Genetic characterization of Salix alba L. and Salix fragilis L. by means of different PCR-derived marker systems

Abstract

Salix alba L. and Salix fragilis L. are two closely related willow species whose phenotypic features, showing a large and continuous variation, have a low diagnostic value for identifying pure species and interspecific hybrids. In this paper, the effectiveness of different multilocus PCR-based molecular markers, such as I-SSRs, RAPDs and AFLPs in detecting genetic polymorphisms able to discriminate the two willow species was evaluated by analysing a set of 12 reference samples. Three genetic similarity indexes, Dice, Jaccard and Simple Matching coefficient, were used for all possible pairwise comparisons of individuals, revealing the same trend of variation within and between species when different marker systems were used. Cluster analysis, based on Dice genetic similarity coefficient, clustered the individuals of S. alba and S. fragilis into two distinct subgroups, indicating that the gene pools are well differentiated. Moreover, a number of private alleles for each marker system allowed the discrimination of the two species because always present only in one of the two. The utility of different marker systems in discriminating willow species was evaluated by the Polymorphism Information Content (PIC) and the Marker Index (MI) parameters. The variation of Dice's indexes obtained from a different number of experiments in relation to the marker systems is discussed.     

柳属(杨柳科)五个Rudolf G(o)rz的植物名称后选模式指定

由于Rudolf Gorz在1935年《静生生物调查所汇报》（植物学）第6卷第1期“中国柳属增志”一文中对2新种、2新变种和1新变型植物名称发表时引证了2号或2号以上的标本,但没指定主模式。根据《国际植物命名法规》规则8．1、9．4．9．9,9．10和辅则9A．1、9A．2、9A．3、9A．4的精神,对这些名称作出后选模式指定。     

本地种云南柳与外来种旱柳（杨柳科）的同倍体自然杂交?

对分布于云南的旱柳( Salix matsudana)和云南柳( Salix cavaleriei)之间的一个自然杂交种进行了研究。野外观察表明疑似杂交种异蕊柳( Salix × heteromera)形态上介于旱柳和云南柳之间,并得到了基于叶形态特征的主成份分析的印证。核基因ITS序列数据表明这三个种存在ITS序列的种内和个体内的多态性,且疑似杂交种的ITS序列的基因型总是疑似亲本的嵌合体。因此可以判定异蕊柳是旱柳和云南柳的自然杂交后代。流式细胞分析表明这三个种均为四倍体,因而,本杂交事件为同倍体杂交。基于四个叶绿体序列片段的数据表明本自然杂交事件是不对称的,云南柳是异蕊柳的母本。常见外来种旱柳与稀有本地种云南柳的杂交可能导致稀有种云南柳的濒危甚至灭绝。研究表明柳属植物的引种应非常谨慎。     

Eighteen microsatellite loci in Salix arbutifolia (Salicaceae) and cross-species amplification in Salix and Populus species

Salix arbutifolia is a riparian dioecious tree species that is of conservation concern in Japan because of its highly restricted distribution. Eighteen polymorphic loci of dinucleotide microsatellites were isolated and characterized. Among these, estimates of the expected heterozygosity ranged from 0.350 to 0.879. Cross-species amplification was successful at 9-13 loci among six Salix species and at three loci in one Populus species.     

Genetic variation and putative hybridization inSalix alba andS. fragilis (Salicaceae): Evidence from allozyme data

Genetic variability was estimated by enzyme electrophoresis in 239 Belgian clones from theSalix alba-S. fragilis complex. This morphological complex suggested a high frequency of hybrids. To test this hypothesis, the clones were pooled as a single co-adapted species complex and secondly as belonging to either species, i.e. beingS. alba-like orS. fragilis-like. The standard genetic variability measures showed higher values for the complex than for the separate species. However, the observed mean heterozygosity was lower in the putative hybrid complex than for each of the species separately. The fixation indices were more variable at the species level and indicated that mostS. fragilis locations appeared fixed forlap-1, whereas no fixation occured inS. alba locations. Averaged at the regional (i.e. catchment) level, this difference between the two species remained and values ranged from 0.457 to 0.617 inS. alba and were much higher, fixed homozygous or monomorph inS. fragilis. Hierarchical F-statistics revealed that most of the differentiation occured at the lower levels of localities and tributaries and that there was no further differentiation between catchments. Tributaries which are 10–25km in length were proposed as the most likely entities for further examination of putative hybridization and events of allelic fixations. By considering the two abovementioned approaches of data input, it could be suggested that most of the allozyme differentiation was between the species and less between the regions.     

Ontogeny of pistillate flowers and inflorescences in Nothofagus subgenus Lophozonia (Nothofagaceae)

  Cupule and pistillate inflorescence morphology is described in detail for the first time for a number of species in Nothofagus subgenus Lophozonia. The architecture and temporal appearance of prophylls, flowers, and cupule valves is dichasial; the cupule valves are third and higher order shoot systems. Comparative studies demonstrate that the dorsal rows of appendages on the cupule valves have a similar arrangement, and each row exhibits the same temporal pattern of development. This suggests that the cupule valves in all species have a modular construction and consist of rows of homologous appendages. This study clearly demonstrates the uniform groundplan of the pistillate inflorescence in this subgenus. Received March 14, 2002; accepted May 8, 2002 Published online: September 13, 2002     

Evolution, phylogeography, and taxonomy within the Viola alba complex (Violaceae)

 Taxa of the Viola alba complex were investigated using allozymes and morphometry. A taxonomic revision is presented. A wide delimitation of V. alba with only three morphological and geographical subspecies is suggested: (1) ssp. dehnhardtii distributed in the Mediterranean eastwards to Turkey; (2) ssp. alba flanking ssp. dehnhardtii in the north and east; and (3) ssp. cretica endemic to Crete. Ssp. cretica, up to now treated as a separate species, is particularly close to ssp. dehnhardtii. Viola cadevallii (NW Mediterranean) is included in the synonymy of ssp. dehnhardtii. Ssp. scotophylla (S Europe), ssp. thessala (Balkan), V. armena (Turkey), and V. besseri (Caucasus) are reduced to synonyms of V. alba ssp. alba. Viola pentelica (Greece) might represent transitional forms between ssp. alba and ssp. dehnhardtii. Glacial refugia for ssp. alba are suggested from the eastern Mediterranean via Turkey to the Caucasus, for ssp. dehnhardtii in the Mediterranean area in general, and for ssp. cretica in Crete. A key to the subspecies is provided. Taxonomic recombination: Viola alba Bess. ssp. cretica (Boiss. & Heldr.) Marcussen, comb. nov. Received June 17, 2002; accepted November 27, 2002 Published online: March 20, 2003     

Pollen vectors and inflorescence morphology in four species of Salix

 Many plant species exhibit inflorescence morphologies intermediate between pollination syndromes and may therefore employ generalist pollination strategies. We studied how wind and insect pollination are related to inflorescence morphology in the floodplain species Salix alba, S. elaeagnos, S. daphnoides and S. triandra. Insect exclusion experiments showed that all four species were primarily pollinated by insects, but were capable of some seed set when wind was the only pollen vector. Such a generalist pollination system may provide reproductive assurance in these pioneer species. High wind pollination success was associated with slender and divided stigmatic lobes and low ovule number per catkin, which may enhance filtering capacity for airborne pollen. In contrast, species that relied more on insect pollination had robust stigmata and many ovules per catkin, which may reduce the number of insect visits necessary for pollination. Received April 18, 2002; accepted July 23, 2002 Published online: November 28, 2002 Addresses of authors: S. Karrenberg (e-mail: karrenberg@bio.indiana.edu), Department of Biology, Indiana University, Jordan Hall, 1001 East Third Street, Bloomington, IN 47405, USA. P. J. Edwards, Geobotanical Institute, ETH, Zürichbergstrasse 38, CH-8044 Zürich, Switzerland. J. Kollmann, Department of Ecology, Royal Veterinary and Agricultural University, Rolighedsvej 21, DK-1958 Frederiksberg, Denmark.     

Hybridization between the peat mosses, Sphagnum capillifolium and S. quinquefarium (Sphagnaceae, Bryophyta) as inferred by morphological characters and isozyme markers

 Isozyme markers and morphological characters were studied in four populations of Sphagnum capillifolium and S. quinquefarium. Recombinant plants were found in three populations, where the two species occur sympatrically. All recombinants possessed different haplotypes and combinations of morphological characters, which show that they are results of independent hybridization events. Strongly male-biased sex ratios were found for Sphagnum capillifolium in all populations where it grew sympatrically with S. quinquefarium. Most of the recombinants were also male fertile. These observations suggest that S. quinquefarium is the female parent in the primary crosses and in subsequent backcrosses. Received September 3, 2001; accepted March 16, 2002 Published online: November 7, 2002 Addresses of the authors: Nils Cronberg (e-mail: Nils.Cronberg@sysbot.lu.se), Department of Systematic Botany, Lund University, S?lvegatan 37, SE-223 62 Lund, Sweden. Rayna Natcheva (e-mail: renimoss@iph.bio.bas.bg), Institute of Botany, Bulgarian Academy of Sciences, 23 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria.     

Morphological and molecular evidence for hybridization and introgression in a willow (Salix) hybrid zone

Hybrid zones provide biologists with the opportunity to examine genetic and ecological interactions between differentiated populations. Accurate identification of hybrid genealogies is considered a necessary prerequisite to understanding observed patterns of hybridization-related phenomena. We analysed molecular and morphological data from individuals in a hybrid zone between two species of willows (Salix sericea Marshall and S. eriocephala Michaux) and report the use of randomly amplified polymorphic DNA (RAPD), chloroplast DNA (cpDNA), and ribosomal DNA (rDNA) markers, as well as vegetative morphology and foliar chemistry data to identify individuals in terms of hybrid genealogy and to infer the direction and extent of backcrossing and introgression within the hybrid zone. A novel version of a maximum likelihood estimate approach (developed for this study) was used to calculate hybrid index scores from RAPD marker data; this method produced results similar to those obtained using traditional arithmetic methods. Distribution of rDNA, cpDNA, and chemistry data were examined within the graphical context of RAPD-based hybrid index score histograms and principal component analyses (PCA) on RAPD and morphology data. Seven of the 21 plants classified as S. eriocephala in the field were possible introgressants. Another plant presented an unequivocal example of backcrossed S. sericea chemistry and RAPD markers. Inter- and intraspecific chloroplast diversity found within the hybrid zone suggests both historic introgression (perhaps in a glacial refugium), and contemporary hybridization. Patterns of inheritance and expression within the hybrid zone suggest that morphological characters are often not expressed in a simple additive fashion, and problems associated with both morphological and molecular data are considered.     

Spatial genetic structure and clonal diversity in an alpine population of Salix herbacea (Salicaceae)

BACKGROUND AND AIMS: Many alpine plant species combine clonal and sexual reproduction to minimize the risks of flowering and seed production in high mountain regions. The spatial genetic structure and diversity of these alpine species is strongly affected by different clonal strategies (phalanx or guerrilla) and the proportion of generative and vegetative reproduction. METHODS: The clonal structure of the alpine plant species Salix herbacea was investigated in a 3 x 3 m plot of an alpine meadow using microsatellite (simple sequence repeat; SSR) analysis. The data obtained were compared with the results of a random amplified polymorphic DNA (RAPD) analysis. KEY RESULTS: SSR analysis, based on three loci and 16 alleles, revealed 24 different genotypes and a proportion of distinguishable genotypes of 0.18. Six SSR clones were found consisting of at least five samples, 17 clones consisting of more than two samples and seven single genotypes. Mean clone size comprising at least five samples was 0.96 m(2), and spatial autocorrelation analysis showed strong similarity of samples up to 130 cm. RAPD analysis revealed a higher level of clonal diversity but a comparable number of larger clones and a similar spatial structure. CONCLUSIONS: The spatial genetic structure as well as the occurrence of single genotypes revealed in this study suggests both clonal and sexual propagation and repeated seedling recruitment in established populations of S. herbacea and is thus suggestive of a relaxed phalanx strategy.     

Floral nectaries and nectar production in brown mustard (Brassica juncea) and white mustard (Sinapis alba) (Brassicaceae)

 The dynamics and abundance of nectar secretion as well as sugar productivity were studied in flowers of brown mustard (Brassica juncea) cv. Małopolska and white mustard (Sinapis alba) cv. Borowska. Moreover, floral nectaries were examined under LM and SEM. In both cultivars lateral and median pairs of nectaries secreted nectar. However, differences were found in morphology and activity of these pairs. The lateral nectaries produced more nectar than the median ones. Nectar secretion started at loose bud and peaked during anther dehiscence. Average amount of nectarsecreted by 100 flowers of cv. Małopolska and cv. Borowska were 119.9 mg and 134 mg. Mean concentration of nectar was 26.7% and 23.4%, respectively. One hundred flowers of cv. Małopolska and cv. Borowska secreted 28.4 mg and 24.9 mg of sugars in nectar. Estimated sugar productivity per 1 ha of crop was 65.5 kg and 71.2 kg, respectively. Received August 28, 2002; accepted November 2, 2002 Published online: June 2, 2003     

Phylogenetic relationships and differentiation among and within populations of Chaenomeles Lindl. (Rosaceae) estimated with RAPDs and isozymes

RAPD and isozyme analyses based on numerous markers have been used for the first time to investigate patterns of phenetic and genetic differentiation among and within nine wild populations of the genus Chaenomeles represented by the species C. japonica, C. speciosa, C. cathayensis and C. thibetica. Highly significant correlations were found between the two different marker systems for both phenetic distances and gene diversity estimates. In agreement with previous studies on cultivated Chaenomeles material, C. japonica was clearly differentiated from C. speciosa and C. cathayensis. The recently recognised species C. thibetica appeared to be rather closely related to C. cathayensis. Populations of C. japonica and C. speciosa were considerably more diverse than populations of C. cathayensis and C. thibetica. Correspondingly, most of the total variability could be attributed to the within-population differentiation in the case of C. japonica and C. speciosa, and to the between-population differentiation in the case of C. cathayensis. Differences in mating systems among the species can be suggested as a possible explanation of the results. A discordant pattern was found between RAPDs and isozymes in the analyses of population structure within C. japonica. This may be explained by a higher proportion of non-neutral markers for isozymes than for RAPDs. This finding also shows the importance of using multiple molecular marker systems in studies of population structure. Received: 23 December 1999 / Accepted: 17 January 2000     

Sex determination and sex ratio in the dioecious shrub Salix viminalis L.

 Various ecological factors (e.g. herbivory, difference between males and females in colonising ability) have been invoked to explain female-biased sex ratios in populations of willow species. It was implicitly assumed that genetic factors would lead to a balanced sex ratio in the absence of ecological disturbances. In an experiment carried out in a homogeneous environment and in the absence of herbivores the progeny sex ratio of 13 crosses of basket willow (Salix viminalis L.) was observed to range from extreme female bias to extreme male bias. The observed sex ratio cannot be explained by the presence of sex chromosomes without assuming that additional loci are also involved in the sex determination. Alternatively, the sex ratios in this study can be explained by a sex determination mechanism governed by multiple independent loci. Received: 1 February 1996 / Accepted: 14 June 1996     

Fungi in ectomycorrhizal associations of silver fir (Abies alba Miller) in Central Italy

 Naturally occurring ectomycorrhizas of silver fir (Abies alba Miller) were studied in two stands, one natural and one artificial, situated in Central Italy. A total of 25 mycorrhizal types was classified, for eight of which the mycobiont was identified at the species level. Analysis of macroscopic and microscopic features and matching of field-collected carpophores with associated mycorrhizas led to the tentative identification of several other types encountered during this study, at least at the genus level. No significant differences were noticed between natural and artificial stands in the relative richness of mycorrhizal types found on A. alba, indicating the maturity of the artificial stand with regard to succession of ectomycorrhizal fungi. Confocal laser scanning microscopy was used for visualization of mycorrhizal structures formed by Lactarius spp., without the need for specific staining with a fluorochrome, thanks to latex autofluorescence. This technique allowed observation of several structures in greater detail than with conventional light microscopy. Accepted: 13 February 1998     

PERMANENT GENETIC RESOURCES: Consensus primers of cyp73 genes discriminate willow species and hybrids (Salix, Salicaceae)

Consensus primers, based on exon sequences of the cyp73 gene family coding for cinnamate 4-hydroxylase (C4H) of the lignin biosynthesis pathway, were designed for the tetraploid willow species Salix alba and Salix fragilis. Diagnostic alleles at species level were observed among introns of three cyp73 genes and allowed unambiguous detection of the first generation and introgressed hybrids in populations. Progeny analysis of a female S. alba with a male introgressed hybrid confirmed the codominant inheritance of each intron. Sequences of the diagnostic alleles of both species were similar to those found in the hybrids.     

Species boundaries and interrelationships of two closely related sympatric diploid wild potato species, Solanum astleyi and S. boliviense, based on RAPDs

 The more than 200 wild and cultivated species relatives of potato (Solanum sect. Petota) present a valuable germplasm base for cultivar improvement. However, species boundaries and interrelationships within sect. Petota are controversial, inhibiting the efficient organization of the many germplasm collections of these species. One controversy involves questions of species boundaries and interrelationships of S. astleyi and S. boliviense. Solanum boliviense is narrowly endemic to two Departments in southern Bolivia, and S. astleyi is known only from one site entirely within the range of this species, where they co-occur. Both species are diploid and morphologically very similar. Artificial hybrids between them are fully fertile, and the species putatively hybridize naturally. These data have been interpreted to designate them as separate species or as S. astleyi an ecotype of S. boliviense. Putative progenitors of S. astleyi are S. boliviense, S. megistacrolobum subsp. megistacrolobum, and S. megistacrolobum subsp. toralapanum. We evaluated interrelationships among these species with random amplified polymorphic DNA’s (RAPDs) generated for 2 accessions of S. astleyi and 14 accessions of S. boliviense. These represent the entire geographic range of the former species and nearly the entire range of the latter. We also analyzed 1 accession each of S. acaule subsp. acaule, S. acaule subsp. aemulans, S. albicans, S. berthaultii, S. megistacrolobum subsp. megistacrolobum, S. megistacrolobum subsp. toralapanum, S. raphanifolium, S. sogarandinum, and S. sparsipilum. Phenetic analyses of the RAPD data show S. astleyi and S. boliviense to form two distinct groups and to be more similar to each other than to any of the other species investigated, suggesting that S. astleyi and S. boliviense are sister taxa. The divergence of S. astleyi and S. boliviense relative to other species examined suggests that they are worthy of taxonomic recognition at the subspecies, rather than species level, and we propose the new combination S. boliviense subsp. astleyi. Received: 21 November 1996 / Accepted: 18 April 1997     

Thlaspi s.str. (Brassicaceae) versus Thlaspi s.l.: morphological and anatomical characters in the light of ITS nrDNA sequence data

 Nuclear ribosomal (ITS) DNA sequence data from representatives of eleven out of 12 genera previously included in Thlaspi sensu lato were analyzed to elucidate relationships within the former genus Thlaspi sensu lato. Sequences from Thlaspi segregates Noccaea, Masmenia, Callothlaspi, Kotschyella, Microthlaspi, and Noccidium were added to the existing data sets, and only some Thlaspi sensu lato segregates formed a monophyletic group. The recently renamed genus Pseudosempervivum, formerly part of the genus Cochlearia, has been shown to be closely related to this group as well. Microthlaspi consists of three independent lineages of which Microthlaspi umbellatum is closely related to Neurotropis. The Thlaspi s.str. taxa including the type species T. arvense, are closely related to Peltaria and Alliaria and they represent a monophyletic group. Kotschyella and Noccidium, which were also integrated into Thlaspi s.l. are not closely related to any Thlaspi lineage. Therefore, we suggest major taxonomical changes which are not in agreement with concepts based on morphological data. Our ITS based phylogeny demonstrates that subtribe Thlaspidinae comprises some additional genera such as Teesdalia, Peltaria, and Alliaria, the latter two even previously classified in other tribes than tribe Lepidieae. Received August 15, 2000 Accepted January 30, 2001     

Cladistic and phenetic analysis of relationships in Vicia subgenus Vicia (Fabaceae) by morphology and isozymes

 Variation of 80 multistate morphological characters and isozymes encoded by 13 loci among 23 vetch species of the type subgenus of the genus Vicia in comparison with V. dumetorum, V. pisiformis and V. sylvatica of the subgenus Cracca is described and analyzed with cladistic parsimony and phenetic neighbour-joining methods by using two different ways of coding. Morphological analyses showed the subgenus Vicia monophyletic and revealed subgroups in a general agreement with traditionally recognized sections, except showing V. faba nested within section Narbonensis and ambiguity in the position of V. lathyroides and V. bithynica. Parsimony analysis of orthozymes as presence/absence characters revealed in the subgenus two basic monophyletic clades: 1) V. faba and three species of the section Peregrinae, V. michauxii, V. aintabensis and V. peregrina, in one subclade linked with species of the Narbonensis and Hyperchusa sections together with V. pisiformis of subgenus Cracca in a second subclade; 2) species belonging to sections Vicia, Sepium, Pseudolathyrus and Lathyroides together with V. sylvatica of the subgenus Cracca. Neighbour-joining analysis of orthozymes revealed the same two basic groups, differing only in the relative position of some species in subclusters. Both isozyme analyses showed paraphyly of the subgenera Vicia and Cracca. Parsimony analysis of orthozymes as character states of isozymes yielded a largely unresolved strict consensus cladogram of 209 most parsimonious trees, and reweighting of characters failed to produce a stable tree. Phylogenetic congruence and discordance among morphological and isozyme analyses, coding ways, homoplasy and weighting of characters are discussed. Received November 20, 2001 Accepted January 31, 2002     

The arctic-alpine polyploids Cerastium alpinum and C. nigrescens (Caryophyllaceae) in a sympatric situation: breakdown of species integrity?

 The high polyploids Cerastium alpinum (8x) and C. nigrescens (12x) were investigated in a mixed population in central Norway to evaluate whether hybridisation has resulted in continuous variation in morphology and genetic markers, a hypothesis previously proposed to account for the extensive taxonomic confusion in this species group. Isozyme, fertility, and morphological (37 characters) variation were examined among 347, 265, and 237 plants, respectively. A PCO analysis based on 23 quantitative morphological characters identified two main groups, corresponding to C. alpinum and C. nigrescens. The groups were also clearly separated in isozyme markers, several qualitative morphological characters, and chromosome numbers. Only 20 plants (8.4%) were more or less intermediate in the PCO analysis. These plants had a parental or hybrid isozyme multilocus phenotype and typically few well-developed anthers, low pollen stainability, and no seed set. Several plants within the C. nigrescens group also had reduced pollen stainability. These results indicate that later-generation hybrids and/or backcrosses towards C. nigrescens are formed. Thus, interspecific, interploidal gene flow probably occurs, but at rates that are insufficient to break down species integrity. Received July 5, 2000 Accepted August 2, 2001