Horsetails are the sister group to all other monilophytes and Marattiales are sister to leptosporangiate ferns

• Introns in two mitochondrial loci, nad2 and rpl2, are highly conserved among monilophytes.
• A nine-gene data set and an indel in rpl2 place horsetails (Equisetales) are sister to all other monilophytes (ferns).
• Marattiales are the sister order to the large clade of leptosporangiate ferns.

The “Monilophyte” clade comprising ferns, horsetails and whisk ferns receives unequivocal support from molecular data as the sister clade to seed plants. However, the branching order of its earliest emerging lineages, the Equisetales (horsetails), the Marattiales, the Ophioglossales/Psilotales and the large group of leptosporangiate ferns has remained dubious. We investigated the mitochondrial nad2 and rpl2 genes as two new, intron-containing loci for a wide sampling of taxa. We found that both group II introns – nad2i542g2 and rpl2i846g2 – are universally present among monilophytes. Both introns have orthologues in seed plants where nad2i542g2 has evolved into a trans-arrangement. In contrast and despite substantial size extensions to more than 5 kb in Psilotum, nad2i542g2 remains cis-arranged in the monilophytes. For phylogenetic analyses, we filled taxonomic gaps in previously investigated mitochondrial (atp1, nad5) and chloroplast (atpA, atpB, matK, rbcL, rps4) loci and created a 9-gene matrix that also included the new mitochondrial nad2 and rpl2 loci. We extended the taxon sampling with two taxa each for all land plant outgroups (liverworts, mosses, hornworts, lycophytes and seed plants) to minimize the risk of phylogenetic artefacts. We ultimately obtained a well-supported molecular phylogeny placing Marattiales as sister to leptosporangiate ferns and horsetails as sister to all remaining monilophytes. In addition, an indel in an exon of the here introduced rpl2 locus independently supports the placement of horsetails. We conclude that under dense taxon sampling, phylogenetic information from a prudent choice of loci is currently superior to character-rich phylogenomic approaches at low taxon sampling. As here shown the selective choice of loci and taxa enabled us to resolve the long-enigmatic diversifications of the earliest monilophyte lineages.

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