Structural rearrangements in plastid genomes of Apiaceae as phylogenetic markers

Apiaceae belong to angiosperm families with freque nt plastome structural rearrangements, some of which are genera lly egarded as synapomorphic for large clades, although typically with limited taxon sampling. Our study aims to improve understanding o f the structural rearrangements in plastome within the Tordylieae tr ibe (ApiaceaeApioideae) with a dense sampling scheme of its spec ies. We showed that presence of psbA pseudogene in inverted repeats near the border wi th a large single-copy region, which is found in the Tor dylieae tribe, may be a clade-specific synapomorphy.


Introduction
Apiaceae are one of the largest and most economically important families of angiosperms. Many species are highly valued as essential oil (coriander, cumin), vegetable (carrot, parsley, celery, dill) and medicinal (dill, ferula) plants. They are also a family with a long history of taxonomic uncertainty. Challenges in accurate species identification persist and phylogenetic relationships among genera in the family continue to be unresolved. Herewith, it is not possible to delimit many molecular clades using morphological traits [1].
Recently, it has been repeatedly stipulated that structural reorganizations in plastid genomes such as the change of gene order, small deletions and duplications in non-coding regions, losses and additions of whole genes or their separate parts, can serve as phylogenetic markers in addition to the results obtained from the analysis of nucleotide substitutions [2][3][4]. Instability in the plastid genome is often associated with the junctions between inverted repeats (IR) and single-copy regions.
Apiaceae plastomes appear to be unusual in angiosperm families due to the frequency and large size of IR junction shifts [5][6]. To date, all major IR expansions and contractions uncovered in the Apiales are restricted to the "apioid superclade," a large, distally branching group within Apiaceae subfamily Apioideae [7]. Specific large expansions of the IR have shown to demarcate large monophyletic groups, whereas small expansions or contractions tend to occur in closely related species.
The aim of our study was to conduct molecular analysis with a dense sampling to elucidate whether IR fluxes could be served as a synapomorphy within a certain group. As a group for our study, Tordylieae tribe was selected. Within this group, occurrence of specific psbA pseudogene was detected in Pastinaca and Heracleum plastomes between ycf2 and trnL-CAA genes ( Fig. 1) [8][9].

Experimental
For study of polymorphism of junction position, 17 Tordylieae taxa were selected. The material was kindly provided by the Botanical Garden of Moscow State University or MW.
Total DNA was isolated from fruits or leaf tissue using the NucleoSpin Plant isolation kit (Macherey-Nagel, Düren, Germany) following manufacturer instructions. To determine the structure of IRb-LSC borders we designed primers Pas3 27248U22: 5'-TTATTTGCAGATGAAGAGATGT-3'; Pas3 27868L20: 5'-GAACTCCCAATTCTCTAGCAAAT-3') based on the sequence of whole plastome of Pastinaca pimpinellifolia M.Bieb. (GenBank accession number KM035850). When the IRb/LSC junctions were typical for Daucus carota subsp. sativus (GenBank accession number DQ898156), PCR amplification products were about 700 bp length. When the pseudogene psbA was located in the IRb as it happened in plastids of Pastinaca, PCR product was about 1,0-1,2kb. PCR products were purified with the Gel Extraction & PCR Cleanup Kit (Evrogen, Russia). Sequencing was performed with ABI Prism BigDye Terminator v. 3.1 reagents with subsequent analysis on an ABI Prism 3100-Avant (Applied Biosystems) automated sequencer. The precise localization of JIRb/LSC in Tordylieae species was defined after the sequencing of PCR-products.  Phylogenetic studies based on sequence data from nuclear ribosomal DNA [e.g., 11] and chloroplast DNA [12][13] have provided important insights into the evolution and diversification of Tordylieae. Molecular phylogenetic analyses have resolved five major lineages within the tribe (Fig. 2). Due to molecular data, the Tordylieae was expanded to include African taxa previously residing in Peucedanum (Notobubon, Dasispermum, and others). Majority of genera traditionally recognized within Tordylieae fall into three clades: (1) Tordylium; (2) Heracleum+Pastinaca; (3) Semenovia+Tetrataenium.

Results and discussion
PCR-based analyses of close relatives of Pastinaca (Heracleum, Leiotulus, Malabaila, Trigonosciadium) detected presence of a psbA pseudogene (Fig. 2) and its absence in Zosima, Ducrosia, Tordylium and Tordyliopsis, making this rearrangement a likely cladespecific synapomorphy. Our screening for presence of the trnH+psbA insertion between ycf2 and trnL-CAA genes showed that this structural feature is characteristic only for Heracleum+Pastinaca clade. The insertion arises as a consequence of a shift of the junction between the IRA and LSC: the inverted repeat A expands and includes the gene trnH-GUG with the 3' part of the psbA, and, eventually, their copies emerge in inverted repeat B, between ycf2 and trnL-CAA genes.
Thus, using representatives of the tribe Tordylieae as an example, we have showed that some changes in the plastid DNA structure (in this case, caused by the shift of IR junctions from the LSC), can be used for testing the hypotheses about taxonomic position at the tribal level.
The work is supported by the Russian Foundation of Basic Research (grant 20-04-01128 A) and carried out as part of the Scientific Projects of the State Order of the Government of Russian Federation to Lomonosov Moscow State University (121031600196-8 and АААА-А17-117120540067-0).