Plastome Structural Evolution and Homoplastic Inversions in Neo-Astragalus (Fabaceae)

Abstract

The plastid genomes of photosynthetic green plants have largely maintained conserved gene content and order as well as structure over hundreds of millions of years of evolution. Several plant lineages, however, have departed from this conservation and contain many plastome structural rearrangements, which have been associated with an abundance of repeated sequences both overall and near rearrangement endpoints. We sequenced the plastomes of 25 taxa of Astragalus L. (Fabaceae), a large genus in the inverted repeat-lacking clade of legumes, to gain a greater understanding of the connection between repeats and plastome inversions. We found plastome repeat structure has a strong phylogenetic signal among these closely related taxa mostly in the New World clade of Astragalus called Neo-Astragalus. Taxa without inversions also do not differ substantially in their overall repeat structure from four taxa each with one large-scale inversion. For two taxa with inversion endpoints between the same pairs of genes, differences in their exact endpoints indicate the inversions occurred independently. Our proposed mechanism for inversion formation suggests the short inverted repeats now found near the endpoints of the four inversions may be there as a result of these inversions rather than their cause. The longer inverted repeats now near endpoints may have allowed the inversions first mediated by shorter microhomologous sequences to propagate, something that should be considered in explaining how any plastome rearrangement becomes fixed regardless of the mechanism of initial formation.

Keywords: chloroplast; inverted repeat-lacking clade; legumes; microhomology-mediated rearrangements; plastid genome.

Fig. 1.

Annotated plastomes of five Astragalus species. Gene order in A. agnicidus is consistent with a plastid genome having the 50-kb inversion (Doyle et al. 1996). A 7-kb inversion is found in A. calycosus (rbcL ∼ trnH-GUG; red), a 40-kb inversion is found in A. mollissimus (trnQ-UUG ∼ trnT-UGU; tan), and 7-kb inversions are found in both A. flexuosus and A. neglectus (trnL-CAA ∼ trnI-CAU; blue). Inversions identified from MUMmer (Marçais et al. 2018) and progressiveMauve (Darling et al. 2010) alignments. The approximate locations of the large single-copy region (LSC), the region ancestrally duplicated as the inverted repeat but now present as a single copy only (“IR”), and the small single-copy region (SSC) are shown at the left. Plastome maps modified from the output of OGDraw (Greiner et al. 2019).

Fig. 2.

Plastome inversions and overdispersed repeats on maximum likelihood phylogram of 30 Astragalus taxa and Oxytropis bicolor from a concatenated alignment of locally colinear blocks (LCBs) identified using progressiveMauve (Darling et al. 2010). The tree is consistent with previous phylogenies of Astragalus at a higher level (Wojciechowski 2005; Scherson et al. 2008; Azani et al. 2019; Su et al. 2021) with Neo-Astragalus a well-supported clade nested within Old World and euploid North American taxa. Bootstrap support values from 1,000 ultrafast bootstrap replicates are shown with color-coded circles. Branch lengths are in units of substitutions per site.

Fig. 3.

Phylogenetic context of plastome repeat content by category (A) and repeat density by position in plastome (B) for 30 Astragalus taxa and Oxytropis bicolor. Cladogram of maximum likelihood topology shown on left. Repeats are categorized by their occurrence among taxa based on Markov clustering. Repeat density in 3-kb sliding windows is averaged over 100-bp steps. The position of repeats colored by category is shown below the horizontal axis in repeat density plots. All plastomes rescaled to the same length. Inversion endpoint locations are shown in all taxa, and colored rectangles are present in taxa with inversions. All inversions were reverted before calculating repeat density.

Fig. 4.

Repeat content within 1 kb of plastome inversion endpoint locations. Each endpoint location for each inversion is designated by the two loci the endpoint lies between. For each of the six inversion endpoints, on the left is a half violin plot showing the distribution of repeat content among all 31 taxa, and on the right is the repeat content at that location for the Astragalus taxon or taxa with the inversion. Inversions were reverted in the four taxa with them before calculating repeat content to make the two endpoints comparable with other taxa without the inversions.

Fig. 5.

Position of repeats and microhomologous sequences ancestrally (upper) and currently (lower) after plastome inversions in Astragalus calycosus (A), A. mollissimus (B), as well as A. flexuosus and A. neglectus (C). In each case, two longer repeats (Acaly1, Acaly2, etc.) that are now in inverted orientation at opposite ends of the inversion are inferred to have ancestrally been direct repeats on the same end of the inversion. Shorter microhomologous sequences (M_c, m_c, etc.) are inferred to have mediated the inversion and are found at the exact inversion endpoints. Different repeats and microhomologous sequences are implicated in the inversions between the same sets of loci in A. flexuosus and A. neglectus. Size and position of features not to scale.

Fig. 6.

Proposed sequence of events for initiation of plastome inversions resulting in short inverted repeats at both ends from ancestrally direct repeats near one endpoint (inspired by Maréchal and Brisson [2010]). MMBIR, microhomology-mediated break-induced replication; RDR, recombination-dependent replication.