Genotyping-by-sequencing-based high-resolution mapping reveals a single candidate gene for the grapevine veraison locus Ver1.

Veraison marks the transition from berry growth to berry ripening and is a crucial phenological stage in grapevine (Vitis vinifera): the berries become soft and begin to accumulate sugars, aromatic substances, and, in red cultivars, anthocyanins for pigmentation, while the organic acid levels begin to decrease. These changes determine the potential quality of wine. However, rising global temperatures lead to earlier flowering and ripening, which strongly influence wine quality. Here, we combined genotyping-by-sequencing with a bioinformatics pipeline on ∼150 F1 genotypes derived from a cross between the early ripening variety 'Calardis Musqué' and the late-ripening variety 'Villard Blanc'. Starting from 20,410 haplotype-based markers, we generated a high-density genetic map and performed a quantitative trait locus analysis based on phenotypic datasets evaluated over 20 years. Through locus-specific-marker-enrichment and recombinant screening of ∼1000 additional genotypes, we refined the originally postulated 5 Mb veraison locus, Ver1, on chromosome 16 to only 112 kb, allowing us to pinpoint the ethylene response factor (ERF) VviERF027 (VCost.v3 gene ID: Vitvi16g00942, CRIBIv1 gene ID: VIT_16s0100g00400) as veraison candidate gene. Furthermore, the early veraison allele could be traced back to a clonal 'Pinot' variant first mentioned in the 17th century. 'Pinot Precoce Noir' passed this allele over 'Madeleine Royale' to the maternal grandparent 'Bacchus Weiss' and, ultimately, to the maternal parent 'Calardis Musqué'. Our findings are crucial for ripening time control, thereby improving wine quality, and for breeding grapevines adjusted to climate change scenarios that have a major impact on agro-ecosystems in altering crop plant phenology.

Chemical imitation of yeast fermentation by the drosophilid-pollinated deceptive trap-flower Aristolochia baetica (Aristolochiaceae).

Deceptive flowers, unlike in mutualistic pollination systems, mislead their pollinators by advertising rewards which ultimately are not provided. Although our understanding of deceptive pollination systems increased in recent years, the attractive signals and deceptive strategies in the majority of species remain unknown. This is also true for the genus Aristolochia, famous for its deceptive and fly-pollinated trap flowers. Representatives of this genus were generally assumed to be oviposition-site mimics, imitating vertebrate carrion or mushrooms. However, recent studies found a broader spectrum of strategies, including kleptomyiophily and imitation of invertebrate carrion. A different deceptive strategy is presented here for the western Mediterranean Aristolochia baetica L. We found that this species is mostly pollinated by drosophilid flies (Drosophilidae, mostly Drosophila spp.), which typically feed on fermenting fruit infested by yeasts. The flowers of A. baetica emitted mostly typical yeast volatiles, predominantly the aliphatic compounds acetoin and 2,3-butandiol, and derived acetates, as well as the aromatic compound 2-phenylethanol. Analyses of the absolute configurations of the chiral volatiles revealed weakly (acetoin, 2,3-butanediol) to strongly (mono- and diacetates) biased stereoisomer-ratios. Electrophysiological (GC-EAD) experiments and lab bioassays demonstrated that most of the floral volatiles, although not all stereoisomers of chiral compounds, were physiologically active and attractive in drosophilid pollinators; a synthetic mixture thereof successfully attracted them in field and lab bioassays. We conclude that A. baetica chemically mimics yeast fermentation to deceive its pollinators. This deceptive strategy (scent chemistry, pollinators, trapping function) is also known from more distantly related plants, such as Arum palaestinum Boiss. (Araceae) and Ceropegia spp. (Apocynaceae), suggesting convergent evolution. In contrast to other studies working on floral scents in plants imitating breeding sites, the present study considered the absolute configuration of chiral compounds.

A comparative analysis of plastome evolution in autotrophic Piperales.

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.

The Mitochondrial Genome of the Holoparasitic Plant Thonningia sanguinea Provides Insights into the Evolution of the Multichromosomal Structure.

Multichromosomal mitochondrial genome (mitogenome) structures have repeatedly evolved in many lineages of angiosperms. However, the underlying mechanism remains unclear. The mitogenomes of three genera of Balanophoraceae, namely Lophophytum, Ombrophytum, and Rhopalocnemis, have already been sequenced and assembled, all showing a highly multichromosomal structure, albeit with different genome and chromosome sizes. It is expected that characterization of additional lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of the plant mitogenome structure and size. Here, we assembled and characterized the mitogenome of Thonningia sanguinea, which, together with Balanophora, forms a clade sister to the clade comprising Lophophytum, Ombrophytum, and Rhopalocnemis. The mitogenome of T. sanguinea possesses a multichromosomal structure of 18 circular chromosomes of 8.7-19.2 kb, with a total size of 246,247 bp. There are very limited shared regions and poor chromosomal correspondence between T. sanguinea and other Balanophoraceae species, suggesting frequent rearrangements and rapid sequence turnover. Numerous medium- and small-sized repeats were identified in the T. sanguinea mitogenome; however, no repeat-mediated recombination was detected, which was verified by Illumina reads mapping and PCR experiments. Intraspecific mitogenome variations in T. sanguinea are mostly insertions and deletions, some of which can lead to degradation of perfect repeats in one or two accessions. Based on the mitogenome features of T. sanguinea, we propose a mechanism to explain the evolution of a multichromosomal mitogenome from a master circle, which involves mutation in organellar DNA replication, recombination and repair genes, decrease of recombination, and repeat degradation.

Plastome phylogenomics reveals an early Pliocene North- and Central America colonization by long-distance dispersal from South America of a highly diverse bromeliad lineage.

Understanding the spatial and temporal frameworks of species diversification is fundamental in evolutionary biology. Assessing the geographic origin and dispersal history of highly diverse lineages of rapid diversification can be hindered by the lack of appropriately sampled, resolved, and strongly supported phylogenetic contexts. The use of currently available cost-efficient sequencing strategies allows for the generation of a substantial amount of sequence data for dense taxonomic samplings, which together with well-curated geographic information and biogeographic models allow us to formally test the mode and tempo of dispersal events occurring in quick succession. Here, we assess the spatial and temporal frameworks for the origin and dispersal history of the expanded clade K, a highly diverse Tillandsia subgenus Tillandsia (Bromeliaceae, Poales) lineage hypothesized to have undergone a rapid radiation across the Neotropics. We assembled full plastomes from Hyb-Seq data for a dense taxon sampling of the expanded clade K plus a careful selection of outgroup species and used them to estimate a time- calibrated phylogenetic framework. This dated phylogenetic hypothesis was then used to perform biogeographic model tests and ancestral area reconstructions based on a comprehensive compilation of geographic information. The expanded clade K colonized North and Central America, specifically the Mexican transition zone and the Mesoamerican dominion, by long-distance dispersal from South America at least 4.86 Mya, when most of the Mexican highlands were already formed. Several dispersal events occurred subsequently northward to the southern Nearctic region, eastward to the Caribbean, and southward to the Pacific dominion during the last 2.8 Mya, a period characterized by pronounced climate fluctuations, derived from glacial-interglacial climate oscillations, and substantial volcanic activity, mainly in the Trans-Mexican Volcanic Belt. Our taxon sampling design allowed us to calibrate for the first time several nodes, not only within the expanded clade K focal group but also in other Tillandsioideae lineages. We expect that this dated phylogenetic framework will facilitate future macroevolutionary studies and provide reference age estimates to perform secondary calibrations for other Tillandsioideae lineages.

Phylogenetic and comparative analyses of Hydnora abyssinica plastomes provide evidence for hidden diversity within Hydnoraceae.

BACKGROUND: To date, plastid genomes have been published for all but two holoparasitic angiosperm families. However, only a single or a few plastomes represent most of these families. Of the approximately 40 genera of holoparasitic angiosperms, a complete plastid genome sequence is available for only about half. In addition, less than 15 species are currently represented with more than one published plastid genome, most of which belong to the Orobanchaceae. Therefore, a significant portion of the holoparasitic plant plastome diversity remains unexplored. This limited information could hinder potential evolutionary pattern recognition as well as the exploration of inter- and intra-species plastid genome diversity in the most extreme holoparasitic angiosperms. RESULTS: Here, we report the first plastomes of Kenyan Hydnora abyssinica accessions. The plastomes have a typical quadripartite structure and encode 24 unique genes. Phylogenetic tree reconstruction recovers the Kenyan accessions as monophyletic and together in a clade with the Namibian H. abyssinica accession and the recently published H. arabica from Oman. Hydnora abyssinica as a whole however is recovered as non-monophyletic, with H. arabica nested within. This result is supported by distinct structural plastome synapomorphies as well as pairwise distance estimates that reveal hidden diversity within the Hydnora species in Africa. CONCLUSION: We propose to increase efforts to sample widespread holoparasitic species for their plastid genomes, as is the case with H. abyssinica, which is widely distributed in Africa. Morphological reinvestigation and further molecular data are needed to fully investigate the diversity of H. abyssinica along the entire range of distribution, as well as the diversity of currently synonymized taxa.

Extreme plastomes in holoparasitic Balanophoraceae are not the norm.

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.

De novo Assembly and Comparative Analyses of Mitochondrial Genomes in Piperales.

The mitochondrial genome of Liriodendron tulipifera exhibits many ancestral angiosperm features and a remarkably slow evolutionary rate, while mitochondrial genomes of other magnoliids remain yet to be characterized. We assembled nine new mitochondrial genomes, representing all genera of perianth-bearing Piperales, as well as for a member of the sister clade: three complete or nearly complete mitochondrial genomes from Aristolochiaceae and six additional draft assemblies including Thottea, Asaraceae, Lactoridaceae, and Hydnoraceae. For comparative purpose, a complete mitochondrial genome was assembled for Saururus, a member of the perianth-less Piperales. The average number of short repeats (50-99 bp) was much larger in genus Aristolochia than in other angiosperm mitochondrial genomes, and approximately 30% of repeats (<350 bp) were found to have the capacity to mediate recombination. We found mitochondrial genomes in perianth-bearing Piperales comprising conserved repertories of protein-coding genes and rRNAs but variable copy numbers of tRNA genes. We identified several shifts from cis- to trans-splicing of the Group II introns of nad1i728, cox2i373, and nad7i209. Two short regions of the cox1 and atp8 genes were likely derived from independent horizontal gene transfer events in perianth-bearing Piperales. We found biased enrichment of specific substitution types in different lineages of magnoliids and the Aristolochiaceae family showed the highest ratio of A:T > T:A substitutions of all other investigated angiosperm groups. Our study reports the first mitochondrial genomes for Piperales and uses this new information for a better understanding of the evolutionary patterns of magnoliids and angiosperms in general.

The use of Anchored Hybrid Enrichment data to resolve higher-level phylogenetic relationships: A proof-of-concept applied to Asterales (Eudicotyledoneae; Angiosperms).

Anchored Hybrid Enrichment (AHE) is a tool for capturing orthologous regions of the nuclear genome shared in low or single copy across lineages. Despite the increasing number of studies using this method, its usefulness to estimate relationships at deeper taxonomic levels in plants has not been fully explored. Here we present a proof of concept about the performance of nuclear loci obtained with AHE to infer phylogenetic relationships and explore the use of gene sampling schemes to estimate divergence times in Asterales. We recovered low-copy nuclear loci using the AHE method from herbarium material and silica-preserved samples. Maximum likelihood, Bayesian inference, and coalescence approaches were used to reconstruct phylogenomic relationships. Dating analyses were conducted under a multispecies coalescent approach by jointly inferring species tree and divergence times with random gene sampling schemes and multiple calibrations. We recovered 403 low-copy nuclear loci for 63 species representing nine out of eleven families of Asterales. Phylogenetic hypotheses were congruent among the applied methods and previously published results. Analyses with concatenated datasets were strongly supported, but coalescence-based analyses showed low support for the phylogenetic position of families Argophyllaceae and Alseuosmiaceae. Estimated family ages were congruent among gene sampling schemes, with the mean age for Asterales around 130 Myr. Our study documents the usefulness of AHE for resolving phylogenetic relationships at deep phylogenetic levels in Asterales. Observed phylogenetic inconsistencies were possibly due to the non-inclusion of families Phellinceae and Pentaphragmataceae. Random gene sampling schemes produced consistent age estimates with coalescence and species tree relaxed clock approaches.

New plastome structural rearrangements discovered in core Tillandsioideae (Bromeliaceae) support recently adopted taxonomy.

Full plastome sequences for land plants have become readily accessible thanks to the development of Next Generation Sequencing (NGS) techniques and powerful bioinformatic tools. Despite this vast amount of genomic data, some lineages remain understudied. Full plastome sequences from the highly diverse (>1,500 spp.) subfamily Tillandsioideae (Bromeliaceae, Poales) have been published for only three (i.e., Guzmania, Tillandsia, and Vriesea) out of 22 currently recognized genera. Here, we focus on core Tillandsioideae, a clade within subfamily Tillandsioideae, and explore the contribution of individual plastid markers and data categories to inform deep divergences of a plastome phylogeny. We generated 37 high quality plastome assemblies and performed a comparative analysis in terms of plastome structure, size, gene content and order, GC content, as well as number and type of repeat motifs. Using the obtained phylogenetic context, we reconstructed the evolution of these plastome attributes and assessed if significant shifts on the evolutionary traits' rates have occurred in the evolution of the core Tillandsioideae. Our results agree with previously published phylogenetic hypotheses based on plastid data, providing stronger statistical support for some recalcitrant nodes. However, phylogenetic discordance with previously published nuclear marker-based hypotheses was found. Several plastid markers that have been consistently used to address phylogenetic relationships within Tillandsioideae were highly informative for the retrieved plastome phylogeny and further loci are here identified as promising additional markers for future studies. New lineage-specific plastome rearrangements were found to support recently adopted taxonomic groups, including large inversions, as well as expansions and contractions of the inverted repeats. Evolutionary trait rate shifts associated with changes in size and GC content of the plastome regions were found across the phylogeny of core Tillandsioideae.

Structural Plastome Evolution in Holoparasitic Hydnoraceae with Special Focus on Inverted and Direct Repeats.

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).

The evolution of the Aristolochia pallida complex (Aristolochiaceae) challenges traditional taxonomy and reflects large-scale glacial refugia in the Mediterranean.

The taxonomy of the Mediterranean Aristolochia pallida complex has been under debate since several decades with the following species currently recognized: A. pallida, A. lutea, A. nardiana, A. microstoma, A. merxmuelleri, A. croatica, and A. castellana. These taxa are distributed from Iberia to Turkey. To reconstruct phylogenetic and biogeographic patterns, we employed cpDNA sequence variation using both noncoding (intron and spacer) and protein-coding regions (i.e., trnK intron, matK gene, and trnK-psbA spacer). Our results show that the morphology-based traditional taxonomy was not corroborated by our phylogenetic analyses. Aristolochia pallida, A. lutea, A. nardiana, and A. microstoma were not monophyletic. Instead, strong geographic signals were detected. Two major clades, one exclusively occurring in Greece and a second one of pan-Mediterranean distribution, were found. Several subclades distributed in Greece, NW Turkey, Italy, as well as amphi-Adriatic subclades, and a subgroup of southern France and Spain, were revealed. The distribution areas of these groups are in close vicinity to hypothesized glacial refugia areas in the Mediterranean. According to molecular clock analyses the diversification of this complex started around 3-3.3 my, before the onset of glaciation cycles, and the further evolution of and within major lineages falls into the Pleistocene. Based on these data, we conclude that the Aristolochia pallida alliance survived in different Mediterranean refugia rarely with low, but often with a high potential for range extension, and a high degree of morphological diversity.

Population genetics and plant growth experiments as prerequisite for conservation measures of the rare European aquatic plant Luronium natans (Alismataceae).

Information provided by population genetic studies is often necessary to effectively protect endangered species. In general, such data is scarce for aquatic plants and this holds also for Luronium natans, an aquatic macrophyte endemic to northwestern and western Europe. It is threatened across its whole distribution range due to human influences, in particular due to eutrophication and intensive fish farming. In spite of habitat protection populations continue to decline and re-introductions are one possibility to prevent the species' extinction. Therefore, insights in genetic diversity and relatedness of source populations is warranted. Thus, we performed Amplified Fragment-Length Polymorphism (AFLP) on two large populations in Saxony, Germany (Großenhainer Pflege and Niederspree), complemented with numerous additional occurrences from Europe. In addition, we conducted experiments on plant growth to assess optimal conditions for ex-situ cultivation taking water temperature, water level and substrate into account. We revealed considerably high levels of genetic diversity within populations (Shannon Indices ranged from 0.367 to 0.416) implying that populations are not restricted to clonal growth only but reproduce also by open-pollinated flowers. Remarkably, the two geographically close Saxon populations were genetically distant to each other but subpopulations within a locality were completely intermingled. Concerning optimal cultivation conditions, longest roots were obtained at temperatures >14°C and saturated, but not submerging water levels. Thus, our findings advocate for a re-introduction scheme from nearby source populations and provide detailed information on successful ex-situ cultivation.

Insights into angiosperm evolution, floral development and chemical biosynthesis from the Aristolochia fimbriata genome.

Aristolochia, a genus in the magnoliid order Piperales, has been famous for centuries for its highly specialized flowers and wide medicinal applications. Here, we present a new, high-quality genome sequence of Aristolochia fimbriata, a species that, similar to Amborella trichopoda, lacks further whole-genome duplications since the origin of extant angiosperms. As such, the A. fimbriata genome is an excellent reference for inferences of angiosperm genome evolution, enabling detection of two novel whole-genome duplications in Piperales and dating of previously reported whole-genome duplications in other magnoliids. Genomic comparisons between A. fimbriata and other angiosperms facilitated the identification of ancient genomic rearrangements suggesting the placement of magnoliids as sister to monocots, whereas phylogenetic inferences based on sequence data we compiled yielded ambiguous relationships. By identifying associated homologues and investigating their evolutionary histories and expression patterns, we revealed highly conserved floral developmental genes and their distinct downstream regulatory network that may contribute to the complex flower morphology in A. fimbriata. Finally, we elucidated the genetic basis underlying the biosynthesis of terpenoids and aristolochic acids in A. fimbriata.

Molecular Phylogeny, Character Evolution, and Biogeography of Hydrangea Section Cornidia, Hydrangeaceae.

Background: Hydrangea section Cornidia consists of 26 currently accepted species and a yet undefined number of new species and erroneously synonymized taxa. This clade consists of (sub)tropical lianas occurring from northern Mexico to southern Chile and Argentina, and one species from Southeast Asia. Currently, no molecular phylogenetic hypothesis is available that includes more than a few species of this section. Hence, a resolved and well-sampled molecular phylogenetic hypothesis may help to enforce taxonomic decisions. In this study, we present a phylogenetic framework based on sequences from two low copy nuclear genes from a comprehensive taxon sampling of H. section Cornidia and a selection of outgroups. Our phylogenetic reconstructions prove the non-monophyly of the traditionally recognized subsections Monosegia and Polysegia and their corresponding series, Speciosae and Aphananthae, and Synstyleae and Chorystyleae, respectively. Three morphologically defined species were recovered with high support as monophyletic, namely, Hydrangea panamensis, Hydrangea serratifolia, and Hydrangea tarapotensis. However, statistical support for some shallow nodes did not allow to refute, with high support, the monophyly of several of the herein recognized species for which more than one individual could be analyzed. Based on the obtained phylogenetic framework, we reconstructed the evolution of selected reproductive characters. Hydrangea section Cornidia is the only genus section for which dioecism has been extensively documented. Our character reconstruction of sexual dimorphism shows that dioecism is the ancestral state in this section and that this was reversed to monoecy in Hydrangea seemannii and Hydrangea integrifolia. Character reconstruction for the enlarged marginal flowers recovered their presence as the ancestral character state in H. section Cornidia, although at least three internal lineages independently lost them; thus, losses were reconstructed to be more likely than gain. With respect to the flower color, more species exhibit white than red flowers, and white is reconstructed as the ancestral state. Cornidia also shows an unusual disjunct geographic distribution between Asia and Central Mesoamerica-South America, as it is not present in the USA and Canada. The origin of Cornidia is reconstructed to be the New World with higher probability, and the presence of one species in Asia is likely due to long-distance dispersal.

Discordant Phylogenomic Placement of Hydnoraceae and Lactoridaceae Within Piperales Using Data From All Three Genomes.

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.

Genome-wide macroevolutionary signatures of key innovations in butterflies colonizing new host plants.

The mega-diversity of herbivorous insects is attributed to their co-evolutionary associations with plants. Despite abundant studies on insect-plant interactions, we do not know whether host-plant shifts have impacted both genomic adaptation and species diversification over geological times. We show that the antagonistic insect-plant interaction between swallowtail butterflies and the highly toxic birthworts began 55 million years ago in Beringia, followed by several major ancient host-plant shifts. This evolutionary framework provides a valuable opportunity for repeated tests of genomic signatures of macroevolutionary changes and estimation of diversification rates across their phylogeny. We find that host-plant shifts in butterflies are associated with both genome-wide adaptive molecular evolution (more genes under positive selection) and repeated bursts of speciation rates, contributing to an increase in global diversification through time. Our study links ecological changes, genome-wide adaptations and macroevolutionary consequences, lending support to the importance of ecological interactions as evolutionary drivers over long time periods.

Evolution of Class II TCP genes in perianth bearing Piperales and their contribution to the bilateral calyx in Aristolochia.

Controlled spatiotemporal cell division and expansion are responsible for floral bilateral symmetry. Genetic studies have pointed to class II TCP genes as major regulators of cell division and floral patterning in model core eudicots. Here we study their evolution in perianth-bearing Piperales and their expression in Aristolochia, a rare occurrence of bilateral perianth outside eudicots and monocots. The evolution of class II TCP genes reveals single-copy CYCLOIDEA-like genes and three paralogs of CINCINNATA (CIN) in early diverging angiosperms. All class II TCP genes have independently duplicated in Aristolochia subgenus Siphisia. Also CIN2 genes duplicated before the diversification of Saruma and Asarum. Sequence analysis shows that CIN1 and CIN3 share motifs with Cyclin proteins and CIN2 genes have lost the miRNA319a binding site. Expression analyses of all paralogs of class II TCP genes in Aristolochia fimbriata point to a role of CYC and CIN genes in maintaining differential perianth expansion during mid- and late flower developmental stages by promoting cell division in the distal and ventral portion of the limb. It is likely that class II TCP genes also contribute to cell division in the leaf, the gynoecium and the ovules in A. fimbriata.

The First Plastid Genome of the Holoparasitic Genus Prosopanche (Hydnoraceae).

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.