Chromosomal-scale genome assembly of Eleutherococcus senticosus provides insights into chromosome evolution in Araliaceae.

Siberian ginseng (Eleutherococcus senticosus, also known as ciwujia) belongs to the Araliaceae family, which contains more than 1,500 species in 41 genera with diverse chromosome numbers and genome sizes. General consensus posits that ancient whole-genome duplication events and rapid evolutionary radiation are the driving forces for this variation in genome properties. In an attempt to generate more genomic information for the Araliaceae family, we report a 1.30 Gb high-quality draft genome assembly (contig N50 = 309.43 kb) of E. senticosus via PacBio long reads and Hi-C chromatin interaction maps. We found that transposable elements accounted for 72.82% of the genome and a total of 36,372 protein-coding genes were predicted. Comparative analyses of the E. senticosus, Panax notoginseng and Daucus carota genomes revealed a burst expansion of Tekay chromoviral elements in Araliaceae after its divergence with Apiaceae. We also found that E. senticosus underwent a lineage-specific whole-genome duplication event Es-α and a whole-genome duplication event Araliaceae-ß that was probably shared by all Araliaceae species. Even though the rediploidization of the E. senticosus genome is evident, pathway analyses show that these two whole-genome duplication events may have contributed to the adaptation of E. senticosus to a cold environment. Taken together, the high-quality genome assembly of E. senticosus provides a valuable genomic resource for future research on the evolution of Araliaceae.

Pre-labelled oligo probe-FISH karyotype analyses of four Araliaceae species using rDNA and telomeric repeat.

BACKGROUND: The family Araliaceae contains many medicinal species including ginseng of which the whole genome sequencing analyses have been going on these days. OBJECTIVE: To characterize the chromosomal distribution of 5S and 45S rDNAs and telomeric repeat in four ginseng related species of Aralia elata (Miq.) Seem., Dendropanax morbiferus H. Lév., Eleutherococcus sessiliflorus (Rupr. Et Maxim.) Seem., Kalopanax septemlobus (Thunb. ex A.Murr.) Koidz. METHOD: Pre-labelled oligoprobe (PLOP)-fluorescence in situ hybridization (FISH) was carried out. RESULTS: The chromosome number of A. elata was 2n = 24, whereas that of the other three species of D. morbiferus, E. sessiliflorus, and K. septemlobus was 2n = 48, corresponding to diploid and tetraploid, respectively, based on the basic chromosome number x = 12 in Araliaceae. In all four species, one pair of 5S signals were detected in the proximal regions of the short arms of chromosome 3, whereas in K. septemlobus, the 5S rDNA signals localized in the subtelomeric region of short arm of chromosome 3, while all the 45S rDNA signals localized at the paracentromeric region of the short arm of chromosome 1. And the telomeric repeat signals were detected at the telomeric region of both short and long arms of most chromosomes. CONCLUSION: The PLOP-FISH was very effective compared with conventional FISH method. These results provide useful comparative cytogenetic information to better understand the genome structure of each species and will be useful to trace the history of ginseng genomic constitution.

Molecular phylogenetics and species delimitation in annual species of Hydrocotyle (Araliaceae) from South Western Australia.

Hydrocotyle L. is a cosmopolitan genus, with approximately 130 species, of mostly perennial herbs in the family Araliaceae. The genus includes around 95 perennial and 35 annual species, with all annual species endemic to Australia. In this study, I used sequences of a nDNA marker (ETS) and two cpDNA markers (psbA-trnH and trnL-trnF) to reconstruct phylogenetic relationships among the annual species of Hydrocotyle. The final sampling included 21 species of Hydrocotyle and seven outgroup taxa. The phylogenetic analyses of the combined molecular dataset (i.e., ETS, psbA-trnH and trnL-trnF) recovered three major clades within Hydrocotyle, defined by annual or perennial life histories, and the presence/absence of floral bracts. The topology reconstructed here indicates that there is insufficient molecular evidence for the formal recognition of two putative new species, Hydrocotyle sp. Hamelinensis and Hydrocotyle sp. Puberula, previously identified on the basis of morphology. More specifically, H. sp. Hamelinensis is conspecific with H. tetragonocarpa Bunge, while H. sp. Puberula is conspecific with H. scutellifera Benth. Morphological studies of H. tetragonocarpa revealed that this species is andromonoecious and has heterocarpic schizocarps. Fertile plants of H. tetragonocarpa bear fruit that are either ovoid and wingless or broadly obcordate and winged. A detailed re-examination of the schizocarp surfaces of H. scutellifera indicated that these schizocarps are either glabrous or papillate, with neither of these two character states co-varying with any other vegetative or reproductive traits. The phylogeny of the annual Hydrocotyle recovered a paraphyletic Hydrocotyle, with Neosciadium glochidiatum (Benth.) Domin nested within the genus. This study is the first to reconstruct a molecular phylogeny of Hydrocotyle using samples of both annual and perennial taxa. The findings from this study provide a phylogenetic framework for future systematic and taxonomic research within the genus.

Purification and structural analysis of volatile sesquiterpenes produced by Escherichia coli carrying unidentified terpene synthase genes from edible plants of the family Araliaceae.

A simple method to purify volatile sesquiterpenes from recombinant Escherichia coli was developed using the cells that carried known sesquiterpene synthase (Tps) genes ZzZss2 (ZSS2) and ZoTps1. This method was applied for the purification and structural analyses of volatile sesquiterpenes produced by E. coli cells that carried unidentified Tps genes, which were isolated from the Aralia-genus edible plants belonging to the family Araliaceae. Recombinant cells carrying each Tps gene were cultured in the two-layer medium (n-octane/TB medium), and volatile sesquiterpenes trapped in n-octane were purified through two-phase partition, silica gel column chromatography, and reversed-phase preparative high-performance liquid chromatography, if necessary. Further, their structures were confirmed by nuclear magnetic resonance, [α]D, and gas chromatography-mass spectrometry analyses. Herein, the products of E. coli cells that carried two Tps gene (named AcTps1 and AcTps2) in Araria cordata "Udo" and a Tps gene (named AeTps1) in Aralia elata "Taranoki" were studied resulting in identifying functionalities of these cryptic Tps genes.

The Identification of Araliaceae Species by ITS2 Genetic Barcoding and Pollen Morphology.

The genetic barcode ITS2 (ITS: internal transcribed spacer) and pollen morphology were used for the identification of the pharmacologically valuable wild Araliaceae species Panax ginseng, Oplopanax elatus, Aralia elata, Aralia continentalis, Eleutherococcus senticosus, and Eleutherococcus sessiliflorus inhabiting the natural forests of Primorye, Russia. The ITS2 locus successfully identified all six species, which supports the use of ITS2 as a standard barcode for medicinal plants. However, the ITS2 locus was insufficient for intra-specific discrimination in these species, neither within Primorye nor from other world representatives within GenBank. Araliaceae pollen was confirmed to undergo size-reducing metamorphosis. The final morphotypes were species-specific for each of the six species but could not discriminate intra-species geographic localities within Primorye. The morphologies of the final pollen morphotypes from homologous species inhabiting other parts of the world are not yet known. Therefore, whether pollen is applicable for Araliaceae intra-species discrimination between Primorye and other world localities could not be established. Based on these findings, we propose that the ITS2 genetic barcode and the final pollen morphotypes are suitable for the identification of Araliaceae species. However, further studies will be needed to determine the suitability of genetic and pollen traits for Araliaceae geographic authentication.

Evolution of the Araliaceae family inferred from complete chloroplast genomes and 45S nrDNAs of 10 Panax-related species.

We produced complete sequences and conducted comparative analysis of the maternally inherited chloroplast (cp) genomes and bi-parentally inherited 45S nuclear ribosomal RNA genes (nrDNA) from ten Araliaceae species to elucidate the genetic diversity and evolution in that family. The cp genomes ranged from 155,993 bp to 156,730 bp with 97.1-99.6% similarity. Complete 45S nrDNA units were about 11 kb including a 5.8-kb 45S cistron. Among 79 cp protein-coding genes, 74 showed nucleotide variations among ten species, of which infA, rpl22, rps19 and ndhE genes showed the highest Ks values and atpF, atpE, ycf2 and rps15 genes showed the highest Ka/Ks values. Four genes, petN, psaJ, psbF, and psbN, related to photosynthesis and one gene, rpl23, related to the ribosomal large subunit remain conserved in all 10 Araliaceae species. Phylogenetic analysis revealed that the ten species could be resolved into two monophyletic lineages, the Panax-Aralia and the Eleutherococcus-Dendropanax groups, which diverged approximately 8.81-10.59 million years ago (MYA). The Panax genus divided into two groups, with diploid species including P. notoginseng, P. vietnamensis, and P. japonicus surviving in Southern Asia and a tetraploid group including P. ginseng and P. quinquefolius Northern Asia and North America 2.89-3.20 MYA.

Gradual vs. abrupt reduction of carpels in syncarpous gynoecia: A case study from Polyscias subg. Arthrophyllum (Araliaceae: Apiales).

PREMISE OF THE STUDY: Revealing the relative roles of gradual and abrupt transformations of morphological characters is an important topic of evolutionary biology. Gynoecia apparently consisting of one carpel have evolved from pluricarpellate syncarpous gynoecia in several angiosperm clades. The process of reduction can involve intermediate stages, with one fertile and one or more sterile carpels (pseudomonomery). The possible origin of monomery directly via an abrupt change of gynoecium merism has been a matter of dispute. We explore the nature of gynoecium reduction in a clade of Araliaceae. METHODS: The anatomy and development of unilocular gynoecia are investigated using light and scanning electron microscopy in two members of Polyscias subg. Arthrophyllum. Gynoecium diversity in the genus is discussed in a phylogenetic framework. KEY RESULTS: Unilocular gynoecia with one fertile ovule have evolved at least four times in Polyscias, including one newly discovered case. The two unilocular taxa investigated are unicarpellate, without any traces of reduced sterile carpels. Carpel orientation is unstable, and the ovary roof and style contain numerous vascular bundles without clearly recognizable dorsals or ventrals. In contrast to pluricarpellate Araliaceae and Apiaceae, the cross zone is apparently oblique in the unicarpellate species. CONCLUSIONS: No support was found for gradual gynoecium reduction via pseudomonomery. The abrupt origin of monomery via direct change of gynoecium merism and the unstable carpel orientation observed are related to the general lability of the flower groundplan in Polyscias. The apparent occurrence of the unusual oblique cross zone in unicarpellate Araliaceae can be explained by developmental constraints.

The complete chloroplast genome sequence of Schefflera octophylla.

The complete chloroplast genome sequence of Schefflera octophylla, a critical Chinese medicine was reported here. The complete chloroplast genome of Schefflera octophylla is 156 685 bp in length with 37.93% overall GC content. A pair of IRs (inverted repeats) of 25 965 bp were separated by LSC (86 609 bp) and SSC (18 146 bp). The phylogenetic analysis of 17 taxa showed a strong sister relationship with Schefflera delavayi. Furthermore the Araliaceae was separated into two major groups.

The complete chloroplast genome sequence of Fatsia japonica (Apiales: Araliaceae) and the phylogenetic analysis.

In this study, we have sequenced the complete chloroplast genome of Fatsia japonica, a well-known ornamental and potential medicinal plant. The complete chloroplast genome of F. japonica is 155 613 bp in length with 62.09% AT content, has a typical quadripartite structure with large (LSC 86 487 bp) and small (SSC 17 866 bp) single-copy regions separated by a pair of inverted repeats (IRs 25 929 bp) and contains 114 unique genes with 18 genes duplicated in the IR making a total of 132 genes. The phylogenetic analysis indicated the position of F. japonica in Apiales and has the potential to facilitate a better understanding of the intergeneric relationships in the family.

The complete chloroplast genome sequence of Dendropanax morbifera (Léveillé).

The complete chloroplast genome sequence of Dendropanax morbifera, an economically and medicinally important endemic tree species in Korea, was obtained by de novo assembly with whole-genome sequence data and manual correction. A circular 156 366-bp chloroplast genome showed typical chloroplast genome structure comprising a large single copy region of 86 475 bp, a small single copy region of 18 125 bp, and a pair of inverted repeats of 25 883 bp. The chloroplast genome harbored 87 protein-coding genes. Phylogenetic analysis with the chloroplast genome revealed that D. morbifera is most closely related to Dendropanax dentiger, an evergreen tree species in China and Southeastern Asia.

The complete chloroplast genome sequence of the evergreen plant Dendropanax dentiger (Araliaceae).

The complete chloroplast genome of the evergreen plant Dendropanax dentiger (Araliaceae) has been reconstructed from the whole-genome Illumina sequencing data. The circular genome is 156,687 bp in size, and comprises a pair of inverted repeat (IR) regions of 25,880 bp each, a large single-copy (LSC) region of 86,680 bp, and a small single-copy (SSC) region of 18,247 bp. The chloroplast genome contains 134 genes, including 89 protein-coding genes (81 PCG species), eight ribosomal RNA genes (four rRNA species), and 37 transfer RNA genes (30 tRNA species). Out of these genes, 16 harbor a single intron, and two contain a couple of introns. The overall A + T content of the whole genome is 62.0%, while the corresponding values of the LSC, SSC, and IR regions are 63.8%, 68.0% and 56.9%, respectively.

The origin of the early differentiation of ivies (Hedera L.) and the radiation of the Asian Palmate group (Araliaceae).

The Asian Palmate group is one of the four major clades of the family Araliaceae that is formed by 18 genera, including ivies (Hedera L.). The Mediterranean diversity centre and temperate affinity of ivies contrast with the inferred Asian centre of diversity of the primarily tropical and subtropical Asian Palmate group. We herein investigated the sister-group relationships of Hedera to reconstruct the evolutionary context for its origin and early diversification. Seven nuclear and plastid DNA regions were analyzed in 61 Araliaceae samples including all the 18 Asian Palmate genera. Maximum Parsimony, Maximum Likelihood and Bayesian Inference were run together with a battery of topology testing analyses constraining the expected Hedera's sister-group relationships. Additionally, Bayesian polytomy resolvability and divergence time analyses were also conducted. Genome incongruence and hard nuclear and plastid basal polytomies are detected for the Asian Palmate group where the lineage of Hedera is placed. Topology testing analyses do not allow rejecting any of the tentative sisters of Hedera. An early radiation with inter-lineage hybridization and genome doubling is suggested for the Asian Palmate group where all the seven temperate genera, including Hedera, seem to have played an important role. The radiation took placed during the Upper Cretaceous in Asia under a general cooling and the eastern Asian mountain uplift that produced new temperate environments and promoted lineage connections. This allows us to hypothesize that the origin of the Hedera lineage may fit in a temperate niche conservatism scenario where the combination of the radiation with lineage admixtures prevents us from discovering its sister-group.

Complete sequencing of five araliaceae chloroplast genomes and the phylogenetic implications.

BACKGROUND: The ginseng family (Araliaceae) includes a number of economically important plant species. Previously phylogenetic studies circumscribed three major clades within the core ginseng plant family, yet the internal relationships of each major group have been poorly resolved perhaps due to rapid radiation of these lineages. Recent studies have shown that phyogenomics based on chloroplast genomes provides a viable way to resolve complex relationships. METHODOLOGY/PRINCIPAL FINDINGS: We report the complete nucleotide sequences of five Araliaceae chloroplast genomes using next-generation sequencing technology. The five chloroplast genomes are 156,333-156,459 bp in length including a pair of inverted repeats (25,551-26,108 bp) separated by the large single-copy (86,028-86,566 bp) and small single-copy (18,021-19,117 bp) regions. Each chloroplast genome contains the same 114 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 80 protein coding genes. Gene size, content, and order, AT content, and IR/SC boundary structure are similar among all Araliaceae chloroplast genomes. A total of 140 repeats were identified in the five chloroplast genomes with palindromic repeat as the most common type. Phylogenomic analyses using parsimony, likelihood, and Bayesian inference based on the complete chloroplast genomes strongly supported the monophyly of the Asian Palmate group and the Aralia-Panax group. Furthermore, the relationships among the sampled taxa within the Asian Palmate group were well resolved. Twenty-six DNA markers with the percentage of variable sites higher than 5% were identified, which may be useful for phylogenetic studies of Araliaceae. CONCLUSION: The chloroplast genomes of Araliaceae are highly conserved in all aspects of genome features. The large-scale phylogenomic data based on the complete chloroplast DNA sequences is shown to be effective for the phylogenetic reconstruction of Araliaceae.

Isolation and characterization of microsatellite loci in the endangered tree Diplopanax stachyanthus (Araliaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Diplopanax stachyanthus to investigate the population genetics of this endangered tree. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, 15 microsatellite markers were developed in D. stachyanthus and evaluated for their variability in 25 samples from a natural population. For the 11 polymorphic loci, the number of alleles ranged from two to eight, while the observed and expected heterozygosities ranged from 0.5200 to 0.7600 and 0.4200 to 0.7813, respectively. Their cross-taxa transferability was also examined in Acanthopanax gracilistylus, Tetrapanax papyrifer, Cornus controversa, and Dendrobenthamia japonica var. chinensis, and four to 15 loci proved amplifiable in these species. CONCLUSIONS: These microsatellite markers could be employed to investigate the population genetics of D. stachyanthus, and may potentially be applicable to other related species.

Applying DNA barcodes for identification of plant species in the family Araliaceae.

An effective DNA marker in authentication of the family Araliaceae was screened out of the five DNA regions (matK, rbcL, ITS2, psbA-trnH and ycf5). In the present study, 1113 sequences of 276 species from 23 genera (Araliaceae) were collected from DNA sequencing and GenBank, in which 16 specimens were from 5 provinces in China and Japan. All of the sequences were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps and efficiency of identification. Compared with other markers, ITS2 showed superiority in species discrimination with an accurate identification of 85.23% and 97.29% at the species and genus levels, respectively, in plant samples from the 589 sequences derived from Araliaceae. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS2 was a powerful barcode for Araliaceae identification.

Reconstructing the species phylogeny of Pseudopanax (Araliaceae), a genus of hybridising trees.

Pseudopanax (Araliaceae) comprises 12 tree species of diverse morphology and ecology, and is endemic to New Zealand. It is notable for the hybridisation that occurs between P. crassifolius and P. lessonii, which have very different leaves and habits. To provide context for the study of this hybridisation and other investigations, we examined the phylogeny of Pseudopanax using chloroplast DNA sequences (c.5900 base-pairs) and AFLP DNA-fingerprinting. Both approaches resolve two principal groups within Pseudopanax--the Arboreus group and the Crassifolius+Lessonii union--but how they are related to other genera remains unclear. There is, nevertheless, little compelling evidence against the monophyly of Pseudopanax, making unnecessary the recent re-segregation of the Arboreus group as Neopanax. The chloroplast data provided minimal additional resolution, although the position of P. linearis was discordant compared to other data. Analyses of the AFLP data strongly recovered each species, aside from the morphologically heterogeneous P. colensoi, and the two mainland species (P. arboreus and P. crassifolius) that each contained a nested island-endemic (P. kermadecensis and P. chathamicus, respectively). However, relationships amongst species within the two principal groups were poorly resolved. An example was the uncertainty of whether P. crassifolius grouped with P. lessonii and its allies, or the morphologically similar species it had been previously placed with. This in turn raises the issue of how hybridisation might affect phylogenies and the ability to reconstruct them, even when using multiple, independent markers.

An improved technique for isolating codominant compound microsatellite markers.

An approach for developing codominant polymorphic markers (compound microsatellite (SSR) markers), with substantial time and cost savings, is introduced in this paper. In this technique, fragments flanked by a compound SSR sequence at one end were amplified from the constructed DNA library using compound SSR primer (AC)6(AG)5 or (TC)6(AC)5 and an adaptor primer for the suppression-PCR. A locus-specific primer was designed from the sequence flanking the compound SSR. The primer pairs of the locus-specific and compound SSR primers were used as a compound SSR marker. Because only one locus-specific primer was needed for design of each marker and only a common compound SSR primer was needed as the fluorescence-labeled primer for analyzing all the compound SSR markers, this approach substantially reduced the cost of developing codominant markers and analyzing their polymorphism. We have demonstrated this technique for Dendropanax trifidus and easily developed 11 codominant markers with high polymorphism for D. trifidus. Use of the technique for successful isolation of codominant compound SSR markers for several other plant species is currently in progress.

[Phylogenetic relationships of the Far Eastern Araliaceae inferred from ITS sequences of nuclear rDNA].

In eight species of the family Araliaceae, inhabiting the territory of the Russian Far East, the sequences of ITS regions of nuclear rDNA were determined. A comparison of these sequences enabled establishment of phylogenetic relationships between the Far Eastern and other members of the family. It was demonstrated that Aralia sensu populations from Primorye and Sakhalin were genetically different and, hereby, could be classified as interspecific taxa. Aralia continentalis along with A. cordata were attributed to the section Aralia sensu. Oplopanax elaus and O. horridus were found to be very close to each other, possibly being the subspecies of one species or relatively young species. Legitimacy of the isolation of two sections within the genus Eleutherococcus was confirmed.

[Genetic relationships among Far Eastern species of the family Araliacea inferred by RAPD analysis]. / Izuchenie geneticheskikh sviazei mezhdu dal'nevostochnymi vidami semeistva Araliaceae metodom RAPD.

A molecular genetic study of Far Eastern species of the family Araliaceae by means of RAPD analysis was conducted. Using 21 primers we assessed variability at 595 loci. Based on matrices of genetic distances D, dendrograms of genetic relationships among eleven species of six genera of this family were constructed. Our results suggest that Acanthopanax sessiliflorus and Eleutherococcus senticosus belong to different genera, Aralia cordata and A. continentalis are different species, and A. elata and A. mandshurica probably cannot be regarded as distinct species. Genetic similarity of Far Eastern A. cordata and American A. hispida is shown.