A Comparative Analysis of Whole Plastid Genomes from the Apiales: Expansion and Contraction of the Inverted Repeat, Mitochondrial to Plastid Transfer of DNA, and Identification of Highly Divergent Noncoding Regions

Previous mapping studies have revealed that the frequency and large size of inverted repeat junction shifts in Apiaceae plastomes are unusual among angiosperms. To further examine plastome structural organization and inverted repeat evolution in the Apiales (Apiaceae + Araliaceae), we have determined the complete plastid genome sequences of five taxa, namely Anthriscus cerefolium (154,719 base pairs), Crithmum maritimum (158,355 base pairs), Hydrocotyle verticillata (153,207 base pairs), Petroselinum crispum (152,890 base pairs), and Tiedemannia filiformis subsp. greenmanii (154,737 base pairs), and compared the results obtained to previously published plastomes of Daucus carota subsp. sativus and Panax schin-seng. We also compared the five Apiaceae plastomes to identify highly variable noncoding loci for future molecular evolutionary and systematic studies at low taxonomic levels. With the exceptions of Crithmum and Petroselinum, which each demonstrate a ~1.5 kilobase shift of its LSC-IRB junction (JLB), all plastomes are typical of most other non-monocot angiosperm plastid DNAs in their structural organization, gene arrangement, and gene content. Crithmum and Petroselinum also incorporate novel DNA in the LSC region adjacent to the LSC-IRA junction (JLA). These insertions (of 1,463 and 345 base pairs, respectively) show no sequence similarity to any other region of their plastid genomes, and BLAST searches of the Petroselinum insert resulted in multiple hits to angiosperm mitochondrial genome sequences, indicative of a mitochondrial to plastid transfer of DNA. A comparison of pairwise sequence divergence values and numbers of variable and parsimony-informative alignment positions (among other sequence characteristics) across all introns and intergenic spacers >150 base pairs in the five Apiaceae plastomes revealed that the rpl32-trnL, trnE-trnT, ndhF-rpl32, 50rps16-trnQ, and trnT-psbD intergenic spacers are among the most fast-evolving loci, with the trnD-trnY-trnE-trnT combined region presenting the greatest number of potentially informative characters overall. These regions are therefore likely to be the best choices for molecular evolutionary and systematic studies at low taxonomic levels. Repeat analysis revealed direct and inverted dispersed repeats of 30 base pairs or more that may be useful in population-level studies. These structural and sequence analyses contribute to a better understanding of plastid genome evolution in the Apiales and provide valuable new information on the phylogenetic utility of plastid noncoding loci, enabling further molecular evolutionary and phylogenetic studies on this economically, ecologically, and medicinally important group of flowering plants. Keywords—Apiaceae, Araliaceae, intergenic spacer regions, intracellular transfer, plastid DNA. The plastid genomes of the majority of photosynthetic angiosperms are highly conserved in structural organization, gene arrangement, and gene content (Palmer 1991; Raubeson and Jansen 2005; Wicke et al. 2011; Jansen and Ruhlman 2012; Ruhlman and Jansen 2014). Their hallmark is the presence of two large duplicate regions in reverse orientation known as the inverted repeat (IR), which separate the remainder of the genome into large single copy (LSC) and small single copy (SSC) regions. Variation in size of this molecule is due most typically to the expansion or contraction of the IR into or out of adjacent single-copy regions and/or changes in sequence complexity due to insertions or deletions of novel sequences. Of the two equimolar structural isomers existing for plastid DNA (ptDNA; Palmer 1983), the structure most commonly presented follows the convention used for Nicotiana tabacum L. (tobacco) in which one copy of the IR is flanked by genes ycf1 and trnH-GUG (and is designated as IRA) and the other copy is flanked by genes rps19 and ndhF (and is designated as IRB; Shinozaki et al. 1986; Yukawa et al. 2005). The junctions between the LSC region and each of these IR copies are designated as JLA (LSC/IRA) and JLB (LSC/IRB), and the junctions flanking the SSC region are designated as JSA (SSC/IRA) and JSB (SSC/IRB; Shinozaki et al. 1986). In most non-monocot angiosperm ptDNAs, JLB lies within the ribosomal protein S10 operon in a more or less fixed position within or near the rps19 gene and JLA lies just downstream of LSC gene trnH-GUG. The IRs of angiosperm plastomes can fluctuate greatly in size, although small contractions and expansions of <100 base pairs (bp) are most frequent, and the positions of all four IR/single-copy junctions can vary even among closely related species (Goulding et al. 1996; Plunkett and Downie 2000). Large IR expansions (>1,000 bp) occur less frequently and outnumber large contractions (Plunkett and Downie 2000; Raubeson and Jansen 2005; Hansen et al. 2007). Because major changes in position of IR junctions can accompany structural rearrangements elsewhere in the plastid genome (Palmer 1991; Boudreau and Turmel 1995; Aii et al. 1997; Cosner et al. 1997; Perry et al. 2002; Chumley et al. 2006; Haberle et al. 2008; Guisinger et al. 2011; Wicke et al. 2011), the availability of sequence data for these genomes can help elucidate the mechanisms responsible for these large-scale IR junction shifts and other major genomic changes. Previous ptDNA restriction site mapping studies have shown that Apiaceae (Umbelliferae) exhibit unprecedented variation in position of their LSC/IR boundary regions (Palmer 1985a; Plunkett and Downie 1999, 2000). While most umbellifer species surveyed possess a JLB indistinguishable from that of Nicotiana tabacum and the vast majority of other eudicots, at least one expansion and seven different contractions of the IR relative to the N. tabacum JLB were detected in 55 species, each ranging in size from ~1–16 kilobase pairs (kb; Plunkett and Downie 2000). As examples, all examined members of the “Aegopodium group,” representatives of tribes Careae and Pyramidoptereae (Downie et al. 2010), have a ~1.1 kb expansion of JLB relative to that of N. tabacum. Careae and Pyramidoptereae are monophyletic sister groups (Banasiak et al. 2013), indicating that IR junction shifts have the potential to demarcate major clades within the family. Coriandrum sativum L. and Bifora radians M. Bieb., both of tribe Coriandreae, have the most contracted IRs, approximately