The Unique Evolutionary Trajectory and Dynamic Conformations of DR and IR/DR-Coexisting Plastomes of the Early Vascular Plant Selaginellaceae (Lycophyte)

GBE The Unique Evolutionary Trajectory and Dynamic Conformations of DR and IR/DR-Coexisting Plastomes of the Early Vascular Plant Selaginellaceae (Lycophyte) Hong-Rui Zhang1,2, Qiao-Ping Xiang1,*, and Xian-Chun Zhang1,* 1 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, The Chinese Academy of Sciences, Beijing, China 2 *Corresponding authors: E-mails: ;. Accepted: March 30, 2019 Data deposition: All the plastomes have been deposited at GenBank under accession numbers MG272483–MG272484, MH598531– MH598537, and MK156800. Abstract Both direct repeats (DR) and inverted repeats (IR) are documented in the published plastomes of Selaginella species indicating the unusual and diverse plastome structure in the family Selaginellaceae. In this study, we newly sequenced complete plastomes of seven species from five main lineages of Selaginellaceae and also resequenced three species (Selaginella tamariscina, Selaginella uncinata, and Selaginella moellendorffii) to explore the evolutionary trajectory of Selaginellaceae plastomes. Our results showed that the plastomes of Selaginellaceae vary remarkably in size, gene contents, gene order, and GC contents. Notably, both DR and IR structures existed in the plastomes of Selaginellaceae with DR structure being an ancestral state. The occurrence of DR structure was at 257 Ma and remained in most subgenera of Selaginellaceae, whereas IR structure only reoccurred in Selaginella sect. Lepidophyllae (143 Ma) and Selaginella subg. Heterostachys (19 Ma). The presence of a pair of large repeats psbK-trnQ, together with DR/IR region in Selaginella bisulcata, Selaginella pennata, S. uncinata, and Selaginella hainanensis, could frequently mediate diverse homologous recombination and create approximately equal stoichiometric isomers (IR/DR-coexisting) and subgenomes. High proportion of repeats is presumably responsible for the dynamic IR/DR-coexisting plastomes, which possess a lower synonymous substitution rate (dS) compared with DR-possessing and IR-possessing plastomes. We propose that the occurrence of DR structure, together with few repeats, is possibly selected to keep the stability of plastomes and the IR/DR-coexisting plastomes also reached an equilibrium in plastome organization through highly efficient homologous recombination to maintain stability. Key words: gene loss, homologous recombination, DR/IR evolution, Selaginella, substitution rate, time divergence. Introduction Plastid genomes (plastomes) of almost all land plants are highly conserved and present the canonical quadripartite structure with a pair of large inverted repeats (termed IRA and IRB) separated by two single-copy regions (termed LSC and SSC) (Mower and Vickrey 2018). Normally, the range of the IR varies through expansion or contraction. Complete loss of the IR is rare but has been observed in some species of Fabaceae (Lavin et al. 1990; Cai et al. 2008), Geraniaceae (Guisinger et al. 2011; Blazier et al. 2016; Ruhlman et al. 2017), and Cactaceae (Sanderson et al. 2015). Remarkably, plastomes with a pair of large direct repeats (termed DRA and DRB) have been documented for four species of Selaginellaceae, Selaginella tamariscina (Xu et al. 2018), Selaginella vardei, Selaginella indica (Zhang et al. 2018), and Selaginella kraussiana (Mower et al. 2018) in land plants. The DR structure in Selaginellaceae was explained to have occurred by 50-kb fragment inversion with a complete IRB being included, compared with the plastome of its sister family Isoetaceae (Mower et al. 2018; Zhang et al. 2018). In addition to the exceptional existence of plastomes with DR structure, a salient fraction of land plants plastomes experienced significant structural rearrangements, with evidence of large inversions and loss of entire gene family, despite the overall conservation in structures and gene order (Mower and Vickrey 2018). A 30-kb inversion (ycf2-psbM) ß The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1258 Genome Biol. Evol. 11(4):1258–1274. doi:10.1093/gbe/evz073 Advance Access publication April 1, 2019 University of Chinese Academy of Sciences, Beijing, China DR and IR/DR-Coexisting Plastomes of the Early Vascular Plant Selaginellaceae Jansen 2014). Three major pathways of gene loss have been detected in land plants: 1) gene transfer to the nucleus (infA, rpl22, rpl32, and accD), 2) substitution by a nuclearencoded, mitochondrial targeted gene product (rps16), and 3) substitution by a nuclear-encoded protein for a plastid gene product (accD, rpl23) (Jansen and Ruhlman 2012). The multiple independent ndh gene loss in different lineages is supposed to belong to the third pathway (Ruhlman et al. 2015). Selaginellaceae, one of the most ancient vascular plants with nearly 400 Myr of evolutionary history (Banks 2009) is the largest family of lycophytes with 750 species classified into the only genus Selaginella (Jermy 1990; Weststrand and Korall 2016b; Zhou et al. 2016). Selaginella species have highly diverse growth forms, including creeping, climbing, prostrate, erect, and rosetted forms, and also inhabit an impressive range of habitats, from tropical rain forests to deserts, alpine, and arctic habitats (Zhang et al. 2013). Both mitochondrial and plastid genomes are more frequently subject to alterations under specific environmental conditions (Marechal and Brisson 2010). With such a high diversity in habitat and growth forms and extremely long evolutionary history, complex plastomes with different structures are inferred in Selaginellaceae (Tsuji et al. 2007). However, only seven species of Selaginella, viz., Selaginella uncinata (Tsuji et al. 2007), Selaginella moellendorffii (Smith 2009), S. tamariscina (Xu et al. 2018), S. vardei, S. indica (Zhang et al. 2018), S. kraussiana, and Selaginella lepidophylla (Mower et al. 2018) have been reported for their plastomes. Compared with the species from Lycopodiaceae and Isoetaceae of lycophytes (Wolf et al. 2005; Karol et al. 2010; Guo et al. 2016; Mower et al. 2018; Zhang et al. 2018), plastomes of Selaginella are, indeed, far less conserved in both structures and gene contents. Both S. uncinata and S. moellendorffii belong to subg. Stachygynandrum based on both morphology-based classification (Jermy 1986) and a recent molecular-based classification (Weststrand and Korall 2016b). However, their plastomes show divergent variation in structure. Several rearrangements, such as a 20-kb fragment inversion, a 17-kb fragment transposition and gene duplications, exist in these two species (Smith 2009). Selaginella kraussiana, belonging to subg. Gymnogynum (sen (...truncated)