Abstract: Three nuclear regions nuclear ribosomal internal transcribed spacer (nrITS) and intron regions of two nuclear low-copy genes—gapCp, and pgiC, in combination with one chloroplast genome region were employed to explore patterns of reticulate evolution in the fern genus Asplenium. This is the one of the first studies using DNA sequences of multiple nuclear markers in ferns. All three nuclear markers amplified well with PCR and several copies were recovered by cloning PCR products. All three nuclear regions showed congruent results by recovering the neo-allotetraploid Asplenium adulterinum as the hybrid of diploid A. trichomanes and diploid A. viride. Conflicting results were obtained for several nodes. First, gapCp did not discriminate between A. aethiopicum and A. praegracile whereas the other markers recovered these two taxa as distinct. Conflicts among gene-trees were found in respect to A. monanthes, chloroplast and pgiC suggested a sister relationship of A. monanthes and A. trichomanes but gapCp and nrITS nested A. monanthes within A. normale. Our results confirm: (i) the usefulness of several nuclear regions, in particular gapCp and pgiC, to unravel reticulate evolution in ferns and species differentiation and (ii) highlights the need to employ more than one nuclear region to obtain reliable hypotheses on reticulate events versus incomplete lineage sorting. Especially, if one assumes that the reticulation event might have occurred in the more distant past. Considering the expected high frequency of reticulate evolution in ferns, the establishment of robust and informative nuclear genomic markers is critical to achieve further progress in our efforts to elucidate fern evolution.

Key words: ferns, gapCp, GAPDH, hybridisation, introgression, nrITS, nuclear genes, pgiC, reticulate evolution.