Chloroplast genome structure analysis of Equisetum unveils phylogenetic relationships to ferns and mutational hotspot region

Abstract

Equisetum is one of the oldest extant group vascular plants and is considered to be the key to understanding vascular plant evolution. Equisetum is distributed almost all over the world and has a high degree of adaptability to different environments. Despite the fossil record of horsetails (Equisetum, Equisetaceae) dating back to the Carboniferous, the phylogenetic relationship of this genus is not well, and the chloroplast evolution in Equisetum remains poorly understood. In order to fill this gap, we sequenced, assembled, and annotated the chloroplast genomes of 12 species of Equisetum, and compared them to 13 previously published vascular plants chloroplast genomes to deeply examine the plastome evolutionary dynamics of Equisetum. The chloroplast genomes have a highly conserved quadripartite structure across the genus, but these chloroplast genomes have a lower GC content than other ferns. The size of Equisetum plastomes ranges from 130,773 bp to 133,684 bp and they encode 130 genes. Contraction/expansion of IR regions and the number of simple sequences repeat regions underlie large genomic variations in size among them. Comparative analysis revealed we also identified 13 divergence hotspot regions. Additionally, the genes accD and ycf1 can be used as potential DNA barcodes for the identification and phylogeny of the genus Equisetum. Twelve photosynthesis-related genes were specifically selected in Equisetum. Comparative genomic analyses implied divergent evolutionary patterns between Equisetum and other ferns. Phylogenomic analyses and molecular dating revealed a relatively distant phylogenetic relationship between Equisetum and other ferns, supporting the division of pteridophyte into Lycophytes, Equisetaceae and ferns. The results show that the chloroplast genome can be used to solve phylogenetic problems within or between Equisetum species, and also provide genomic resources for the study of Equisetum systematics and evolution.

Keywords: divergent hotspot; evolutionary; phylogenomics; pteridophytes; sequence characteristic.

Figure 1

Summary data on the assembly of chloroplast genomes in Equisetum. (A) Size (bp). (B) Number of genes. (C) GC content (%).

Figure 2

The comprehensive arrangement of the chloroplast genome in Equisetum. The large (LSC) and small (SSC) single copy regions are separated by the inverted repeats (IRa, IRb), represented by bold black lines on the inner circle. Genes located outside the circle undergo transcription in a counter-clockwise fashion, while those inside undergo transcription in a clockwise direction.

Figure 3

Phylogenetic tree based on Genes, CDSs and whole chloroplast genome concatenated analyses sequences from 28 species. The construction of phylogenetic trees was accomplished by employing both Bayesian inference (BI) and maximum likelihood (ML) methodologies. To present the level of support above the branches, we showcase Bayesian posterior probabilities (PP) and bootstrap percentages obtained from maximum likelihood analyses (BP).

Figure 4

The maximum clade credibility tree of Equisetum was constructed using the BEAST method, based on the chloroplast genome sequences. This tree provided information on the mean ages and 95% highest posterior density (HPD) intervals for the node ages. (A) Secondary calibration 488.0 Mya (B) Fossil calibration 429.0 Mya (C) Fossil calibration 325.0 Mya (D) Fossil calibration 386.0 Mya (E) Fossil calibration 136.5 Mya (F) Fossil calibration 72 Mya.

Figure 5

The classification and distribution of SSRs in the chloroplast genomes of Equisetum. (A) The difference of repeated sequence in chloroplast genomes. (B) Occurrence rate of SSRs. (C) Count of identified SSR motifs across various types of repeat classes.

Figure 6

Forms of mutation in the Equisetum chloroplast genomes. (A) Location of the all indels from 12 species. (B) Number of indel, inversion and substitution sequences in the chloroplast genomes.

Figure 7

Depicts the comparison among the border regions of 25 distinct species chloroplast genomes. Compare the boundaries of large orders (LSC), small single copies (SSC), and the border between the reverse repetition (IR) region.

Figure 8

Visualization of comparison of Equisetum chloroplast genome sequences. (A) Assessment of nucleotide variability (Pi) within the chloroplast genome among 12 Equisetum species. (B) Color-coded representation to indicate genomic regions encompassing protein-coding sequences, rRNA, tRNA coding sequences, and conserved noncoding sequences (CNS). The vertical axis presents the percentage similarity, spanning from 50% to 100%.

Figure 9

Codon bias analysis of chloroplast genomes of Equisetum. (A) ENC-plot analysis. (B) PR2-plot analysis. (C) Neutrality plot analysis.

Figure 10

A Pairwise Ka/Ks ratios 12 Equisetum species. The genes of Ka/Ks > 1 from 12 chloroplast genome of Equisetum..