RESEARCH ARTICLE The First Complete Plastid Genome from Joinvilleaceae (J. ascendens; Poales) Shows Unique and Unpredicted Rearrangements William P. Wysocki1,2*, Sean V. Burke2, Wesley D. Swingley2, Melvin R. Duvall2 1 Center for Data Intensive Sciences, University of Chicago, 5454 South Shore Dr., Chicago, IL 60615, United States of America, 2 Northern Illinois University; 1425 W. Lincoln Hwy, DeKalb, IL 60115, United States of America * [email protected] a11111 Abstract Joinvilleaceae is a family of tropical grass-like monocots that comprises only the genus Joinvillea. Previous studies have placed Joinvilleaceae in close phylogenetic proximity to the well-studied grass family. A full plastome sequence was determined and characterized for J. ascendens. The plastome was sequenced with next generation methods, fully assem- OPEN ACCESS bled de novo and annotated. The assembly revealed two novel inversions specific to the Citation: Wysocki WP, Burke SV, Swingley WD, Duvall MR (2016) The First Complete Plastid Joinvilleaceae lineage and at least one novel plastid inversion in the Joinvilleaceae-Poa- Genome from Joinvilleaceae (J. ascendens; ceae lineage. Two previously documented inversions in the Joinvilleaceae-Poaceae line- Poales) Shows Unique and Unpredicted age and one previously documented inversion in the Poaceae lineage were also verified. Rearrangements. PLoS ONE 11(9): e0163218. Inversion events were identified visually and verified computationally by simulation muta- doi:10.1371/journal.pone.0163218 tions. Additionally, the loss and subsequent degradation of the accD gene in order Poales Editor: Chih-Horng Kuo, Academia Sinica, TAIWAN was explored extensively in Poaceae and J. ascendens. The two novel inversions along Received: June 10, 2016 with changes in gene composition between families better delimited lineages in the Poales. Accepted: September 6, 2016 The presence of large inversions and subsequent reversals in this small family suggested a Published: September 22, 2016 high potential for large-scale rearrangements to occur in plastid genomes. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Introduction The work is made available under the Creative Commons CC0 public domain dedication. Among monocot Poales, the grass family is most conspicuous ecologically and important agro- nomically. Close relatives to the grasses are found in two families, Joinvilleaceae and Ecdeioco- Data Availability Statement: All plastome files are available from the NCBI Genbank database leaceae [1], which are not as well studied, but suggest biogeographical, morphological, and (KX035098). phylogenomic aspects that bear on the evolutionary origins of the large grass radiation [1, 2, 3]. The two genera of Ecdeiocoleaceaeare narrowly endemic to a restricted region of sand plains Funding: This work was supported by DEB- 1120761 and DEB-1342782 to MRD from NSF in extreme western Australia. Joinvilleaceae, which is more accessible, was selected for this (http://www.nsf.gov). The funders had no role in study because of its previously reported and detailed plastome molecular evolutionary history study design, data collection and analysis, decision [4]. to publish, or preparation of the manuscript. Joinvilleaceae includes the single genus Joinvillea, which originally contained six species [5], Competing Interests: The authors have declared but The Plant List [6] now recognizes four species: J. ascendens, J. borneensis, J. bryanii, and J. that no competing interests exist. plicata. These species are found in often inaccessible localities of Malaysia, western Indonesia, PLOS ONE | DOI:10.1371/journal.pone.0163218 September 22, 2016 1 / 10 Joinvillea Plastome Rearrangements and the Philippines. Joinvillea is also sparsely distributed in the Hawaiian archipelago and other remote Pacific islands where they may be introduced [6, 7]. Joinvillea species are robust, grass-like herbs with strongly plicate leaves and flowers that are borne in large, multibranched terminal panicles. The small, perfect flowers themselves are distinguished from those of grasses in having a conspicuous perianth of six tepals and non-plumose stigmas, both of which suggest insect pollination, possibly by ants [1]. After fertilization the ovaries develop into fleshy drupes [8], again contrasting with the caryopsis typical of Poaceae. Most angiosperm plastid chromosomes (plastomes) are structured with two single copy regions, referred to as the large and small single copy (LSC and SSC) regions, flanked by two inverted perfectly repeated regions (IR) [9]. Compared to grasses, the plastomes of Joinvillea spp. were suggested to have undergone major kilobase-sizedrearrangements indicative of large scale inversion events, suggested by PCR analyses primed from alternative sites in the LSC region [4, 10, 11]. Small inversions are often associated with stem-loop structures suggesting recombination in the stem-forming region as the causal mechanism [12]. However, larger inversions, like those in graminid Poales (Poaceae, Joinvilleaceae, Ecdeiocoleaceae,and Flagel- lariaceae), have been proposed to be the result of recombination. Hiratsuka et al. [13] proposed a mechanism of intermolecular recombination between homologous trnA loci with the simul- taneous formation of chimeric pseudogenes to explain major inversions in rice. Whatever the causes, the distribution of plastome inversions can now be re-explored using full plastome sequences obtained by next-generation sequencing (NGS) technology. While over 140 full grass plastomes have been assembled using traditional Sanger sequenc- ing ([14–17]; and many others) and NGS [18–21], a complete plastome from other families within the graminid clade has yet to be sequenced. Here, the complete plastid chromosome of Joinvillea ascendens is sequenced and described. Evident in the arrangement of subgenomic sections are several large-scale molecular evolutionary events that are not present in grasses and the common cattail (Typha latifolia). Materials and Methods DNA extraction and plastome assembly Silica-dried leaf material was provided by Dr. Lynn Clark, Iowa State University, Ames, IA under voucher (Clark & Attigala 1714). The tissue was homogenized in liquid nitrogen and DNA extrac- tion was performed using the DNeasy plant minikit (Qiagen, Valencia, CA, USA) according to manufacturer instructions. Extractions were quantified using the Qubit fluorometric assay (Life Technologies, Grand Island, NY, USA) and diluted to 2.5 ng/ μL in 20 μL. Illumina libraries were prepared using the Nextera kit (Illumina, San Diego, California, USA) and sequenced single-end on a HiSeq 2000 platform at the Iowa State University DNA core facility (Ames, IA, USA). Quality-trimmed reads were assembled into contiguous sequences (contigs) using Velvet v. 1.2.08 [22] iteratively by using a range of kmer sizes (19–85 by steps of six) and running Velvet using assembled contigs as input [23]. Since no reference plastome exists for any species of Join- villeaceae, contigs could not be scaffolded by an automated method. Extensive in silico genome walking was needed, which follows a process of traversing a decision tree. This entailed manually following all possible branching decisions at the terminus of each contig to extend contigs into linear segments that ultimately formed a circular genetic map. A correct assembly would not lead to premature assembly of repeat regions, including the major inverted repeats, early termination, or incorrect insertion of repetitive sequences. Contigs were then extended by mapping reads and other assembled contigs in Geneious Pro version 7.1.8 (Biomatters Ltd., Auckland, New Zealand) until perfect overlap of at least 20 base pairs (bp) with other contigs or reads was obtained. This was repeated until the quadripartite plastome structure was completed. PLOS ONE | DOI:10.1371/journal.pone.0163218 September 22, 2016 2 / 10 Joinvillea Plastome Rearrangements The assembly was verified by mapping the quality-trimmed reads to the draft assembly in Geneious Pro. The consensus of that mapping was then compared to the sequence of the draft assembly to 1) detect inconsistencies in base composition 2) areas of discontinuity in the read mapping and 3) regions of low read depth. Read depth was found to exceed our minimum acceptable threshhold of 10 across the entire plastome assembly. The plastome was annotated by aligning plastid genes from two reference plastomes: Anomochloa marantoidea (NC014062), which is the sister lineage to all other grasses, and Typha latifolia (NC013823). A. marantoidea was used as the representative grass plastome because of its previous designa- tion as a deeply diverging grass [24]. T. latifolia was used as a reference plastome for gene order (see below). The full plastome for J. ascendens was deposited in Genbank (KX035098). This was compared to the draft plastome of J. plicata [16], which comprises 20 contigs. The draft plastome had only 74.5% coverage of the complete plastome, and homologous portions were 95.2% identical as expected in congeneric species. BLASTN [25] was used to identify regions of significant sequence similarity between the J. ascendens, A. marantoidea, and T. latifolia plastomes. These regions were annotated and plastome rearrangements were first manually identified. The Mauve alignment algorithm [26] was then used with default settings to verify these rearrangements