Insights Into Chloroplast Genome Evolution Across Opuntioideae (Cactaceae) Reveals Robust Yet Sometimes Conflicting Phylogenetic Topologies

Abstract

Chloroplast genomes (plastomes) are frequently treated as highly conserved among land plants. However, many lineages of vascular plants have experienced extensive structural rearrangements, including inversions and modifications to the size and content of genes. Cacti are one of these lineages, containing the smallest plastome known for an obligately photosynthetic angiosperm, including the loss of one copy of the inverted repeat (∼25 kb) and the ndh gene suite, but only a few cacti from the subfamily Cactoideae have been sufficiently characterized. Here, we investigated the variation of plastome sequences across the second-major lineage of the Cactaceae, the subfamily Opuntioideae, to address (1) how variable is the content and arrangement of chloroplast genome sequences across the subfamily, and (2) how phylogenetically informative are the plastome sequences for resolving major relationships among the clades of Opuntioideae. Our de novo assembly of the Opuntia quimilo plastome recovered an organelle of 150,347 bp in length with both copies of the inverted repeat and the presence of all the ndh gene suite. An expansion of the large single copy unit and a reduction of the small single copy unit was observed, including translocations and inversion of genes, as well as the putative pseudogenization of some loci. Comparative analyses among all clades within Opuntioideae suggested that plastome structure and content vary across taxa of this subfamily, with putative independent losses of the ndh gene suite and pseudogenization of genes across disparate lineages, further demonstrating the dynamic nature of plastomes in Cactaceae. Our plastome dataset was robust in resolving three tribes with high support within Opuntioideae: Cylindropuntieae, Tephrocacteae and Opuntieae. However, conflicting topologies were recovered among major clades when exploring different assemblies of markers. A plastome-wide survey for highly informative phylogenetic markers revealed previously unused regions for future use in Sanger-based studies, presenting a valuable dataset with primers designed for continued evolutionary studies across Cactaceae. These results bring new insights into the evolution of plastomes in cacti, suggesting that further analyses should be carried out to address how ecological drivers, physiological constraints and morphological traits of cacti may be related with the common rearrangements in plastomes that have been reported across the family.

Keywords: Opuntia; cacti; de novo assembly; plastid structural rearrangements; plastome; pseudogenization; reference-guided assembly.

FIGURE 1

Circular map of chloroplast genome of Opuntia quimilo with annotated genes. The genes transcribed clockwise are shown inside of the circle, whereas genes transcribed counter clockwise are shown outside of the circle. The borders of chloroplast genome are defined with LSC, SSC, IRa, and IRb. The dashed gray color of inner circle shows the GC content.

FIGURE 2

Plastid genome structure and gene order in Opuntia quimilo compared with purslane (Portulaca oleracea). Purslane has the canonical order typical of most angiosperms. For simplicity, the circular map has been linearized. Green line highlights the trnM^CAU-rbcL synapomorphic inversion of Cactaceae, which in O. quimilo also includes the trnV^UAC gene. Regions I, IV, V, VI, and VII are colinear in both plastomes. Region II is colinear but is translocated in the O. quimilo plastome, while region III is inverted and translocated. Region V comprise the genes that are typically in the IR region but are translocated to the large single copy in O. quimilo. Genes highlighted in orange are those typically found in the SSC but transferred to the IR region in O. quimilo. Orange dashed line indicate the double inversion on the ycf1-rpl32 genes, placing ycf1 gene adjacent to rpl32. Black triangles represent duplicated genes present in purslane but absent in O. quimilo; LSC, large single-copy region; SSC, small single-copy region; IR, Inverted repeat.

FIGURE 3

Visualized alignment of the Opuntioideae chloroplast genome sequences (one IR stripped) with annotations using mVISTA. Each horizontal lane shows the graph for the sequence pairwise identity with Opuntia quimilo as reference. The x-axis represents the base sequence of the alignment and the y-axis represents the pairwise percent identity within 50–100%. Gray arrows represent the genes and their orientations. Dark-blue boxes represent exon regions; light-blue boxes represent tRNA and rRNA regions; red boxes represent non-coding sequence (CNS) regions.

FIGURE 4

Nucleotide diversity graphs of the 17 Opuntioideae chloroplast genome sequences from the sliding windows analysis performed in DnaSP (windows length: 800 bp, step size: 200 bp). The x-axis represents the base sequence of the alignment, and the y-axis represents the nucleotide diversity (π value). Each variation hotspot for the chloroplast genome sequences of the Opuntioideae alignment is annotated on the graph.

FIGURE 5

Maximum likelihood phylogenetic tree from RAxML analysis transformed in cladogram with the phylogram represented in small size with substitution rate scaled. All nodes have total bootstrap values (bs = 100) with exception for those that are shown above the branch. Each tip is represented with the assembly map of raw read coverages from Geneious mapper to the Opuntia quimilo chloroplast genome (one IR stripped, represented on the top with annotated genes). Red stars represent low coverage mapping and putative losses associated with the ndh gene suite; green stars represent partial low coverages associated with putative pseudogenization of ycf1, ycf2, and accD genes. Tribe Opuntieae is highlighted in orange, Tephrocacteae in green and Cylindropuntieae in yellow.

FIGURE 6

Topological comparisons of different datasets based on ML analyses. (A) Plastome dataset topology, (B) top 10 marker dataset topology, (C) top five marker dataset topology, and (D) five marker dataset topology for which primers were designed. The Cylindropuntieae sister to Tephrocacteae + Opuntieae topology was recovered only in the five-marker primer dataset (D). Generic relationships are highly variable in Tephrocacteae among the datasets used. ^∗bootstrap support.