Genetic Variability of Thermal <Emphasis Type="Italic">Nymphaea</Emphasis> (Nymphaeaceae) Populations Based on ISSR Markers: Implications on Relationships,Hybridization, and Conservation

The globally widespread genus Nymphaea exhibits a wide range of morphological and taxonomical diversity. The intrusion of a cultivated variety by progressive propagation and its affect on aquatic habitat is demonstrated in this case study. We have studied the genetic diversity, population, and stand structure of the neophyte Nymphaea × ‘Panama Pacific’ as well as other species found in Lake Hévíz and dikes nearby using inter-simple sequence repeat (ISSR) markers. The ISSR assay revealed a low genetic variability for the small populations of Nymphaea caerulea, Nymphaea lotus var. thermalis, and a medium level for Nymphaea alba, Nymphaea rubra var. longiflora, and Nymphaea × ‘Panama Pacific’. The evolutionary genetic status of individuals found in the overlapping cultivation area of Nymphaea × ‘Panama Pacific’ and N. caerulea was affirmed to be of hybrid origin by reticulate network analysis and with morphological parameters. The Bayesian analysis of hybrid classes and the segregation of the ISSR markers also confirmed the hybrid origin of the individuals in question and revealed that they are falling into F2 or latter genotype frequency classes, indicating the viability and fertility of the hybrids. The set of analyzed species by phylogenetic network analysis of ISSR data has been divided into three major groups according to their evolutionary patterns (subg. Barachyceras, Lotos, and Nymphaea). Our results are in accordance with these three major subgenera within Nymphaea.     

Do they really hybridize? A field study in artificially established mixed populations of Euphrasia minima and E. salisburgensis (Orobanchaceae) in the Swiss Alps

Hybridization and introgression in the European species of Euphrasia depend on the relationships between the species, on flower size and habitat. Hybridization between Euphrasia minima and Euphrasia salisburgensis was investigated in their natural habitat using artificial sympatric populations of both species in the Swiss Alps. The insect behavior in the populations suggests, that cross-pollination is likely to occur. A number of putative hybrids were detected by morphological characteristics, and their hybrid origin was verified using RAPD analysis. The predominance of RAPD bands in one of the species and the occurrence of these bands in some plants of the second species point to earlier introgression events. The number of hybrids found in the artificial populations together with results of earlier studies indicate that insect visits and cross-pollination in small-flowered Euphrasia species in lower alpine regions may be more common than has been suggested in the past.     

Hybridization between insular endemic and widespread species of Viola in non-disturbed environments assessed by nuclear ribosomal and cpDNA sequences

Viola jaubertiana Marès & Vigin. is a narrow endemic violet of the Balearic Islands, restricted to small, fragmented, and scattered populations living in inaccessible rocky places and calcareous overhangs. V. jaubertiana is entirely glabrous and morphologically very uniform. However, several authors have reported hairy individuals collected at the type locality, suggesting that these rupicolous, pubescent plants are putative hybrids with V. alba Bess. subsp. dehnhardtii (Ten.) W. Becker, a woodland violet growing in the area. Ribosomal ITS sequences of the putative hybrids analysed showed additive species-specific sites of V. alba subsp. dehnhardtii and V. jaubertiana, strongly supporting its hybrid origin from these progenitors. CpDNA sequences of all putative hybrids were uniform, and identical to those present in V. jaubertiana accessions. This suggests that the gene flow between V. alba subsp. dehnhardtii and V. jaubertiana is unidirectional and identifies the endemic V. jaubertiana as the unique ovule donor. The additivity of the ITS sequences, together with the pollen and ovule sterility, suggests that the sampled individuals are primary F₁ hybrids, whereas no trace of introgressive hybridization or hybrid zone has been evidenced by the nuclear and plastid markers used. Judging from herbarium sheets, hybridization between V. alba subsp. dehnhardtii and V. jaubertiana is recurrent and dates back from the XIXth century. Hybrids between these species are not linked to disturbed environments. In fact, they have been always reported in rupicolous habitats, where the maternal species is restricted.     

Molecular confirmation of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea

The hypothesis of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea, two mangrove species distributed in the Indo-West Pacific region, was proposed in 1970s based on morphological traits; however, no molecular evidence has been reported to support it. In this study, we sequenced two low-copy nuclear genes and one chloroplast intergenic spacer (trnS-trnG) in the two Lumnitzera species and their putative hybrid to test this hypothesis. Our results revealed that there were 9 and 27 nucleotide substitutions at the two nuclear loci, respectively, between one haplotype of L. racemosa and L. littorea, and that the putative hybrid showed additivity in chromatograms at these sites. Sequencing the chloroplast intergenic region trnS-trnG showed that the two Lumnitzera species differed by seven fixed nucleotide substitutions and four fixed insertions/deletions in this region, while the putative hybrid had identical sequences to L. racemosa. Molecular data clearly demonstrated that there indeed existed natural hybridization between L. racemosa and L. littorea and that L. racemosa was the maternal parent in this hybridization event. The uncommon direction of hybridization and F₁ nature of hybrids in this case, and in mangroves in general, is discussed.     

Molecular and cytological evidences for the natural wheatgrass hybrids occurrence and origin in west China

Interspecies hybridization has been frequently observed in the tribe Triticeae. Natural hybridization between Kengyilia and Roegneria or Elymus species has not been reported as yet. Several sterile wheatgrass individuals exhibiting intermediately morphological traits between Kengyilia and Roegneria or Elymus species were identified in the meadow of Sichuan and Gansu provinces in China, suggesting their natural hybrid origin. The putative hybrids were analyzed by using the sequences of ITS and trnH-psbA together with cytological observation in order to assess the origin of hybrids. Both ITS and cytological data revealed the evidence of allopolyploid origin and confirmed the presence of StStYYP and StStYYHP genomes in the putative natural hybrids. The data suggest that the StStYYP hybrid originated from hybridization between Kengyilia and Roegneria and the hybrid with StStYYHP originated from hybridization between Kengyilia and Elymus. Chloroplast sequence data demonstrated that K. rigidula and K. melanthera were the likely maternal donors in the hybridization events.     

Multilocus sequence and microsatellite identification of intra-specific hybrids and ancestor-like donors among natural Ethiopian isolates of Leishmania donovani

Protozoan parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis is endemic in Ethiopia where it has also been responsible for fatal epidemics. It is postulated that genetic exchange in Leishmania has implications for heterosis (hybrid vigour), spread of virulent strains, resistance to chemotherapeutics, and exploitation of different hosts and vectors. Here we analyse 11 natural Ethiopian Leishmania donovani isolates consisting of four putative hybrids, seven parent-like isolates and over 90 derived biological clones. We apply a novel combination of high resolution multilocus microsatellite typing (five loci) and multilocus sequence typing (four loci) that together distinguish parent-like and hybrid L. donovani strains. Results indicate that the four isolates (and their associated biological clones) are genetic hybrids, not the results of mixed infections, each possessing heterozygous markers consistent with inheritance of divergent alleles from genetically distinct Ethiopian L. donovani lineages. The allelic profiles of the putative hybrids may have arisen from a single hybridisation event followed by inbreeding or gene conversion, or alternatively from two or more hybridisation events. Mitochondrial sequencing showed uniparental maxicircle inheritance for all of the hybrids, each possessing a single mitochondrial genotype. Fluorescence activated cell sorting analysis of DNA content demonstrated that all hybrids and their associated clones were diploid. Together the data imply that intra-specific genetic exchange is a recurrent feature of natural L. donovani populations, with substantial implications for the phyloepidemiology of Leishmania.     

Nuclear and chloroplast DNA sequences data support the origin of Potamogeton intortusifolius J.B. He in China as a hybrid between P. perfoliatus Linn. and P. wrightii Morong

Internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequence data were used to test the hypothesis that natural populations of Potamogeton intortusifolius J.B. He in China originated from hybridization between P. perfoliatus Linn. and P. wrightii Morong. Based on ITS sequences data, P. intortusifolius possessed heterozygous rDNA genotypes which confirmed the hybrid origin of P. intortusifolius. Chloroplast rbcL gene sequences of P. intortusifolius from Yichang population revealed the same chloroplast haplotype as P. perfoliatus and the samples of P. intortusifolius from Weinan population had the same chloroplast haplotype as P. wrightii, which indicated that both putative parental species had been the maternal parent and that the two populations of P. intortusifolius had independent origins. This study confirms P. intortusifolius as a reciprocal hybrid. Because P. × intortusifolius in China has the same hybrid origin as P. × anguillanus Koidz. in Japan, it is suggested that P. × intortusifolius should be a synonym of P. × anguillanus.     

Anthocyanins in Salix species: A new anthocyanin in salix purpurea bark

Cyanidin 3-glucoside and delphinidin 3-glucoside (minor component) occurred in the bark of Salix purpurea (29 cultivars examined), S. fragilis (3 cvs), S. americana (4 cvs), S. rubra (1 cv.), S. incana (1 cv.), S. aegyptica (1 cv.) and S. alba (3 cvs) and several hybrids. A previously unrecorded anthocyanin was also present in appreciable amounts in the bark of eleven S. purpurea cultivars, notably those with small leaves. In other large-leaved cultivars of S. purpurea (18) it occurred only in traces or was not detected with certainty. A trace was found in one purpurea hybrid, but it was not detected in four others. Small amounts of the new anthocyanin were also found in S. incana (1 cv.), S. fragilis cv. Basfordiana, S. americana cv. Cordata and one hybrid, but were absent from other cultivars of S. fragilis (2) and S. americana (2), and also from S. alba (3), S. aegyptica (1), S. daphnoides (1), S. rubra (1), S. triandra (1), S. viminalis (1) and three hybrids. The new anthocyanin is unique in containing fructose as well as glucose and is based upon a previously undescribed anthocyanidin, possibly dimeric in nature, which is provisionally named purpurinidin.     

Geographic distribution and genotypic composition of invasive hybrid watermilfoil (<Emphasis Type="Italic">Myriophyllum spicatum</Emphasis> × <Emphasis Type="Italic">M. sibiricum</Emphasis>) populations in North America

The recent recognition of invasive hybrid watermilfoil (Myriophyllum spicatum × M. sibiricum) in North America has necessitated a more thorough evaluation of its overall distribution and occurrence in natural populations. A comprehensive survey of watermilfoil populations was conducted in five Minnesota lakes, three of which were suspected a priori to contain hybrid watermilfoil. DNA sequence data verified that hybrid plants between the nonindigenous M. spicatum L. and indigenous M. sibiricum Kom. occurred in three of the five lakes sampled. Myriophyllum spicatum was not detected in lakes where hybrids were prevalent. Further sampling of lakes in Idaho, Michigan, Minnesota, Wisconsin and Washington identified 30 additional hybrid watermilfoil populations. In only three of these populations the hybrid watermilfoil was found to co-occur with M. spicatum. To facilitate the field identification of the two parental species and their hybrid, morphological data from watermilfoil specimens collected across the United States were evaluated. We determined that leaf segment/leaf length measurements can effectively distinguish M. spicatum and M. sibiricum; however, hybrids are intermediate for these characters and such measurements frequently overlap with respect to their parental taxa. By incorporating a combined molecular and morphological approach to identifying watermilfoils, the hybrids can be identified readily and their distributions elucidated both within and between lakes. Because hybrids may respond differently to local ecological conditions than their parents, information on their presence and distribution should be of particular importance to management and conservation programs.     

Chemical characterisation of wild populations of Thymus from different climatic regions in southeast Spain

Thymus is taxonomically a very complex genus with a high frequency of hybridisation and introgression among sympatric species. The variation in accumulation of leaf-surface flavonoids was investigated in 71 wild populations of Thymus from different putative hybrid swarm areas in Andalucia, Spain. Twenty-two flavones, five flavanones, two dihydroflavonols, a flavonol and two unknowns were detected by HPLC–DAD combined with LC–APCI-MS analysis. The majority of compounds were flavones with a luteolin-type substitution of the B-ring, in contrast to previous reports on Macedonian taxa, which predominantly accumulate flavones with apigenin-type substitution of the B-ring. Anatomical and morphometric studies, supported by cluster analysis, identified pure Thymus hyemalis and Thymus baeticus populations, and a large number of putative hybrids. Flavonoid variation was closely related to morphological variation in all populations and is suspected to be a result of genetic polymorphism. Principal component analysis identified the presence of species-specific and geographically linked chemotypes and putative hybrids with mixed morphological and chemical characteristics. Qualitative and quantitative flavonoid accumulation appears to be genetically regulated, while external factors play a secondary role. Flavonoid profiles can thus provide diagnostic markers for the taxonomy of Thymus and are also useful in detecting hybridising taxa.     

Extensive Clonal Assemblies in Populus alba and Populus x canescens from the Iberian Peninsula

Riparian vegetation in temperate zones holds great environmental importance and thus its conservation should be a priority. Among riverine tree species, genus Populus stands out, being also a model organism in research. In this work, we present a genetic analysis of the hybrid Populus x canescens and its parent species Populus alba and Populus tremula, with special emphasis in P. alba with which backcrosses frequently occur. This study focuses on the river Douro basin (Spain) where the presence of hybrids has been previously reported. Nuclear microsatellite markers and Bayesian statistical analysis have been used for the detection of hybrids and purebred stands. This methodology has also made possible the study of clonality in the taxonomic continuum P. alba–P. x canescens. Our results highlight the existence of a small number of genotypes accounting for most of the individual trees and stands. Possible causes leading to this situation are discussed. Also, the presence of individuals genetically close to ornamental cultivars is reported for the first time. Finally, concern about the present situation of P. alba genetic resources is brought up.     

Nuclear cyp73 intron fragment length polymorphism supports morphological analysis of Salix species and hybrids

Abstract

The polyploid Salix alba L.–Salix fragilis L. hybrid complex still presents major difficulties in morphological identification. Most of the measured characters show a low diagnostic value for unambiguously identifying the parental species and their hybrid Salix × rubens Schrank due to continuous variation creating a large overlap in leaf and catkin morphology. Fragment length polymorphism of nuclear cyp73 intron markers was used to identify species and hybrids. This multilocus genotyping could be applied in a morphological analysis of trees from hybrid zones and allowed to demonstrate that morphological features of leaves and catkins clearly separated S. alba from S. fragilis. The hybrid individuals largely overlapped with both parental species but appeared to be morphologically more similar to S. fragilis than to S. alba. Cyp73 analysis of 11 Salix taxa revealed intermediate positions of two hybrid taxa with S. alba, namely S. × rubens and S. × sepulcralis Simonkai with their respective parental species S. fragilis and S. babylonica L. Additionally, the cyp73 intron multilocus genotypes clustered tetraploid taxa separately from diploid willows. Cyp73 introns are valuable markers for fast, reliable and straightforward genotyping in willow species and hybrids.     

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus ?×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.     

Analysis of a hybrid swarm involving Eucalyptus crenulata and E. ovata using leaf oils and morphology

The morphology and leaf oils of naturally occurring Eucalyptus crenulata, E. ovata and presumed hybrids between them were analysed. The nine hybrid trees found all showed intermediacy between the parents in either morphology or oils or both. Morphological and oils characters were not always strongly linked. Field distribution data also suggest hybrid origin. It is suggested that leaf oils may be much more valuable as characters in Eucalyptus taxonomy than has previously been thought.     

Morphological Characters Useful for the Delimitation of Taxa Within Viola Subsect. Viola (Violaceae): A Morphometric Study from the West Carpathians

Forty-nine morphological characters were scored or measured on 44 populations (376 individuals) of Viola subsect. Viola from the West Carpathians and adjacent areas (Slovakia, Czech Republic, Austria and Hungary). The presence of six species, namely V. alba (represented by subsp. alba), V. ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. was revealed based on pollen fertility, cytological and morphometric analyses. The morphological characters traditionally used to delimit taxa within the subsection and those revealed by our study as most reliable are widely discussed. A key for identifying the taxa and most common hybrids of subsection Viola occurring in the West Carpathians is presented. Chromosome counting and flow cytometry were used to determine the ploidy levels of the populations studied. All individuals of V. alba subsp. alba, V. collina, V. hirta and V. odorata were tetraploid, while those of V. ambigua and V. suavis s.l. were octoploid.     

Characterisation and inheritance of nuclear microsatellite loci for use in population studies of the allotetraploid <Emphasis Type="Italic">Salix alba</Emphasis>–<Emphasis Type="Italic">Salix fragilis</Emphasis> complex

We present nine polymorphic di- and tri-nucleotide repeat nuclear microsatellite markers selected specifically for their use in high throughput studies concerning the dioecious allotetraploid Salix alba–Salix fragilis willow complex. These taxa and their hybrids are difficult to discriminate using morphological characters. Thus, multiplex reactions were developed for these microsatellite loci and their effectiveness to distinguish individuals, especially hybrids, and their inheritance patterns in controlled crosses were determined. All loci displayed disomic–monogenic inheritance which allowed for the genotype data to be analysed as for a diploid organism. The nine loci produced a total of 67 alleles (mean, 7.4 alleles per locus; range, 3–11 alleles) in a reference panel of 57 individuals from two germplasm collections and natural populations. Gene diversity values (as measured by the expected heterozygosity) ranged from 0.000–0.820. A total of 53 distinct multilocus genotypes were observed, and ordination analysis revealed three separate clusters corresponding to S. alba, S. fragilis and hybrids. The microsatellite loci described here will be used in population genetic studies to investigate genetic variation, gene flow, levels of hybridisation and the extent of introgression in natural populations of the S. alba–S. fragilis complex. They are also useful for clonal identification, conservation and sustainable management of germplasm collections, genetic mapping and the selection of individuals and/or certification of controlled crosses for breeding programmes.     

New evidence for a postglacial homoploid hybrid origin of the widespread Central European Scabiosa columbaria L. s. str. (Dipsacaceae)

We have tested whether Scabiosa columbaria s. str. could have originated by recent homoploid hybrid speciation because it is of intermediate morphology and is distributed in areas under glacial influence. Existing morphological and new RAPD data for 11 ingroup populations were analysed using multivariate statistics. Most methods indicated that S. columbaria s. str. could be the first example for a homoploid hybrid origin from Central Europe although a rigorous proof was missing partly because of ongoing introgression. Sequence variation was absent for 5,000 bp from 11 regions supporting a postglacial origin of S. columbaria s. str. An analysis of realized macroclimatic niches using GIS data showed that the niches of S. columbaria s. str. were partly intermediate between its putative parental species rather than extreme as often found in other homoploid hybrids. This could be due to geographical preconditions: large new habitats were available in postglacial Europe while in the case of most other homoploid hybrids only ecologically challenging habitats were not occupied by parental taxa.     

Hybridisations between Mytilus edulis and Mytilus galloprovincialis and performance of pure species and hybrid veliger larvae at different temperatures

The mussels Mytilus edulis L. and Mytilus galloprovincialis Lamark hybridise naturally in the wild along the Atlantic coast of Europe producing a patchwork of mixed pure species and hybrid populations. Individuals of both species were spawned in the laboratory and were hybridised in a series of reciprocal crosses. After 72 h, the proportion of eggs which developed into larvae (%yield) and the proportion of those larvae which had a normal veliger morphology (%normality) were estimated and compared between pure species and hybrid families. There were no significant differences in %yield or %normality between pure species and hybrids, but significant differences were evident between the offspring from different parents irrespective of whether they were hybrids or pure species. Therefore confirmation of hybrid heterosis in laboratory studies should not be based on a single, or a few reciprocal crosses. Hybrid and pure species veliger larvae were grown for approximately 4 weeks at 10, 14 or 20 °C. In all trials, pure M. galloprovincialis larvae grew significantly faster at 20 °C than either reciprocal hybrid or pure M. edulis larvae. Irrespective of temperature, in general, hybrid larvae grew slower than larvae of either pure species. Increased exposure to planktonic predation due to slow growth can be interpreted as selection against hybrids and this may play a role in the structure and distribution of mixed pure species and hybrid populations.     

Genome size and base composition variation in natural and experimental Narcissus (Amaryllidaceae) hybrids

Background and Aims

Although there is evidence that both allopolyploid and homoploid hybridization lead to rapid genomic changes, much less is known about hybrids from parents with different basic numbers without further chromosome doubling. Two natural hybrids, Narcissus × alentejanus (2n = 19) and N. × perezlarae (2n = 29), originated by one progenitor (N. cavanillesii, 2n = 28) and two others (N. serotinus, 2n = 10 and N. miniatus, 2n = 30, respectively) allow us to study how DNA content and composition varies in such hybrids.

Methods

Flow cytometry measurements with two staining techniques, PI and DAPI, were used to estimate 2C values and base composition (AT/GC ratio) in 390 samples from 54 wild populations of the two natural hybrids and their parental species. In addition, 20 synthetic F₁ hybrid individuals were also studied for comparison.

Key Results

Natural hybrids presented 2C values intermediate between those found in their parental species, although intra-population variance was very high in both hybrids, particularly for PI. Genome size estimated from DAPI was higher in synthetic hybrids than in hybrids from natural populations. In addition, differences for PI 2C values were detected between synthetic reciprocal crosses, attributable to maternal effects, as well as between natural hybrids and those synthetic F₁ hybrids in which N. cavanillesii acted as a mother.

Conclusions

Our results suggest that natural hybrid populations are composed of a mixture of markedly different hybrid genotypes produced either by structural chromosome changes, consistent with classic cytogenetic studies in Narcissus, or by transposon-mediated events.