Genome-Wide Identification and Characterization of miRNAs and Natural Antisense Transcripts Show the Complexity of Gene Regulatory Networks for Secondary Metabolism in Aristolochia contorta.

Aristolochia contorta Bunge is an academically and medicinally important plant species. It belongs to the magnoliids, with an uncertain phylogenetic position, and is one of the few plant species lacking a whole-genome duplication (WGD) event after the angiosperm-wide WGD. A. contorta has been an important traditional Chinese medicine material. Since it contains aristolochic acids (AAs), chemical compounds with nephrotoxity and carcinogenicity, the utilization of this plant has attracted widespread attention. Great efforts are being made to increase its bioactive compounds and reduce or completely remove toxic compounds. MicroRNAs (miRNAs) and natural antisense transcripts (NATs) are two classes of regulators potentially involved in metabolism regulation. Here, we report the identification and characterization of 223 miRNAs and 363 miRNA targets. The identified miRNAs include 51 known miRNAs belonging to 20 families and 172 novel miRNAs belonging to 107 families. A negative correlation between the expression of miRNAs and their targets was observed. In addition, we identified 441 A. contorta NATs and 560 NAT-sense transcript (ST) pairs, of which 12 NATs were targets of 13 miRNAs, forming 18 miRNA-NAT-ST modules. Various miRNAs and NATs potentially regulated secondary metabolism through the modes of miRNA-target gene-enzyme genes, NAT-STs, and NAT-miRNA-target gene-enzyme genes, suggesting the complexity of gene regulatory networks in A. contorta. The results lay a solid foundation for further manipulating the production of its bioactive and toxic compounds.

A draft reference genome assembly of California Pipevine, Aristolochia californica Torr.

The California Pipevine, Aristolochia californica Torr., is the only endemic California species within the cosmopolitan birthwort family Aristolochiaceae. It occurs as an understory vine in riparian and chaparral areas and in forest edges and windrows. The geographic range of this plant species almost entirely overlaps with that of its major specialized herbivore, the California Pipevine Swallowtail Butterfly Battus philenor hirsuta. While this species pair is a useful, ecologically well-understood system to study co-evolution, until recently, genomic resources for both have been lacking. Here, we report a new, chromosome-level assembly of A. californica as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin proximity sequencing technology to produce a de novo assembled genome. Our genome assembly, the first for any species in the genus, contains 531 scaffolds spanning 661 megabase (Mb) pairs, with a contig N50 of 6.53 Mb, a scaffold N50 of 42.2 Mb, and BUSCO complete score of 98%. In combination with the recently published B. philenor hirsuta reference genome assembly, the A. californica reference genome assembly will be a powerful tool for studying co-evolution in a rapidly changing California landscape.

Two Novel Betarhabdovirins Infecting Ornamental Plants and the Peculiar Intracellular Behavior of the Cytorhabdovirus in the Liana Aristolochia gibertii.

Two novel members of the subfamily Betarhabdovirinae, family Rhabdoviridae, were identified in Brazil. Overall, their genomes have the typical organization 3'-N-P-P3-M-G-L-5' observed in mono-segmented plant-infecting rhabdoviruses. In aristolochia-associated cytorhabdovirus (AaCV), found in the liana aristolochia (Aristolochia gibertii Hook), an additional short orphan ORF encoding a transmembrane helix was detected between P3 and M. The AaCV genome and inferred encoded proteins share the highest identity values, consistently < 60%, with their counterparts of the yerba mate chlorosis-associated virus (Cytorhabdovirus flaviyerbamate). The second virus, false jalap virus (FaJV), was detected in the herbaceous plant false jalap (Mirabilis jalapa L.) and represents together with tomato betanucleorhabdovirus 2, originally found in tomato plants in Slovenia, a tentative new species of the genus Betanucleorhabdovirus. FaJV particles accumulate in the perinuclear space, and electron-lucent viroplasms were observed in the nuclei of the infected cells. Notably, distinct from typical rhabdoviruses, most virions of AaCV were observed to be non-enclosed within membrane-bounded cavities. Instead, they were frequently seen in close association with surfaces of mitochondria or peroxisomes. Unlike FaJV, AaCV was successfully graft-transmitted to healthy plants of three species of the genus Aristolochia, while mechanical and seed transmission proved unsuccessful for both viruses. Data suggest that these viruses belong to two new tentative species within the subfamily Betarhabdovirinae.

Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta.

Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.

The complete chloroplast genome of Aristolochia fangchi provided insights into the phylogeny and species identification of Aristolochia.

Aristolochia fangchi is an important species within the family Aristolochiaceae, most of which contain nephrotoxic aristolochic acid. The inadvertent use of Aristolochiaceae plants as raw ingredients in the manufacturing of patent medicine poses a significant risk warranting considerable attention. In this study, we assembled and analyzed the complete chloroplast genome of Aristolochia fangchi, which is a 159 867 bp long circular molecule. Functional annotation of the A. fangchi plastome unveiled a total of 113 genes, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Subsequently, a series of genome structure and characteristic evaluations were conducted against the A. fangchi plastome. Further phylogenetic analysis suggested that a plausible phylogenetic relationship among Aristolochiaceae derived from the concatenated sequences of shared conserved genes rather than from the entire chloroplast genome with one IR copy. Finally, a DNA polymorphism assessment against a dozen Aristolochia plastomes yielded multiple potential regions for biomarker designation. Six pairs of primers were generated and underwent both in silico and actual PCR validations. In conclusion, this study identified the unique characteristics of the A. fangchi plastome, providing invaluable insights for further investigations on species identification and the phylogeny evolution between A. fangchi and its related species.

Comparative morphoanatomy and transcriptomic analyses reveal key factors controlling floral trichome development in Aristolochia (Aristolochiaceae).

Trichomes are specialized epidermal cells in aerial plant parts. Trichome development proceeds in three stages, determination of cell fate, specification, and morphogenesis. Most genes responsible for these processes have been identified in the unicellular branched leaf trichomes from the model Arabidopsis thaliana. Less is known about the molecular basis of multicellular trichome formation across flowering plants, especially those formed in floral organs of early diverging angiosperms. Here, we aim to identify the genetic regulatory network (GRN) underlying multicellular trichome development in the kettle-shaped trap flowers of Aristolochia (Aristolochiaceae). We selected two taxa for comparison, A. fimbriata, with trichomes inside the perianth, which play critical roles in pollination, and A. macrophylla, lacking specialized trichomes in the perianth. A detailed morphoanatomical characterization of floral epidermis is presented for the two species. We compared transcriptomic profiling at two different developmental stages in the different perianth portions (limb, tube, and utricle) of the two species. Moreover, we present a comprehensive expression map for positive regulators and repressors of trichome development, as well as cell cycle regulators. Our data point to extensive modifications in gene composition, expression, and putative roles in all functional categories when compared with model species. We also record novel differentially expressed genes (DEGs) linked to epidermis patterning and trichome development. We thus propose the first hypothetical genetic regulatory network (GRN) underlying floral multicellular trichome development in Aristolochia, and pinpoint key factors responsible for the presence and specialization of floral trichomes in phylogenetically distant species of the genus.

A draft reference genome assembly of the Pipevine Swallowtail butterfly, Battus philenor hirsuta.

The California Pipevine Swallowtail Butterfly, Battus philenor hirsuta, and its host plant, the California Pipevine or Dutchman's Pipe, Aristolochia californica Torr., are an important California endemic species pair. While this species pair is an ideal system to study co-evolution, genomic resources for both are lacking. Here, we report a new, chromosome-level assembly of B. philenor hirsuta as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin proximity sequencing technology to produce a de novo assembled genome. Our genome assembly, the first for any species in the genus, contains 109 scaffolds spanning 443 mega base (Mb) pairs, with a contig N50 of 14.6 Mb, a scaffold N50 of 15.2 Mb, and BUSCO complete score of 98.9%. In combination with the forthcoming A. californica reference genome, the B. philenor hirsuta genome will be a powerful tool for documenting landscape genomic diversity and plant-insect co-evolution in a rapidly changing California landscape.

Characterization of two putative norlaudanosoline methyltransferases from Aristolochia debilis.

In view of the nephrotoxicity, hepatotoxicity, and carcinogenicity of aristolochic acids (AAs), the removal of AAs from plants becomes an urgent priority for ensuring the safety of Aristolochia herbal materials. In this study, based on the root-predominant distribution of aristolochic acid I (AAI) in Aristolochia debilis, transcriptome sequencing, in combination with phylogenetic analyses, and gene expression pattern analysis together provided five candidate genes for investigating AAI biosynthesis. Comprehensive in vitro and in vivo enzymatic assays revealed that Ab6OMT1 (6-O-methyltransferase) and AbNMT1 (N-methyltransferase) exhibit promiscuity in substrate recognition, and they could act in a cooperative fashion to achieve conversion of norlaudanosoline, a predicted intermediate in AAI biosynthetic route, into 3'-hydroxy-N-methylcoclaurine through two different methylation reaction sequences. These results shed light on the molecular basis for AAI biosynthesis in Aristolochia herbs. More importantly, Ab6OMT1 and AbNMT1 may be employed as targets for the metabolic engineering of AAI biosynthesis to produce AAs-free Aristolochia herbal materials.

Genetic diversity assessment and biotechnological aspects in Aristolochia spp.

Aristolochia, belonging to the family Aristolochiaceae, has immense ecological significance due to its large size and huge geographic distribution. In the context of dealing with a genus with a huge number of species like Aristolochia, these markers come in handy to precisely identify a particular species and enumerate the genetic diversity. Also, certain species of Aristolochia are economically important due to the presence of secondary metabolites and vast use in traditional and modern medicine. But, the presence of profitable biochemical constituents in Aristolochia is very low and the breeding process of the plant is highly dependable on pollinators. Hence, identifying different biotechnological approaches to fasten the reproductive cycle of Aristolochia and increase the secondary metabolites is of great interest to the researchers. In this study, a comprehensive review has been established on different types of morphological/anatomical markers (starch grains with "Maltese cross"), phytochemical markers (aristolochic acid, triterpenoid, aristolactam etc.) and genetic markers (ISSR, SSR, DNA bar-coding) for various Aristolochia spp. We have also discussed the applications of different biotechnological tools in Aristolochia spp. which include discrete approaches to promote in vitro germination, in vitro shooting, root induction, somatic embryogenesis, synthetic seed production, acclimatization and hardening and sustainable production of secondary metabolites. In a nutshell, the present review is a first of kind approach to comprehensively demonstrate the genetic diversity studies and biotechnological aspects in Aristolochia spp. KEY POINTS: • Insights into the in vitro propagation of Aristolochia spp. • In vitro production and optimization of secondary metabolites. • Assessment of genetic diversity by molecular markers.

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.

Gene expression underlying floral epidermal specialization in Aristolochia fimbriata (Aristolochiaceae).

BACKGROUND AND AIMS: The epidermis constitutes the outermost tissue of the plant body. Although it plays major structural, physiological and ecological roles in embryophytes, the molecular mechanisms controlling epidermal cell fate, differentiation and trichome development have been scarcely studied across angiosperms, and remain almost unexplored in floral organs. METHODS: In this study, we assess the spatio-temporal expression patterns of GL2, GL3, TTG1, TRY, MYB5, MYB6, HDG2, MYB106-like, WIN1 and RAV1-like homologues in the magnoliid Aristolochia fimbriata (Aristolochiaceae) by using comparative RNA-sequencing and in situ hybridization assays. KEY RESULTS: Genes involved in Aristolochia fimbriata trichome development vary depending on the organ where they are formed. Stem, leaf and pedicel trichomes recruit most of the transcription factors (TFs) described above. Conversely, floral trichomes only use a small subset of genes including AfimGL2, AfimRAV1-like, AfimWIN1, AfimMYB106-like and AfimHDG2. The remaining TFs, AfimTTG1, AfimGL3, AfimTRY, AfimMYB5 and AfimMYB6, are restricted to the abaxial (outer) and the adaxial (inner) pavement epidermal cells. CONCLUSIONS: We re-evaluate the core genetic network shaping trichome fate in flowers of an early-divergent angiosperm lineage and show a morphologically diverse output with a simpler genetic mechanism in place when compared to the models Arabidopsis thaliana and Cucumis sativus. In turn, our results strongly suggest that the canonical trichome gene expression appears to be more conserved in vegetative than in floral tissues across angiosperms.

Chloroplast genome features of an important medicinal and edible plant: Houttuynia cordata (Saururaceae).

Houttuynia cordata (Saururaceae), an ancient and relic species, has been used as an important medicinal and edible plant in most parts of Asia. However, because of the lack of genome information and reliable molecular markers, studies on its population structure, or phylogenetic relationships with other related species are still rare. Here, we de novo assembled the complete chloroplast (cp) genome of H. cordata using the integration of the long PacBio and short Illumina reads. The cp genome of H. cordata showed a typical quadripartite cycle of 160,226 bp. This included a pair of inverted repeats (IRa and IRb) of 26,853 bp, separated by a large single-copy (LSC) region of 88,180 bp and a small single-copy (SSC) region of 18,340 bp. A total of 112 unique genes, including 79 protein-coding genes, 29 tRNA genes, and four rRNA genes, were identified in this cp genome. Eighty-one genes were located on the LSC region, 13 genes were located on the SSC region, and 17 two-copy genes were located on the IR region. Additionally, 48 repeat sequences and 86 SSR loci, which can be used as genomic markers for population structure analysis, were also detected. Phylogenetic analysis using 21 cp genomes of the Piperales family demonstrated that H. cordata had a close relationship with the species within the Aristolochia genus. Moreover, the results of mVISTA analysis and comparisons of IR regions demonstrated that the cp genome of H. cordata was conserved with that of the Aristolochia species. Our results provide valuable information for analyzing the genetic diversity and population structure of H. cordata, which can contribute to further its genetic improvement and breeding.

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.

Complete Chloroplast Genomes and Comparative Analysis of Sequences Evolution among Seven Aristolochia (Aristolochiaceae) Medicinal Species.

Aristolochiaceae, comprising about 600 species, is a unique plant family containing aristolochic acids (AAs). In this study, we sequenced seven species of Aristolochia, and retrieved eleven chloroplast (cp) genomes published for comparative genomics analysis and phylogenetic constructions. The results show that the cp genomes had a typical quadripartite structure with conserved genome arrangement and moderate divergence. The cp genomes range from 159,308 bp to 160,520 bp in length and have a similar GC content of 38.5%⁻38.9%. A total number of 113 genes were identified, including 79 protein-coding genes, 30 tRNAs and four rRNAs. Although genomic structure and size were highly conserved, the IR-SC boundary regions were variable between these seven cp genomes. The trnH-GUG genes, are one of major differences between the plastomes of the two subgenera Siphisia and Aristolochia. We analyzed the features of nucleotide substitutions, distribution of repeat sequences and simple sequences repeats (SSRs), positive selections in the cp genomes, and identified 16 hotspot regions for genomes divergence that could be utilized as potential markers for phylogeny reconstruction. Phylogenetic relationships of the family Aristolochiaceae inferred from the 18 cp genome sequences were consistent and robust, using maximum parsimony (MP), maximum likelihood (ML), and Bayesian analysis (BI) methods.

Evolution of RADIALIS and DIVARICATA gene lineages in flowering plants with an expanded sampling in non-core eudicots.

PREMISE OF THE STUDY: Bilateral symmetry in core eudicot flowers is established by the differential expression of CYCLOIDEA (CYC), DICHOTOMA (DICH), and RADIALIS (RAD), which are restricted to the dorsal portion of the flower, and DIVARICATA (DIV), restricted to the ventral and lateral petals. Little is known regarding the evolution of these gene lineages in non-core eudicots, and there are no reports on gene expression that can be used to assess whether the network predates the diversification of core eudicots. METHODS: Homologs of the RAD and DIV lineages were isolated from available genomes and transcriptomes, including those of three selected non-core eudicot species, the magnoliid Aristolochia fimbriata and the monocots Cattleya trianae and Hypoxis decumbens. Phylogenetic analyses for each gene lineage were performed. RT-PCR was used to evaluate the expression and putative contribution to floral symmetry in dissected floral organs of the selected species. KEY RESULTS: RAD-like genes have undergone at least two duplication events before eudicot diversification, three before monocots and at least four in Orchidaceae. DIV-like genes also duplicated twice before eudicot diversification and underwent independent duplications specific to Orchidaceae. RAD-like and DIV-like genes have differential dorsiventral expression only in C. trianae, which contrasts with the homogeneous expression in the perianth of A. fimbriata. CONCLUSIONS: Our results point to a common genetic regulatory network for floral symmetry in monocots and core eudicots, while alternative genetic mechanisms are likely driving the bilateral perianth symmetry in the early-diverging angiosperm Aristolochia.

Floral MADS-box protein interactions in the early diverging angiosperm Aristolochia fimbriata Cham. (Aristolochiaceae: Piperales).

Floral identity MADS-box A, B, C, D, E, and AGL6 class genes are predominantly single copy in Magnoliids, and predate the whole genome duplication (WGD) events in monocots and eudicots. By comparison with the model species Arabidopsis thaliana, the expression patterns of B-, C-, and D-class genes in stamen, carpel, and ovules are conserved in Aristolochia fimbriata, whereas A-, E-class, and AGL6 genes have different expression patterns. Nevertheless, the interactions of these proteins that act through multimeric complexes remain poorly known in early divergent angiosperms. This study evaluates protein interactions among all floral MADS-box A. fimbriata proteins using the Yeast Two Hybrid System (Y2H). We found no homodimers and less heterodimers formed by AfimFUL when compared to AfimAGL6, which allowed us to suggest AGL6 homodimers in combination with AfimSEP2 as the most likely tetramer in sepal identity. We found AfimAP3-AfimPI obligate heterodimers and AfimAG-AfimSEP2 protein interactions intact suggesting conserved stamen and carpel tetrameric complexes in A. fimbriata. We observed a broader interaction partner set for AfimSEP2 than for its paralog AfimSEP1. We show conserved and exclusive MADS-box protein interactions in A. fimbriata in comparison with other eudicot and monocot model species in order to establish plesiomorphic MADS-box protein floral networks in angiosperms.

DNA barcoding of Aristolochia plants and development of species-specific multiplex PCR to aid HPTLC in ascertainment of Aristolochia herbal materials.

The anecdotal evidence is outstanding on the uses of Aristolochia plants as traditional medicines and dietary supplements in many regions of the world. However, herbal materials derived from Aristolochia species have been identified as potent human carcinogens since the first case of severe renal disease after ingesting these herbal preparations. Any products containing Aristolochia species have thus been banned on many continents, including Europe, America and Asia. Therefore, the development of a method to identify these herbs is critically needed for customer safety. The present study evaluated DNA barcoding of the rbcL, matK, ITS2 and trnH-psbA regions among eleven Aristolochia species collected in Thailand. Polymorphic sites were observed in all four DNA loci. Among those eleven Aristolochia species, three species (A. pierrei, A. tagala and A. pothieri) are used as herbal materials in Thai folk medicine, namely, in Thai "Krai-Krue". "Krai-Krue" herbs are interchangeably used as an admixture in Thai traditional remedies without specific knowledge of their identities. A species-specific multiplex PCR based on nucleotide polymorphisms in the ITS2 region was developed as an identification tool to differentiate these three Aristolochia species and to supplement the HPTLC pattern in clarifying the origins of herbal materials. The combination of multiplex PCR and HPTLC profiling achieves accurate herbal identification with the goal of protecting consumers from the health risks associated with product substitution and contamination.

Molecular Structure and Phylogenetic Analyses of Complete Chloroplast Genomes of Two Aristolochia Medicinal Species.

The family Aristolochiaceae, comprising about 600 species of eight genera, is a unique plant family containing aristolochic acids (AAs). The complete chloroplast genome sequences of Aristolochia debilis and Aristolochia contorta are reported here. The results show that the complete chloroplast genomes of A. debilis and A. contorta comprise circular 159,793 and 160,576 bp-long molecules, respectively and have typical quadripartite structures. The GC contents of both species were 38.3% each. A total of 131 genes were identified in each genome including 85 protein-coding genes, 37 tRNA genes, eight rRNA genes and one pseudogene (ycf1). The simple-sequence repeat sequences mainly comprise A/T mononucletide repeats. Phylogenetic analyses using maximum parsimony (MP) revealed that A. debilis and A. contorta had a close phylogenetic relationship with species of the family Piperaceae, as well as Laurales and Magnoliales. The data obtained in this study will be beneficial for further investigations on A. debilis and A. contorta from the aspect of evolution, and chloroplast genetic engineering.

Recalcitrant deep and shallow nodes in Aristolochia (Aristolochiaceae) illuminated using anchored hybrid enrichment.

Recalcitrant relationships are characterized by very short internodes that can be found among shallow and deep phylogenetic scales all over the tree of life. Adding large amounts of presumably informative sequences, while decreasing systematic error, has been suggested as a possible approach to increase phylogenetic resolution. The development of enrichment strategies, coupled with next generation sequencing, resulted in a cost-effective way to facilitate the reconstruction of recalcitrant relationships. By applying the anchored hybrid enrichment (AHE) genome partitioning strategy to Aristolochia using an universal angiosperm probe set, we obtained 231-233 out of 517 single or low copy nuclear loci originally contained in the enrichment kit, resulting in a total alignment length of 154,756bp to 160,150bp. Since Aristolochia (Piperales; magnoliids) is distantly related to any angiosperm species whose genome has been used for the plant AHE probe design (Amborella trichopoda being the closest), it serves as a proof of universality for this probe set. Aristolochia comprises approximately 500 species grouped in several clades (OTUs), whose relationships to each other are partially unknown. Previous phylogenetic studies have shown that these lineages branched deep in time and in quick succession, seen as short-deep internodes. Short-shallow internodes are also characteristic of some Aristolochia lineages such as Aristolochia subsection Pentandrae, a clade of presumably recent diversification. This subsection is here included to test the performance of AHE at species level. Filtering and subsampling loci using the phylogenetic informativeness method resolves several recalcitrant phylogenetic relationships within Aristolochia. By assuming different ploidy levels during bioinformatics processing of raw data, first hints are obtained that polyploidization contributed to the evolution of Aristolochia. Phylogenetic results are discussed in the light of current systematics and morphology.

Deep into the Aristolochia Flower: Expression of C, D, and E-Class Genes in Aristolochia fimbriata (Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae) is a member of an early diverging lineage of flowering plants and a promising candidate for evo-devo studies. Aristolochia flowers exhibit a unique floral synorganization that consists of a monosymmetric and petaloid calyx formed by three congenitally fused sepals, and a gynostemium formed by the congenital fusion between stamens and the stigmatic region of the carpels. This floral ground plan atypical in the magnoliids can be used to evaluate the role of floral organ identity MADS-box genes during early flower evolution. In this study, we present in situ hybridization experiments for the homologs of the canonical C-, D-, and E-class genes. Spatiotemporal expression of the C-class gene AfimAG is restricted to stamens, ovary, and ovules, suggesting a conserved stamen and carpel identity function, consistent with that reported in core-eudicots and monocots. The D-class gene AfimSTK is detected in the anthers, the stigmas, the ovary, the ovules, the fruit, and the seeds, suggesting conserved roles in ovule and seed identity and unique roles in stamens, ovary, and fruit development. In addition, AfimSTK expression patterns in areas of organ abscission and dehiscence zones suggest putative roles linked to senescence processes. We found that both E-class genes are expressed in the anthers and the ovary; however, AfimSEP2 exhibits higher expression compared to AfimSEP1. These findings provide a comprehensive picture of the ancestral expression patterns of the canonical MADS-box floral organ identity genes and the foundations for further comparative analyses in other magnoliids.