Plastome (plastid genome) sequences provide valuable information for understanding the phylogenetic relationships and evolutionary history of plants. Although the rapid development of high-throughput sequencing technology has led to an explosion of plastome sequences, annotation remains a significant bottleneck for plastomes. User-friendly batch annotation of multiple plastomes is an urgent need. We introduce Plastid Genome Annotator (PGA), a standalone command line tool that can perform rapid, accurate, and flexible batch annotation of newly generated target plastomes based on well-annotated reference plastomes. In contrast to current existing tools, PGA uses reference plastomes as the query and unannotated target plastomes as the subject to locate genes, which we refer to as the reverse query-subject BLAST search approach. PGA accurately identifies gene and intron boundaries as well as intron loss. The program outputs GenBank-formatted files as well as a log file to assist users in verifying annotations. Comparisons against other available plastome annotation tools demonstrated the high annotation accuracy of PGA, with little or no post-annotation verification necessary. Likewise, we demonstrated the flexibility of reference plastomes within PGA by annotating the plastome of Rosa roxburghii using that of Amborella trichopoda as a reference. The program, user manual and example data sets are freely available at https://github.com/quxiaojian/PGA . PGA facilitates rapid, accurate, and flexible batch annotation of plastomes across plants. For projects in which multiple plastomes are generated, the time savings for high-quality plastome annotation are especially significant.

The use of transcriptomic and genomic datasets for phylogenetic reconstruction has become increasingly common as researchers attempt to resolve recalcitrant nodes with increasing amounts of data. The large size and complexity of these datasets introduce significant phylogenetic noise and conflict into subsequent analyses. The sources of conflict may include hybridization, incomplete lineage sorting, or horizontal gene transfer, and may vary across the phylogeny. For phylogenetic analysis, this noise and conflict has been accommodated in one of several ways: by binning gene regions into subsets to isolate consistent phylogenetic signal; by using gene-tree methods for reconstruction, where conflict is presumed to be explained by incomplete lineage sorting (ILS); or through concatenation, where noise is presumed to be the dominant source of conflict. The results provided herein emphasize that analysis of individual homologous gene regions can greatly improve our understanding of the underlying conflict within these datasets. Here we examined two published transcriptomic datasets, the angiosperm group Caryophyllales and the aculeate Hymenoptera, for the presence of conflict, concordance, and gene duplications in individual homologs across the phylogeny. We found significant conflict throughout the phylogeny in both datasets and in particular along the backbone. While some nodes in each phylogeny showed patterns of conflict similar to what might be expected with ILS alone, the backbone nodes also exhibited low levels of phylogenetic signal. In addition, certain nodes, especially in the Caryophyllales, had highly elevated levels of strongly supported conflict that cannot be explained by ILS alone. This study demonstrates that phylogenetic signal is highly variable in phylogenomic data sampled across related species and poses challenges when conducting species tree analyses on large genomic and transcriptomic datasets. Further insight into the conflict and processes underlying these complex datasets is necessary to improve and develop adequate models for sequence analysis and downstream applications. To aid this effort, we developed the open source software phyparts ( https://bitbucket.org/blackrim/phyparts ), which calculates unique, conflicting, and concordant bipartitions, maps gene duplications, and outputs summary statistics such as internode certainy (ICA) scores and node-specific counts of gene duplications.

Although Pentapetalae (comprising all core eudicots except Gunnerales) include ≈70% of all angiosperms, the origin of and relationships among the major lineages of this clade have remained largely unresolved. Phylogenetic analyses of 83 protein-coding and rRNA genes from the plastid genome for 86 species of seed plants, including new sequences from 25 eudicots, indicate that soon after its origin, Pentapetalae diverged into three clades: (i) a “superrosid” clade consisting of Rosidae, Vitaceae, and Saxifragales; (ii) a “superasterid” clade consisting of Berberidopsidales, Santalales, Caryophyllales, and Asteridae; and (iii) Dilleniaceae. Maximum-likelihood analyses support the position of Dilleniaceae as sister to superrosids, but topology tests did not reject alternative positions of Dilleniaceae as sister to Asteridae or all remaining Pentapetalae. Molecular dating analyses suggest that the major lineages within both superrosids and superasterids arose in as little as 5 million years. This phylogenetic hypothesis provides a crucial historical framework for future studies aimed at elucidating the underlying causes of the morphological and species diversity in Pentapetalae.

Although great progress has been made in clarifying deep-level angiosperm relationships, several early nodes in the angiosperm branch of the Tree of Life have proved difficult to resolve. Perhaps the last great question remaining in basal angiosperm phylogeny involves the branching order among the five major clades of mesangiosperms (Ceratophyllum, Chloranthaceae, eudicots, magnoliids, and monocots). Previous analyses have found no consistent support for relationships among these clades. In an effort to resolve these relationships, we performed phylogenetic analyses of 61 plastid genes (≈42,000 bp) for 45 taxa, including members of all major basal angiosperm lineages. We also report the complete plastid genome sequence ofCeratophyllum demersum. Parsimony analyses of combined and partitioned data sets varied in the placement of several taxa, particularlyCeratophyllum, whereas maximum-likelihood (ML) trees were more topologically stable. Total evidence ML analyses recovered a clade of Chloranthaceae + magnoliids as sister to a well supported clade of monocots + (Ceratophyllum+ eudicots). ML bootstrap and Bayesian support values for these relationships were generally high, although approximately unbiased topology tests could not reject several alternative topologies. The extremely short branches separating these five lineages imply a rapid diversification estimated to have occurred between 143.8 ± 4.8 and 140.3 ± 4.8 Mya.

• Premise of the study: Recent analyses employing up to five genes have provided numerous insights into angiosperm phylogeny, but many relationships have remained unresolved or poorly supported. In the hope of improving our understanding of angiosperm phylogeny, we expanded sampling of taxa and genes beyond previous analyses.• Methods: We conducted two primary analyses based on 640 species representing 330 families. The first included 25260 aligned base pairs (bp) from 17 genes (representing all three plant genomes, i.e., nucleus, plastid, and mitochondrion). The second included 19846 aligned bp from 13 genes (representing only the nucleus and plastid).• Key results: Many important questions of deep‐level relationships in the nonmonocot angiosperms have now been resolved with strong support. Amborellaceae, Nymphaeales, and Austrobaileyales are successive sisters to the remaining angiosperms (Mesangiospermae), which are resolved into Chloranthales + Magnoliidae as sister to Monocotyledoneae + [Ceratophyllaceae + Eudicotyledoneae]. Eudicotyledoneae contains a basal grade subtending Gunneridae. Within Gunneridae, Gunnerales are sister to the remainder (Pentapetalae), which comprises (1) Superrosidae, consisting of Rosidae (including Vitaceae) and Saxifragales; and (2) Superasteridae, comprising Berberidopsidales, Santalales, Caryophyllales, Asteridae, and, based on this study, Dilleniaceae (although other recent analyses disagree with this placement). Within the major subclades of Pentapetalae, most deep‐level relationships are resolved with strong support.• Conclusions: Our analyses confirm that with large amounts of sequence data, most deep‐level relationships within the angiosperms can be resolved. We anticipate that this well‐resolved angiosperm tree will be of broad utility for many areas of biology, including physiology, ecology, paleobiology, and genomics.