Incongruent phylogeographies in spite of similar morphology, ecology, and distribution:Phippsia algida andP. concinna (Poaceae) in the North Atlantic region

Phylogeographic and taxonomic relationships among 54 North Atlantic populations of the snowbed grass genusPhippsia were investigated based on isozymes and genetically based morphological variation. The results support recognition of two distinct species,P. algida andP. concinna, the latter with at least two subspecies. Both of these self-fertilizing arctic pioneer species were genetic allotetraploids almost without intrapopulational variation. The two species showed strikingly different phylogeographies in the North Atlantic region in spite of their similarity in morphology, habitat ecology, mating system, and dispersal ecology, and in spite of their present cooccurrence in many geographic areas, sometimes even in the same snowbeds. The same electrophoretic multilocus phenotype was observed in all populations ofP. algida, and although this species showed considerable morphological variation, the variation was unstructured geographically. Thus,P. algida showed a pattern similar to other arctic species investigated in the North Atlantic region; it has probably dispersed postglacially across the sea barriers among Greenland, Svalbard, Iceland, and Scandinavia. In contrast,P. concinna was virtually fixed for different multilocus phenotypes in the three main geographic areas analyzed (S Norway, Svalbard, Greenland), corresponding to fairly distinct divergence in morphology. This pattern suggests absence of postglacial among-area dispersal ofP. concinna in spite of all of its similarities with its congener; it may have immigrated to the North Atlantic from different source areas and/or survived the last glaciationin situ.     

The relationship between Callitriche L. clones and environmental variables using genotyping

The genetic variation and clonality in Callitriche L. populations from 16 headwater rivers in Belgium were determined by random amplified polymorphic DNA (RAPD). It was shown that samples of morphological C. obtusangula can be separated into at least two genetically distinct groups. Both gene pools occurred within each considered river basin, i.e. Nete and Meuse. A multivariate analysis showed that the variability among different clones, as characterized by their genotypes, can be explained through ecological indicator values such as diatom saprobity and environmental variables related to water quality type. This study showed that multilocus genotypes or gene pools might have a non-random distribution in the rivers and in their particular environments.     

Glacial survival does not matter: RAPD phylogeography of Nordic Saxifraga oppositifolia

The arctic-alpine Saxifraga oppositifolia has recently been suggested to have survived the last glaciation in high-arctic refugia, based on a finding of more genetic (RFLP) variation in Svalbard compared with more southern areas. To elucidate the migration history of this allogamous species, we analysed 18 populations from Norway, Svalbard and Novaya Zemlya using random amplified polymorphic DNAs (RAPDs). There was no more RAPD variation in the high Arctic than further south. In an analysis of molecular variance (AMOV A), most of the RAPD variation was found within populations (64%). There was less intrapopulational variation in Svalbard (65%) than in northern Norway (78%) and southern Norway (86%), suggesting that there is more inbreeding towards the north, probably because of lower pollinator activity. Twenty-eight per cent of the RAPD variation was found among populations within these geographical regions, and only 9% was found among the regions. In PCO and UPGMA analyses, plants and populations of different geographical origins were to a large extent intermingled. There was, however, a distinct, south-north clinal geographical structuring of the RAPD variation both in the PCO analysis and in a spatial autocorrelation (Mantel) analysis. These results suggest that there has been extensive gene flow among more or less continuously distributed populations of S. oppositifolia during the Weichselian, and that the extant Nordic populations were established after massive, centripetal immigration from these genetically variable, periglacial populations. The postglacial period may not have been sufficiently long for the subsequently isolated populations of this long-lived, allogamous perennial to diverge. Given the high levels of migration inferred from this study, genetic differentiation of glacial survivor populations, if any existed, would most likely have been swamped in the postglacial period. Thus, our molecular data support recent conclusions based on palaeobotanical and biogeographical data that the glacial survival hypothesis is superfluous.     

Clonal diversity of Phragmites australis propagating along water depth gradient

Clonal composition of a large, expanding reed stand was studied with two genetic profiling methods. Sampling was carried out along two parallel, and two perpendicular transects at the shore of Lake Balaton, Hungary, in a 900 m long reed stand, with distances between sampling points ranging from 15 to 30 m. The four primer pairs involved in microsatellite comparisons provided a total of 45 polymorphic allelic variants that determined 61 multilocus phenotypes. Along the transects, clone number decreased towards the deep water. RAPD analysis involved eleven random decamer primers and 115 repeatably amplifying and polymorphic RAPD fragments. Applying our new data evaluation method, highly similar information was gained from RAPD investigation and the microsatellite method on the number and extension of the clones. The results demonstrated that clone competition along a water depth gradient where generative reproduction is allowed only at the lakeshore edge brings about decreasing genetic diversity irrespectively of the health status of reed stand.