The plastid genome has remained remarkably conserved throughout the evolution of land plants (reviewed in
[1–3]). Genomes from diverse land plant lineages—including seed plants, ferns, lycophytes, hornworts, mosses, and liverworts—have a similar repertoire of genes that generally encode for proteins involved in photosynthesis or gene expression. The order of these plastid genes has remained consistent for most species, such that large syntenic tracks can be easily identified between genomes. Furthermore, most plastid genomes have a quadripartite structure involving a large single-copy (LSC) and a small single-copy (SSC) region separated by two copies of an inverted repeat (IR). Although these generalities apply to most land plants, exceptions certainly exist, such as the convergent loss of photosynthetic genes from parasitic plants
[4–6] or ndh genes from several lineages
[7, 8], the highly rearranged genomes of some species
[9–11], and the independent loss of one copy of the IR in several groups
Because of the conserved structure and content of plastid genomes, its sequences have been favored targets for many plant phylogenetic analyses (e.g.,
[14, 15]). Through extensive sequencing from phylogenetically diverse species, our understanding of the relationships between the major groups of land plants has greatly improved in recent years
[15–19]. However, there are a few nodes whose position remains elusive, most notably that of the Gnetales
[7, 20] and the horsetails
[16, 18, 21]. Horsetails (Equisetopsida) are particularly enigmatic because until recently
 their morphology had been considered to be ‘primitive’ among vascular plants, and consequently they were grouped with the “fern allies” rather than with the “true” ferns. Recent molecular and morphological evidence now unequivocally support the inclusion of horsetails in ferns sensu lato (Monilophyta or Moniliformopses), which also encompasses whisk ferns and ophioglossoid ferns (Psilotopsida), marattioid ferns (Marattiopsida), and leptosporangiate ferns (Polypodiopsida)
[16, 18, 21].
Despite this progress, the relationships among fern groups, especially horsetails, have been difficult to resolve with confidence. Many molecular phylogenetic analyses have suggested that horsetails are sister to marattioid ferns
[16, 21–23], while other analyses using different data sets and/or optimality criteria have suggested a position either with leptosporangiate ferns, with Psilotum, or as the sister group to all living monilophytes
[3, 18, 21, 24, 25]. However, these various analyses rarely place Equisetum with strong statistical support. This phylogenetic uncertainty stems from at least two main issues. First, Equisetopsida is an ancient lineage dating back more than 300 million years, but extant (crown group) members are limited to Equisetum, which diversified only within the last 60 million years
. Second, substitution rates in the plastid (and mitochondrial) genome appear to be elevated in horsetails compared with other early diverging ferns (note the long branches in
[21, 22, 25, 27]). Consequently, molecular phylogenetic analyses produce a long evolutionary branch leading to Equisetum, a problem that can lead to long-branch attraction artifacts (reviewed in
In cases where molecular phylogenetic results are inconsistent, the use of rare genomic structural changes, such as large-scale inversions and the presence or absence of genes and introns, can provide independent indications of organismal relationships
. One notable example used the differential distribution of three mitochondrial introns to infer that liverworts were the earliest diverging land plant lineage
. Other studies have identified diagnostic inversions in the plastid genomes of euphyllophytes
 and monilophytes
. Unfortunately, complete plastid genomes are currently lacking from several important fern clades, preventing a comprehensive study of the utility of plastid structural changes in resolving fern relationships.
In this study, we sequenced three additional fern plastid genomes: the ophioglossoid fern Ophioglossum californicum, the horsetail Equisetum hyemale, and the whisk fern Psilotum nudum. By sequencing the first ophioglossoid fern and a second horsetail (E. hyemale belongs to a different subgenus than the previously sequenced E. arvense[26, 32]), we expected that this increased sampling would allow us to evaluate diversity in plastid genome structure and content and to resolve fern relationships using sequence and structural characters.