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Comparative Chloroplast Genomics at Low Taxonomic Levels: A Case Study Using Amphilophium (Bignonieae, Bignoniaceae).

ABSTRACT: Chloroplast (cp) genome organization, gene order, and content have long been considered conserved among land plants. Despite that, the generation of thousands of complete plastomes through next-generation sequencing (NGS) has challenged their conserved nature. In this study, we analyze 11 new complete plastomes of Amphilophium (Bignonieae, Bignoniaceae), a diverse genus of Neotropical lianas, and that of Anemopaegma prostratum. We explored the structure and content of the assembled plastomes and performed comparative analyses within Amphilophium and among other plastomes available for Bignoniaceae. The overall gene content and orientation of plastomes is similar in all species studied. Plastomes are not conserved among Amphilophium, showing significant differences in length (155,262-164,786 bp), number of genes duplicated in the IRs (eight, 18, or 19), and location of the SC/IR boundaries (i.e., LSC/IRa junction between rps19 and rpl2 genes, within petD, or within petB). Length differences reflect expansions of the IRs and contractions of the LSC regions. The plastome of A. prostratum is 168,172 bp, includes 19 duplicated genes, and has the LSC/IRa boundary located within the petB gene. Amphilophium plastomes show high nucleotide diversity, with many hypervariable regions, and 16 genes with signatures of positive selection. Multiple SSRs and repeat regions were identified for Amphilophium and Anemopaegma prostratum. The differences in structure detected within Amphilophium plastomes in terms of LSC/IR and IR/SSC boundaries, number of duplicated genes, and genome sizes are mostly shared between taxa that belong to the same clade. Our results bring new insights into the evolution of plastomes at low taxonomic levels.

SUBMITTER: Thode VA

PROVIDER: S-EPMC6594259 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Molecular evolutionary studies share the common goal of elucidating historical relationships, and the common challenge of adequately sampling taxa and characters. Particularly at low taxonomic levels, recent divergence, rapid radiations, and conservative genome evolution yield limited sequence variation, and dense taxon sampling is often desirable. Recent advances in massively parallel sequencing make it possible to rapidly obtain large amounts of sequence data, and multiplexing makes extensive sampling of megabase sequences feasible. Is it possible to efficiently apply massively parallel sequencing to increase phylogenetic resolution at low taxonomic levels? RESULTS:We reconstruct the infrageneric phylogeny of Pinus from 37 nearly-complete chloroplast genomes (average 109 kilobases each of an approximately 120 kilobase genome) generated using multiplexed massively parallel sequencing. 30/33 ingroup nodes resolved with > or = 95% bootstrap support; this is a substantial improvement relative to prior studies, and shows massively parallel sequencing-based strategies can produce sufficient high quality sequence to reach support levels originally proposed for the phylogenetic bootstrap. Resampling simulations show that at least the entire plastome is necessary to fully resolve Pinus, particularly in rapidly radiating clades. Meta-analysis of 99 published infrageneric phylogenies shows that whole plastome analysis should provide similar gains across a range of plant genera. A disproportionate amount of phylogenetic information resides in two loci (ycf1, ycf2), highlighting their unusual evolutionary properties. CONCLUSION:Plastome sequencing is now an efficient option for increasing phylogenetic resolution at lower taxonomic levels in plant phylogenetic and population genetic analyses. With continuing improvements in sequencing capacity, the strategies herein should revolutionize efforts requiring dense taxon and character sampling, such as phylogeographic analyses and species-level DNA barcoding.

Project description:The chloroplast is one of the most important organelles of plants. This organelle has a circular DNA with approximately 130 genes. The use of plastid genomic data in phylogenetic and evolutionary studies became possible with high-throughput sequencing methods, which allowed us to rapidly obtain complete genomes at a reasonable cost. Here, we use high-throughput sequencing to study the "Adenocalymma-Neojobertia" clade (Bignonieae, Bignoniaceae). More specifically, we use Hi-Seq Illumina technology to sequence 10 complete plastid genomes. Plastomes were assembled using selected plastid reads and de novo approach with SPAdes. The 10 assembled genomes were analyzed in a phylogenetic context using five different partition schemes: (1) 91 protein-coding genes ("coding"); (2) 76 introns and spacers with alignment manually edited ("non-coding edited"); (3) 76 non-coding regions with poorly aligned regions removed using T-Coffee ("non-coding filtered"); (4) 91 coding regions plus 76 non-coding regions edited ("coding + non-coding edited"); and, (5) 91 protein-coding regions plus the 76 filtered non-coding regions ("coding + non-coding filtered"). Fragmented regions were aligned using Mafft. Phylogenetic analyses were conducted using Maximum Likelihood (ML) and Bayesian Criteria (BC). The analyses of the individual plastomes consistently recovered an expansion of the Inverted Repeated (IRs) regions and a compression of the Small Single Copy (SSC) region. Major genomic translocations were observed at the Large Single Copy (LSC) and IRs. ML phylogenetic analyses of the individual datasets led to the same topology, with the exception of the analysis of the "non-coding filtered" dataset. Overall, relationships were strongly supported, with the highest support values obtained through the analysis of the "coding + non-coding edited" dataset. Four regions at the LSC, SSC, and IR were selected for primer development. The "Adenocalymma-Neojobertia" clade shows an unusual pattern of plastid structure variation, including four major genomic translocations. These rearrangements challenge the current view of conserved plastid genome architecture in terms of gene order. It also complicates both genomic assemblies using reference genomes and sequence alignments using whole plastomes. Therefore, strategies that employ de novo assemblies and manual evaluation of sequence alignments are required to prevent assembly and alignment errors.

Project description:BACKGROUND: At present, plant molecular systematics and DNA barcoding techniques rely heavily on the use of chloroplast gene sequences. Because of the relatively low evolutionary rates of chloroplast genes, there are very few choices suitable for molecular studies on angiosperms at low taxonomic levels, and for DNA barcoding of species. METHODOLOGY/PRINCIPAL FINDINGS: We scanned the entire chloroplast genomes of 12 genera to search for highly variable regions. The sequence data of 9 genera were from GenBank and 3 genera were of our own. We identified nearly 5% of the most variable loci from all variable loci in the chloroplast genomes of each genus, and then selected 23 loci that were present in at least three genera. The 23 loci included 4 coding regions, 2 introns, and 17 intergenic spacers. Of the 23 loci, the most variable (in order from highest variability to lowest) were intergenic regions ycf1-a, trnK, rpl32-trnL, and trnH-psbA, followed by trnS(UGA)-trnG(UCC), petA-psbJ, rps16-trnQ, ndhC-trnV, ycf1-b, ndhF, rpoB-trnC, psbE-petL, and rbcL-accD. Three loci, trnS(UGA)-trnG(UCC), trnT-psbD, and trnW-psaJ, showed very high nucleotide diversity per site (? values) across three genera. Other loci may have strong potential for resolving phylogenetic and species identification problems at the species level. The loci accD-psaI, rbcL-accD, rpl32-trnL, rps16-trnQ, and ycf1 are absent from some genera. To amplify and sequence the highly variable loci identified in this study, we designed primers from their conserved flanking regions. We tested the applicability of the primers to amplify target sequences in eight species representing basal angiosperms, monocots, eudicots, rosids, and asterids, and confirmed that the primers amplified the desired sequences of these species. SIGNIFICANCE/CONCLUSIONS: Chloroplast genome sequences contain regions that are highly variable. Such regions are the first consideration when screening the suitable loci to resolve closely related species or genera in phylogenetic analyses, and for DNA barcoding.

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Comparative Chloroplast Genomics at Low Taxonomic Levels: A Case Study Using Amphilophium (Bignonieae, Bignoniaceae).

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