Phylogenetic relationships within the magnoliid order Piperales have been studied extensively, yet the relationships of the monotypic family Lactoridaceae and the holoparasitic Hydnoraceae to the remainder of the order remain a matter of debate. Since the first confident molecular phylogenetic placement of Hydnoraceae among Piperales, different studies have recovered various contradictory topologies. Most phylogenetic hypotheses were inferred using only a few loci and have had incomplete taxon sampling at the genus level. Based on these results and an online survey of taxonomic opinion, the Angiosperm Phylogeny Group lumped both Hydnoraceae and Lactoridaceae in Aristolochiaceae; however, the latter family continues to have unclear relationships to the aforementioned taxa. Here we present extensive phylogenomic tree reconstructions based on up to 137 loci from all three subcellular genomes for all genera of Piperales. We infer relationships based on a variety of phylogenetic methods, explore instances of phylogenomic discordance between the subcellular genomes, and test alternative topologies. Consistent with these phylogenomic results and a consideration of the principles of phylogenetic classification, we propose to exclude Hydnoraceae and Lactoridaceae from the broad circumscription of Aristolochiaceae, and instead favor recognition of four monophyletic and morphologically well circumscribed families in the perianth-bearing Piperales: Aristolochiaceae, Asaraceae, Hydnoraceae, and Lactoridaceae, with a total of six families in the order.

Plastome condensation during adaptation to a heterotrophic lifestyle is generally well understood and lineage-independent models have been derived. However, understanding the evolutionary trajectories of comparatively old heterotrophic lineages that are on the cusp of a minimal plastome, is essential to complement and expand current knowledge. We study Hydnoraceae, one of the oldest and least investigated parasitic angiosperm lineages. Plastome comparative genomics, using seven out of eight known species of the genus Hydnora and three species of Prosopanche, reveal a high degree of structural similarity and shared gene content; contrasted by striking dissimilarities with respect to repeat content [inverted and direct repeats (DRs)]. We identified varying inverted repeat contents and positions, likely resulting from multiple, independent evolutionary events, and a DR gain in Prosopanche. Considering different evolutionary trajectories and based on a fully resolved and supported species-level phylogenetic hypothesis, we describe three possible, distinct models to explain the Hydnoraceae plastome states. For comparative purposes, we also report the first plastid genomes for the closely related autotrophic genera Lactoris (Lactoridaceae) and Thottea (Aristolochiaceae).

Background: Balanophoraceae plastomes are known for their highly condensed and re-arranged nature alongside the most extreme nucleotide compositional bias known to date, culminating in two independent reconfigurations of their genetic code. Currently, a large portion of the Balanophoraceae diversity remains unexplored, hindering, among others, evolutionary pattern recognition. Here, we explored newly sequenced plastomes of Sarcophyte sanguinea and Thonningia sanguinea. The reconstructed plastomes were analyzed using various methods of comparative genomics based on a representative taxon sampling. Results: Sarcophyte, recovered sister to the other sampled Balanophoraceae s. str., has plastomes up to 50% larger than those currently published. Its gene set contains five genes lost in any other species, including matK. Five cis-spliced introns are maintained. In contrast, the Thonningia plastome is similarly reduced to published Balanophoraceae and retains only a single cis-spliced intron. Its protein-coding genes show a more biased codon usage compared to Sarcophyte, with an accumulation of in-frame TAG stop codons. Structural plastome comparison revealed multiple, previously unknown, structural rearrangements within Balanophoraceae. Conclusions: For the “minimal plastomes” of Thonningia, we propose a genetic code change identical to sister genus Balanophora. Sarcophyte however differs drastically from our current understanding on Balanophoraceae plastomes. With a less-extreme nucleotide composition, there is no evidence for an altered genetic code. Using comparative genomics, we identified a hotspot for plastome reconfiguration in Balanophoraceae. Based on previously published and newly identified structural reconfigurations, we propose an updated model of evolutionary plastome trajectories for Balanophoraceae, illustrating a much greater plastome diversity than previously known.

Plastomes of parasitic and mycoheterotrophic plants show different degrees of reduction depending on the plants’ level of heterotrophy and host dependence in comparison to photoautotrophic sister species, and the amount of time since heterotrophic dependence was established. In all but the most recent heterotrophic lineages, this reduction involves substantial decrease in genome size and gene content and sometimes alterations of genome structure. Here, we present the first plastid genome of the holoparasitic genus Prosopanche, which shows clear signs of functionality. The plastome of Prosopanche americana has a length of 28,191 bp and contains only 24 unique genes, i.e., 14 ribosomal protein genes, four ribosomal RNA genes, five genes coding for tRNAs and three genes with other or unknown function (accD, ycf1, ycf2). The inverted repeat has been lost. Despite the split of Prosopanche and Hydnora about 54 MYA ago, the level of genome reduction is strikingly congruent between the two holoparasites although highly dissimilar nucleotide sequences are observed. Our results lead to two possible evolutionary scenarios that will be tested in the future with a larger sampling: 1) a Hydnoraceae plastome, similar to those of Hydnora and Prosopanche today, existed already in the most recent common ancestor and has not changed much with respect to gene content and structure, or 2) the genome similarities we observe today are the result of two independent evolutionary trajectories leading to almost the same endpoint. The first hypothesis would be most parsimonious whereas the second would point to taxon dependent essential gene sets for plants released from photosynthetic constraints.

Premise: Many plastomes of autotrophic Piperales have been reported to date, describing a variety of differences. Most studies focused only on a few species or a single genus, and extensive, comparative analyses have not been done. Here, we reviewed publicly available plastome reconstructions for autotrophic Piperales, reanalyzed publicly available raw data, and provided new sequence data for all previously missing genera. Comparative plastome genomics of >100 autotrophic Piperales were performed.Methods: We performed de novo assemblies to reconstruct the plastomes of newly generated sequence data. We used Sanger sequencing and read mapping to verify the assemblies and to bridge assembly gaps. Furthermore, we reconstructed the phylogenetic relationships as a foundation for comparative plastome genomics.Results: We identified a plethora of assembly and annotation issues in published plastome data, which, if unattended, will lead to an artificial increase of diversity. We were able to detect patterns of missing and incorrect feature annotation and determined that the inverted repeat (IR) boundaries were the major source for erroneous assembly. Accounting for the aforementioned issues, we discovered relatively stable junctions of the IRs and the small single‐copy region (SSC), whereas the majority of plastome variations among Piperales stems from fluctuations of the boundaries of the IR and the large single‐copy (LSC) region.Conclusions: This study of all available plastomes of autotrophic Piperales, expanded by new data for previously missing genera, highlights the IR‐LSC junctions as a potential marker for discrimination of various taxonomic levels. Our data indicates a pseudogene‐like status for cemA and ycf15 in various Piperales. Based on a review of published data, we conclude that incorrect IR‐SSC boundary identification is the major source for erroneous plastome assembly. We propose a gold standard for assembly and annotation of high‐quality plastomes based on de novo assembly methods and appropriate references for gene annotation.