Leen Leus

Host adaptation and speciation through hybridization and polyploidy in Phytophthora

It is becoming increasingly evident that interspecific hybridization is a common event in phytophthora evolution. Yet, the fundamental processes underlying interspecific hybridization and the consequences for its ecological fitness and distribution are not well understood. We studied hybridization events in phytophthora clade 8b. This is a cold-tolerant group of plant pathogenic oomycetes in which six host-specific species have been described that mostly attack winter-grown vegetables. Hybrid characterization was done by sequencing and cloning of two nuclear (ITS and Ypt1) and two mitochondrial loci (Cox1 and Nadh1) combined with DNA content estimation using flow cytometry. Three different mtDNA haplotypes were recovered among the presumed hybrid isolates, dividing the hybrids into three types, with different parental species involved. In the nuclear genes, additivity, i.e. the presence of two alleles coming from different parents, was detected. Hybrid isolates showed large variations in DNA content, which was positively correlated with the additivity in nuclear loci, indicating allopolyploid hybridization followed by a process of diploidization. Moreover, indications of homeologous recombination were found in the hybrids by cloning ITS products. The hybrid isolates have been isolated from a range of hosts that have not been reported previously for clade 8b species, indicating that they have novel pathogenic potential. Next to this, DNA content measurements of the non-hybrid clade 8b species suggest that polyploidy is a common feature of this clade. We hypothesize that interspecific hybridization and polyploidy are two linked phenomena in phytophthora, and that these processes might play an important and ongoing role in the evolution of this genus.

Reproductive isolation and hybridization in sympatric populations of three Dactylorhiza species (Orchidaceae) with different ploidy levels

BACKGROUND AND AIMS The potential for gene exchange between species with different ploidy levels has long been recognized, but only a few studies have tested this hypothesis in situ and most of them focused on not more than two co-occurring species. In this study, we examined hybridization patterns in two sites containing three species of the genus Dactylorhiza (diploid D. incarnata and D. fuchsii and their allotetraploid derivative D. praetermissa). METHODS To compare the strength of reproductive barriers between diploid species, and between diploid and tetraploid species, crossing experiments were combined with morphometric and molecular analyses using amplified fragment length polymorphism markers, whereas flow cytometric analyses were used to verify the hybrid origin of putative hybrids. KEY RESULTS In both sites, extensive hybridization was observed, indicating that gene flow between species is possible within the investigated populations. Bayesian assignment analyses indicated that the majority of hybrids were F(1) hybrids, but in some cases triple hybrids (hybrids with three species as parents) were observed, suggesting secondary gene flow. Crossing experiments showed that only crosses between pure species yielded a high percentage of viable seeds. When hybrids were involved as either pollen-receptor or pollen-donor, almost no viable seeds were formed, indicating strong post-zygotic reproductive isolation and high sterility. CONCLUSIONS Strong post-mating reproductive barriers prevent local breakdown of species boundaries in Dactylorhiza despite frequent hybridization between parental species. However, the presence of triple hybrids indicates that in some cases hybridization may extend the F(1) generation.

Patterns of hybridization between diploid and derived allotetraploid species of Dactylorhiza (Orchidaceae) co-occurring in Belgium

PREMISE OF THE STUDY Although the potential for gene flow between species with large differences in chromosome numbers has long been recognized, only few studies have thoroughly investigated in situ hybridization across taxa with different ploidy levels. We combined morphological, cytological, and genetic marker data with pollination experiments to investigate the degree, direction, and spatial pattern of hybridization between the diploid Dactylorhiza incarnata and its tetraploid derivative, D. praetermissa. METHODS To identify hybrids, 169 individuals were genotyped using AFLPs and morphologically characterized. Individuals were clustered on the basis of their AFLP profile using the program Structure. To reduce the dimensionality of the plant-trait matrix, PCA was applied. The origin of suspected hybrid individuals was verified using flow cytometry. An AMOVA and spatial autocorrelation analysis were used to indirectly infer the extent of gene flow. KEY RESULTS Only five individuals were regarded as putative hybrids on the basis of the AFLP data; all had been assigned to the D. praetermissa morphotype. Only one had deviating DNA content and was presumably a triploid. High Φ(ST) values between different subpopulations and significant spatial genetic structure were observed, suggesting localized gene flow. CONCLUSIONS Using combined data to study hybridization between D. incarnata and D. praetermissa, very few unequivocal hybrids were observed. We propose several non-mutually exclusive explanations. Localized pollen flow, in combination with different microhabitat preferences, is probably one of the reasons for the low frequency of hybrids. Also, the triploid first-generation hybrids may experience difficulties in successful establishment, as a result of genic incompatibilities.

Development of an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the simultaneous determination of salicylic acid, jasmonic acid, and abscisic acid in rose leaves.

This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in hybrid rose leaf matrices. Deuterium-labeled [(2)H6] (+)-2-cis-4-trans-abscisic acid, [(2)H6] (±)-jasmonic acid, and [(2)H4]-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm(3) Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (-)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 μg/g, 0.00015 and 0.00030 μg/g, and 0.0089 and 0.018 μg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.