Next generation DNA sequencing reveals allopolyploid origin of decaploid Isoëtes lacustris (Isoëtaceae)

Isoetes L. is a cosmopolitan genus comprising about 200 species, almost half of them being allo- or autopolyploids. We investigated the origin of decaploid I. lacustris using next generation sequencing (NGS). We sequenced the second intron of LEAFY (LFYint2) and analysed the morphology of seven samples of I. lacustris from five localities from East Europe and West Siberia. We have shown that I. lacustris is an allopolyploid having in its genome the subgenomes of the North American diploid I. prototypus, tetraploid I. tuckermanii (which contains subgenomes of diploid I. viridimontana and unknown species), and one more unknown diploid progenitor. NGS showed high reproducibility, demonstrating the same sets of LFYint2 copies in all the samples. The potential fifth subgenome has not been found, most likely due to a loss of the LEAFY gene as a result of subgenome reduction, as is characteristic of polyploids. These data imply that the ancestor of I. lacustris most likely originated in the north-east of North America (New England and Acadian region) where I. prototypus, I. tuckermanii and I. viridimontana co-occur, and that I. lacustris then spread to Eurasia via the North Atlantic Land Bridge. NGS-based amplicon sequencing has the potential to evaluate the ratio of frequency of different copies and to distinguish allopolyploidy and autopolyploidy (if the PCR efficiency of each of the copies is taken into account). This is especially useful, given that both play a prominent role in Isoetes evolution. We also demonstrate that widely adopted cloning of LFYint2 in Isoetes leads to biased results due to the amplification errors.