The Increase of Simple Sequence Repeats during Diversification of Marchantiidae, An Early Land Plant Lineage, Leads to the First Known Expansion of Inverted Repeats in the Evolutionarily-Stable Structure of Liverwort Plastomes

Abstract

The chloroplast genomes of liverworts, an early land plant lineage, exhibit stable structure and gene content, however the known resources are very limited. The newly sequenced plastomes of Conocephalum, Riccia and Sphaerocarpos species revealed an increase of simple sequence repeats during the diversification of complex thalloid liverwort lineage. The presence of long TA motifs forced applying the long-read nanopore sequencing method for proper and dependable plastome assembly, since the length of dinucleotide repeats overcome the length of Illumina short reads. The accumulation of SSRs (simple sequence repeats) enabled the expansion of inverted repeats by the incorporation of rps12 and rps7 genes, which were part of large single copy (LSC) regions in the previously sequenced plastomes. The expansion of inverted repeat (IR) at the genus level is reported for the first time for non-flowering plants. Moreover, comparative analyses with remaining liverwort lineages revealed that the presence of SSR in plastomes is specific for simple thalloid species. Phylogenomic analysis resulted in trees confirming monophyly of Marchantiidae and partially congruent with previous studies, due to dataset-dependent results of Dumortiera-Reboulia relationships. Despite the lower evolutionary rate of Marchantiales plastomes, significant barcoding gap was detected, even for recently divergent holarctic Conocephalum species. The sliding window analyses revealed the presence of 18 optimal (500 bp long) barcodes that enable the molecular identification of all studied species.

Keywords: chloroplast genome; cpSSR; inverted repeats expansion; liverworts; nanopore sequencing; phylogeny; super-barcoding.

Figure 1

The chloroplast genome of Conocephalum salebrosum. Genes inside and outside the outer circle are transcribed in counterclockwise and clockwise directions, respectively. The genes are color-coded based on their function. The dashed area in the inner circle visualizes the G/C content.

Figure 2

The differences in inverted repeat (IR) borders among Marchantiidae.

Figure 3

Phylogram based on coding regions of liverworts plastomes derived from a Bayesian analysis. The posterior probabilities values were equal to 1 for each of the clade. Right side of the figure provides data on SSR abundance in liverworts plastomes.

Figure 4

Phylogram of Marchantiidae derived from a Bayesian analysis of complete large single copy (LSC) and small single copy (SSC) regions. The posterior probabilities values lower than 1 are shown. The IR region was excluded from analysis due to incongruent phylogenetic signal.

Figure 5

Nucleotide diversity (Pi) by sliding window analysis in the aligned whole plastomes (excluding second IR) of 14 Marchantiidae specimens. Window length 1000 bp, step size 100bp.

Figure 6

Results of several analyses across the plastome sequences of five Marchantiidae species with at least two sequenced specimens using the sliding window method. (A)—the plot of the mean Kimura 2-parameter distance matrix for each 500 bp-long window. (B)—the proportion of zero non-conspecific distances. (C)—the proportion of zero cells in the distance matrix. The unbroken horizontal line crossing the y-axis at 0 is the proportion of zero cells in the distance matrix created from the full dataset. (D)—the proportion of clades that are identical between the windows and the full dataset. (E)—the sum of diagnostic nucleotide positions for all species. (F)—the proportion of species that are monophyletic.