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Genomes of Conopholis americana and Epifagus virginiana: two holoparasitic plants (Orobanchaceae).

Conopholis americana (American cancer-root) and Epifagus virginiana (beechdrops) are sister genera of holoparasitic plants (Orobanchaceae) native to eastern North America, parasitizing oaks and American beech, respectively. Both have served as models for plastid genome reduction, yet no nuclear genomes exist for either genus or any New World holoparasitic Orobanchaceae. Here we present the first nuclear genome assemblies for both species using PacBio HiFi sequencing. The C. americana assembly totals 1.82 Gb and E. virginiana totals 440 Mb, representing an approximately 4-fold difference in genome size between these sister genera. We observed a BUSCO completeness of 79% to 80% in both species, which is typical of holoparasites. While gene prediction identified 33,889 genes in C. americana and 21,031 in E. virginiana, repeat annotation revealed that LTR retrotransposons account for 78% of the genome size difference. These assemblies reveal contrasting mechanisms of genome evolution in sister holoparasitic genera and provide foundational resources for comparative genomics of parasitic plants.

Genome, Plant↗

Diversity and evolution of Ty1-copia and Ty3-gypsy retroelements in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae).

We present the first study on the diversity and evolution of Ty1-copia and Ty3-gypsy retroelements in a group of non-photosynthetic flowering plants. To this end partial sequences of the reverse transcriptase (rt) gene were obtained from 20 clones for each retroelement type from seven and six accessions of Orobanche and Phelipanche (Orobanchaceae), respectively. Overall sequence similarity is higher in Ty3-gypsy elements than in Ty1-copia elements in agreement with the results from other angiosperm groups. Higher sequence diversity and stronger phylogenetic structure, especially of Ty1-copia sequences, in Orobanche species compared to Phelipanche species support the previously suggested hypothesis (based on karyological and cytological data) that genomes of Orobanche species are more dynamic than those of Phelipanche species. No evidence was found for intraspecific differences of retroelement diversity nor for differences between pest taxa and their putative wild relatives, e.g., O. crenata and O. owerini. The occurrence of a few sequences from Phelipanche species in clades otherwise comprising sequences from Orobanche species might be due to horizontal gene transfer, but the alternative of vertical transmission cannot be rejected unambiguously.

Chromosome Mapping↗

Section-level relationships of North American Agalinis (Orobanchaceae) based on DNA sequence analysis of three chloroplast gene regions.

BACKGROUND: The North American Agalinis are representatives of a taxonomically difficult group that has been subject to extensive taxonomic revision from species level through higher sub-generic designations (e.g., subsections and sections). Previous presentations of relationships have been ambiguous and have not conformed to modern phylogenetic standards (e.g., were not presented as phylogenetic trees). Agalinis contains a large number of putatively rare taxa that have some degree of taxonomic uncertainty. We used DNA sequence data from three chloroplast genes to examine phylogenetic relationships among sections within the genus Agalinis Raf. (=Gerardia), and between Agalinis and closely related genera within Orobanchaceae. RESULTS: Maximum likelihood analysis of sequences data from rbcL, ndhF, and matK gene regions (total aligned length 7323 bp) yielded a phylogenetic tree with high bootstrap values for most branches. Likelihood ratio tests showed that all but a few branch lengths were significantly greater than zero, and an additional likelihood ratio test rejected the molecular clock hypothesis. Comparisons of substitution rates between gene regions based on linear models of pairwise distance estimates between taxa show both ndhF and matK evolve more rapidly than rbcL, although the there is substantial rate heterogeneity within gene regions due in part to rate differences among codon positions. CONCLUSIONS: Phylogenetic analysis supports the monophyly of Agalinis, including species formerly in Tomanthera, and this group is sister to a group formed by the genera Aureolaria, Brachystigma, Dasistoma, and Seymeria. Many of the previously described sections within Agalinis are polyphyletic, although many of the subsections appear to form natural groups. The analysis reveals a single evolutionary event leading to a reduction in chromosome number from n = 14 to n = 13 based on the sister group relationship of section Erectae and section Purpureae subsection Pedunculares. Our results establish the evolutionary distinctiveness of A. tenella from the more widespread and common A. obtusifolia. However, further data are required to clearly resolve the relationship between A. acuta and A. tenella.

Chloroplasts↗

Phylogeny of holoparasitic Orobanche (Orobanchaceae) inferred from nuclear ITS sequences.

Orobanche is the largest genus among the holoparasitic members of Orobanchaceae. We present the first molecular phylogenetic analysis (using nuclear ITS sequences) that includes members of all sections of Orobanche, Gymnocaulis, Myzorrhiza, Trionychon, and Orobanche. Orobanche is not monophyletic, but falls into two lineages: (1) the Orobanche group comprises Orobanche sect. Orobanche and the small Near Asian genus Diphelypaea and is characterized by a chromosome base number of x=19 and (2) the Phelipanche group contains Orobanche sects. Gymnocaulis, Myzorrhiza, and Trionychon and possesses a chromosome base number of x=12. The relationships between these two groups and to other genera such as Boschniakia or Cistanche remain unresolved. Within the Orobanche group, Orobanche macrolepis and Orobanche anatolica (including Orobanche colorata) constitute two phylogenetically distinct lineages. Intrasectional structurings proposed by some authors for O. sect. Orobanche are not confirmed by the molecular data. In most cases, intraspecific sequence divergence between accessions, if present, is negligible and not correlated with morphological or ecological traits. In a few cases, however, there is evidence for the presence of cryptic taxa.

Biological Evolution↗

Horizontal gene transfer of a plastid gene in the non-photosynthetic flowering plants Orobanche and Phelipanche (Orobanchaceae).

Plastid sequences are among the most widely used in phylogenetic and phylogeographic studies in flowering plants, where they are usually assumed to evolve like non-recombining, uniparentally transmitted, single-copy genes. Among others, this assumption can be violated by intracellular gene transfer (IGT) within cells or by the exchange of genes across mating barriers (horizontal gene transfer, HGT). We report on HGT of a plastid region including rps2, trnL-F, and rbcL in a group of non-photosynthetic flowering plants. Species of the parasitic broomrape genus Phelipanche harbor two copies of rps2, a plastid ribosomal gene, one corresponding to the phylogenetic position of the respective species, the other being horizontally acquired from the related broomrape genus Orobanche. While the vertically transmitted copies probably reside within the plastid genome, the localization of the horizontally acquired copies is not known. With both donor and recipient being parasitic plants, a possible pathway for the exchange of genetic material is via a commonly attacked host.

Amino Acid Sequence↗

Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences.

The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.

Base Sequence↗

Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: many losses of photosynthesis and complex patterns of rate variation.

The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism.

Arabidopsis Proteins↗

Rate variation in parasitic plants: correlated and uncorrelated patterns among plastid genes of different function.

BACKGROUND: The analysis of synonymous and nonsynonymous rates of DNA change can help in the choice among competing explanations for rate variation, such as differences in constraint, mutation rate, or the strength of genetic drift. Nonphotosynthetic plants of the Orobanchaceae have increased rates of DNA change. In this study 38 taxa of Orobanchaceae and relatives were used and 3 plastid genes were sequenced for each taxon. RESULTS: Phylogenetic reconstructions of relative rates of sequence evolution for three plastid genes (rbcL, matK and rps2) show significant rate heterogeneity among lineages and among genes. Many of the non-photosynthetic plants have increases in both synonymous and nonsynonymous rates, indicating that both (1) selection is relaxed, and (2) there has been a change in the rate at which mutations are entering the population in these species. However, rate increases are not always immediate upon loss of photosynthesis. Overall there is a poor correlation of synonymous and nonsynonymous rates. There is, however, a strong correlation of synonymous rates across the 3 genes studied and the lineage-speccific pattern for each gene is strikingly similar. This indicates that the causes of synonymous rate variation are affecting the whole plastid genome in a similar way. There is a weaker correlation across genes for nonsynonymous rates. Here the picture is more complex, as could be expected if there are many causes of variation, differing from taxon to taxon and gene to gene. CONCLUSIONS: The distinctive pattern of rate increases in Orobanchaceae has at least two causes. It is clear that there is a relaxation of constraint in many (though not all) non-photosynthetic lineages. However, there is also some force affecting synonymous sites as well. At this point, it is not possible to tell whether it is generation time, speciation rate, mutation rate, DNA repair efficiency or some combination of these factors.

Cell Nucleus↗

Disintegration of the scrophulariaceae.

A molecular systematic study of Scrophulariaceae sensu lato using DNA sequences of three plastid genes (rbcL, ndhF, and rps2) revealed at least five distinct monophyletic groups. Thirty-nine genera representing 24 tribes of the Scrophulariaceae s.l. (sensu lato) were analyzed along with representatives of 15 other families of Lamiales. The Scrophulariaceae s.s. (sensu stricto) include part or all of tribes Aptosimeae, Hemimerideae, Leucophylleae, Manuleae, Selagineae, and Verbasceae (= Scrophularieae) and the conventional families Buddlejaceae and Myoporaceae. Veronicaceae includes all or part of tribes Angelonieae, Antirrhineae, Cheloneae, Digitaleae, and Gratioleae and the conventional families Callitrichaceae, Globulariaceae, Hippuridaceae, and Plantaginaceae. The Orobanchaceae include tribes Buchnereae, Rhinantheae, and the conventional Orobanchaceae. All sampled members of Orobanchaceae are parasitic, except Lindenbergia, which is sister to the rest of the family. Family Calceolariaceae Olmstead is newly erected herein to recognize the phylogenetic distinctiveness of tribe Calceolarieae. The Calceolariaceae are close to the base of the Lamiales. The Stilbaceae are expanded by the inclusion of Halleria. Mimulus does not belong in any of these five groups.

Journal Article↗

Painting new pathways: Castilleja enters the genomic era.

Castilleja (Orobanchaceae), commonly known as Indian paintbrush, is a genus of approximately 200 hemiparasitic species found primarily across the Americas. Long studied for its taxonomic complexity, vibrant floral displays, and ecological interactions with host plants, Castilleja has recently emerged as a versatile research system spanning parasitic biology, specialized metabolism, conservation genetics, and pharmacology. The availability of whole-genome sequencing is transforming the field, revealing expanded gene families involved in host recognition, enabling genomic species delimitation of cryptic taxa, and providing frameworks for mapping biosynthetic pathways of bioactive compounds, including iridoid glycosides and phenylethanoid glycosides. This review synthesizes advances across these disciplines and highlights how genomics serves as an integrative force connecting taxonomy, ecology, phytochemistry, and parasitic biology in this genus.

Castilleja↗

Pscroph, a parasitic plant EST database enriched for parasite associated transcripts.

BACKGROUND: Parasitic plants in the Orobanchaceae develop invasive root haustoria upon contact with host roots or root factors. The development of haustoria can be visually monitored and is rapid, highly synchronous, and strongly dependent on host factor exposure; therefore it provides a tractable system for studying chemical communications between roots of different plants. DESCRIPTION: Triphysaria is a facultative parasitic plant that initiates haustorium development within minutes after contact with host plant roots, root exudates, or purified haustorium-inducing phenolics. In order to identify genes associated with host root identification and early haustorium development, we sequenced suppression subtractive libraries (SSH) enriched for transcripts regulated in Triphysaria roots within five hours of exposure to Arabidopsis roots or the purified haustorium-inducing factor 2,6 dimethoxybenzoquinone. The sequences of over nine thousand ESTs from three SSH libraries and their subsequent assemblies are available at the Pscroph database http://pscroph.ucdavis.edu. The web site also provides BLAST functions and allows keyword searches of functional annotations. CONCLUSION: Libraries prepared from Triphysaria roots treated with host roots or haustorium inducing factors were enriched for transcripts predicted to function in stress responses, electron transport or protein metabolism. In addition to parasitic plant investigations, the Pscroph database provides a useful resource for investigations in rhizosphere interactions, chemical signaling between organisms, and plant development and evolution.

Arabidopsis↗

Divergent evolution of two plastid genes, rbcL and atpB, in a non-photosynthetic parasitic plant.

Plastid DNA (ptDNA) regions for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubiso) (rbcL) and the beta-subunit of ATP synthase (atpB) genes of the holoparasite Lathraea clandestina L. were sequenced. These regions were obtained by cloning either a Bam HI endonuclease generated fragment from the Lathraea ptDNA or polymerase chain reaction (PCR) amplified products. The Lathraea ptDNA contains the entire sequence for the rbcL gene which shares 94.5% homology with the Nicotiana tabacum gene, whereas atpB is maintained as a pseudogene. The intergenic region between divergently transcribed rbcL and atpB genes is shorter (758 bp) in L. clandestina plastid genome in comparison with N. tabacum (823 bp), however they have a noticeable similarity, mainly in the rbcL 5'-upstream region. A low level of the rbcL gene transcription was detected whereas no atpB transcripts were found in Latraea. The plasmid rbcL gene of the hemiparasite Melampyrum pratense and the autotroph Digitalis purpurea both from the Scrophulariaceae were cloned by PCR amplification and then sequenced. The L. clandestina rbcL gene is highly homologous to the M. pratense and D. purpurea genes. The data indicate that the evolution of the plastid atpB-rbcL region was different in parasites from the Scrophulariaceae and Orobanchaceae families.

Amino Acid Sequence↗

Lack of a functional plastid tRNA(Cys) gene is associated with loss of photosynthesis in a lineage of parasitic plants.

We recently reported that the gene for chloroplast tRNA(Cys)(GCA) is a pseudogene in the plastid DNA of Epifagus virginiana, a non-photosynthetic parasitic flowering plant in the family Orobanchaceae. Since this is the only tRNA(Cys) gene in the plastid genome, and since Epifagus appears to possess a functional plastid translational apparatus, it seems probable that nuclear-encoded tRNAs are imported into plastids to effect translation. In this study we have surveyed species closely related to Epifagus to establish how widespread the loss of this tRNA gene has been. We find that Conopholis americana, another non-photosynthetic parasite, lacks the gene altogether, but that seven closely-related photosynthetic plants (both parasitic and free-living) maintain an intact chloroplast tRNA(Cys) gene. Thus, the tRNA(Cys) gene appears to have become non-functional at the same time that photosynthetic ability was lost. This may be because the levels of putatively imported tRNAs are sufficient to meet the demands of plastid gene expression under nonphotosynthetic conditions only.

Animals↗

Loss of transfer RNA genes from the plastid 16S-23S ribosomal RNA gene spacer in a parasitic plant.

The plastid 16S-23S intergenic spacer region in Conopholis americana, a totally heterotrophic angiosperm in the family Orobanchaceae, has undergone large deletions, including the entire tRNA(Ile) gene and all but small remnants of the tRNA(Ala) gene. The length of the region is less than 20% of that of other land plants which have been investigated, making it the smallest 16S-23S intergenic spacer reported thus far for any land plant. The remaining sequences in the spacer are 90.1% identical to tobacco, indicating that, while the region is well conserved at the sequence level, it is evolving rapidly by deletion. Experiments using the polymerase chain reaction and hybridization to DNA gel blots have failed to reveal either of the two missing tRNA genes elsewhere in the Conopholis cell.

Base Sequence↗

Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence.

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria versicolor Fischer and C. Meyer. Agrobacterium tumefaciens and Agrobacterium rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A. tumefaciens and A. rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will allow an in planta assessment of genes predicted to function in plant parasitism.

Agrobacterium tumefaciens↗

Pollination niche overlap between a parasitic plant and its host.

Niche theory predicts that species which share resources should evolve strategies to minimise competition for those resources, or the less competitive species would be extirpated. Some plant species are constrained to co-occur, for example parasitic plants and their hosts, and may overlap in their pollination niche if they flower at the same time and attract the same pollinators. Using field observations and experiments between 1996 and 2006, we tested a series of hypotheses regarding pollination niche overlap between a specialist parasitic plant Orobanche elatior (Orobanchaceae) and its host Centaurea scabiosa (Asteraceae). These species flower more or less at the same time, with some year-to-year variation. The host is pollinated by a diverse range of insects, which vary in their effectiveness, whilst the parasite is pollinated by a single species of bumblebee, Bombus pascuorum, which is also an effective pollinator of the host plant. The two species therefore have partially overlapping pollination niches. These niches are not finely subdivided by differential pollen placement, or by diurnal segregation of the niches. We therefore found no evidence of character displacement within the pollination niches of these species, possibly because pollinators are not a limiting resource for these plants. Direct observation of pollinator movements, coupled with experimental manipulations of host plant inflorescence density, showed that Bombus pascuorum only rarely moves between inflorescences of the host and the parasite and therefore the presence of one plant is unlikely to be facilitating pollination in the other. This is the first detailed examination of pollination niche overlap in a plant parasite system and we suggest avenues for future research in relation to pollination and other shared interactions between parasitic plants and their hosts.

Animals↗

Anti-nociceptive and anti-inflammatory activity caused by Cistanche deserticola in rodents.

In the present study, the rhizomes of Cistanche deserticola (Orobanchaceae, abbreviated as CD) were extracted with 50% ethanol and isolated orderly by ethyl acetate, n-butanol and water. The analgesic and anti-inflammatory effects of CD extract and three layers were evaluated in several animal models. CD extracts effectively inhibited writhing response induced by 1% acetic acid and biphasic licking responses caused by 1% formalin, and also reduced the edema induced by 1% carrageenan but not zymosan. Furthermore, the butanolic and aqueous layers of CD extract not only reduced the pain induced by acetic acid and formalin, but also decreased the edema that induced by carrageenan. Effects of the butanolic layer of CD extract are better than that of the aqueous layer. In addition, the effect of the butanolic layer of CD extract was not abolished by naloxone. These results revealed that CD has analgesic and anti-inflammatory effects, and the butanolic and aqueous layers are mainly active constituents. Furthermore, the analgesic and anti-inflammatory effects of the butanolic layer of CD extract were not related to opioid receptors and immune system.

Analgesics, Non-Narcotic↗