Molecular evidence for the monophyly of the monocots is consistent across several chloroplast and mitochondrial loci. Most of these studies also find the monogeneric Acoraceae J. G. Agardh (Acoranae Reveal) to be the sister lineage of all other extant monocots in what has been called the Acoranan Hypothesis. Phylogenetic studies of nuclear 18S loci, conducted broadly across the angiosperms, find the monocots to be paraphyletic because Acorus calamus is anomalously found in a grade of basal dicots, either at the base of a clade of selected Piperales, or flanked by representative Nymphaeaceae and Annonaceae, depending on the sampling. This nuclear/cytoplasmic incongruence was investigated herein. Further sampling among paleoherbs failed to resolve the monocots as monophyletic in 18S gene trees. The 18S locus was then sequenced from a second species, A. gramineus, in two different laboratories from independent sources of plant material and found to be over 99% identical to the published 18S sequence of A. calamus. 18S loci were then cloned from A. calamus to explore the possibility that an underepresented 18S paralogue existed in the Acoraceae genome that, when found and sequenced, would cluster with monocots. Sequences of 20, 18S clones were compared to published sequences and again found to be nearly identical. 18S sequences of 32 paleoherb species were then extensively reanalyzed using Bayesian Inference. Analyses were performed both including and excluding short ambiguous regions corresponding to helix termini. Several substitution models, rates of among-site variation, and different base frequencies were selected in consecutive analyses. Although Acoraceae varied somewhat in phylogenetic position, it was most frequently found at the base of Chloranthaceae (clade credibility values: 49-57%). Monocots were never recovered as monophyletic. The inability to demonstrate the monophyly of a group with so many synapomorphies utilizing 18S sequences clearly reflects a singular history for this locus among paleoherbs.

Key words: 18S locus, Acoranae, Acoranan Hypothesis, Acorus, Bayesian Inference