Complete plastid genomes from Ophioglossum californicum, Psilotum nudum, and Equisetum hyemale reveal an ancestral land plant genome structure and resolve the position of Equisetales among monilophytes

Abstract

Background: Plastid genome structure and content is remarkably conserved in land plants. This widespread conservation has facilitated taxon-rich phylogenetic analyses that have resolved organismal relationships among many land plant groups. However, the relationships among major fern lineages, especially the placement of Equisetales, remain enigmatic.

Results: In order to understand the evolution of plastid genomes and to establish phylogenetic relationships among ferns, we sequenced the plastid genomes from three early diverging species: Equisetum hyemale (Equisetales), Ophioglossum californicum (Ophioglossales), and Psilotum nudum (Psilotales). A comparison of fern plastid genomes showed that some lineages have retained inverted repeat (IR) boundaries originating from the common ancestor of land plants, while other lineages have experienced multiple IR changes including expansions and inversions. Genome content has remained stable throughout ferns, except for a few lineage-specific losses of genes and introns. Notably, the losses of the rps16 gene and the rps12i346 intron are shared among Psilotales, Ophioglossales, and Equisetales, while the gain of a mitochondrial atp1 intron is shared between Marattiales and Polypodiopsida. These genomic structural changes support the placement of Equisetales as sister to Ophioglossales + Psilotales and Marattiales as sister to Polypodiopsida. This result is augmented by some molecular phylogenetic analyses that recover the same relationships, whereas others suggest a relationship between Equisetales and Polypodiopsida.

Conclusions: Although molecular analyses were inconsistent with respect to the position of Marattiales and Equisetales, several genomic structural changes have for the first time provided a clear placement of these lineages within the ferns. These results further demonstrate the power of using rare genomic structural changes in cases where molecular data fail to provide strong phylogenetic resolution.

Figure 1

Plastome maps for newly sequenced monilophytes. Boxes on the inside and outside of the outer circle represent genes transcribed clockwise and anti-clockwise, respectively. The inner circle displays the GC content represented by dark gray bars. The location of the IRs are marked on the inner circle and represented by a thicker black line in the outer circle. The large euphyllophyte LSC inversion and the small monilophyte LSC inversion are highlighted on the outer circle by blue and purple bars, respectively.

Figure 2

Comparison of the IR and adjacent sequences from monilophytes. A section of the plastid genome from clpP to trnQ-UUG is presented for selected monilophytes. The section includes the IR, SSC, and parts of the LSC. Genes shown above or below the lines indicate direction of transcription to the right or the left, respectively. The IR is marked by gray boxes, inferred IR extensions are shown by red arrows, and inferred inversions leading to the specific gene arrangement in Polypodiopsida are denoted by black bars. Molecular apomorphies based on gene and intron losses are highlighted by vertical gray lines. Maps are drawn approximately to scale. Color coding of genes corresponds to the legend shown in Figure  1.

Figure 3

Evolution of inverted repeat borders in selected land plants. Species names are abbreviated in circles. Vertical lines depict the borders of the IR relative to the detailed gene map from E. arvense shown at bottom. Thick, solid vertical lines in dark blue mark the putative ancestral IR borders. Thin, dashed vertical lines and circles indicate the IR borders in species that deviate from the ancestral position. Horizontal arrows indicate the extent and direction of IR expansion. Numbers at the arrow tails define the order of successive expansions. All non-seed plant cpDNAs were included, except for Isoetes, Selaginella, and Polypodiopsida because their genomes have gene order rearrangements that make an alignment impossible. Included species: Cycas taitungensis (Cta), Angiopteris evecta (Aev), Psilotum nudum (Pnu), Equisetum arvense (Ear), Equisetum hyemale (Ehy), Ophioglossum californicum (Oca), Huperzia lucidula (Hlu), Anthoceros formosae (Afo), Physcomitrella patens (Ppa), Syntrichia ruralis (Sru), Aneura mirabilis (Ami), Marchantia polymorpha (Mpo), Ptilidium pulcherrimum (Ppu). Higher group names: seed plants (SP), monilophytes (MP), lycophytes (LP), hornworts (HW), mosses (MS), liverworts (LW).

Figure 4

Distribution of intron rps12i346 in monilophytes. All available lycophyte and monilophyte plastid rps12 genes were aligned, and excerpts of the alignment covering the rps12i346 intron sequences and adjacent rps12 exons are shown. Numbers display the total size of the intron if present in the respective taxon.

Figure 5

Phylogenetic analysis of monilophyte plastid genes. The trees shown were generated by maximum likelihood (left) or Bayesian (right) inference of a data set containing 49 plastid protein genes from 32 vascular plants. Thick branches represent clades with 100% bootstrap support or >0.99 posterior probability. Lower support values are indicated near each node. Trees were rooted on lycophytes. Both trees were drawn to the same scale shown at bottom right.

Figure 6

Phylogenetic history of genomic changes during monilophyte evolution. The most parsimonious reconstruction of genomic changes was plotted onto the ML topology from Figure 5. Homoplasious changes are boxed. All genomic changes involve the plastid genome, except for the gain of the mitochondrial atp1i361 intron. Genomic changes listed for Polypodiopsida indicate that they are synapomorphic for the four complete cpDNA sequences (Alsophila spinulosa, Adiantum capillus-veneris, Pteridium aquilinum and Cheilanthes lindheimeri), but many of them will not necessarily be synapomorphic for all Polypodiopsida.