Abstract
The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the Ranunculus auricomus complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict R. variabilis is an ancient hybrid of the diploid sexual species R. notabilis and the sexual species R. cassubicifolius. The additivity pattern shared by R. variabilis and the synthetic hybrids supports an evolutionary and biogeographical scenario that R. variabilis originated from ancient hybridization. Concerted evolution of ITS copies in R. variabilis is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.
Highlights
Hybridization and polyploidy are creative evolutionary forces in plant radiations, and most or perhaps all angiosperms are either polyploid or of ancient polyploid origin [1]
All 14 polymorphic sites detected in the ITS1-ITS2 of experimental hybrids were additive, either presenting a combination of maternal (R. carpaticola or R. cassubicifolius) and paternal (R. notabilis) contributions, or identical to polymorphisms already existing in parental genomes
The three sexual species exhibited intraspecific polymorphisms, especially R. notabilis, which showed nine polymorphisms shared with R. variabilis
Summary
Hybridization and polyploidy are creative evolutionary forces in plant radiations, and most or perhaps all angiosperms are either polyploid or of ancient polyploid origin [1]. The reconstruction of hybrid relationships and parentage in natural systems is still a methodological challenge. The internal transcribed spacer (ITS) of the nrDNA has been established as a standard molecular marker to infer generic and interspecific relationships in flowering plants [6,7,8] and to infer hybridization events in other eukaryotes including algae, heterotrophic protists, invertebrates and vertebrates [9,10,11,12,13,14,15]. ITS nrDNA has proved to be a helpful non-coding marker to infer hybridization events because of its biparental inheritance and its occurrence in hundreds to thousands of copies within a single genome [8,16,17]. The majority of systematics studies aim at reconstruction of phylogenetic relationships using ITS in combination with maternally inherited plastid marker (e.g., [18,19,20,21,22,23])
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