Systematic studies on some species of the genus Artemisia: biomolecular analysis

ABSTRACT

The internal transcribed spacers (ITS) of the ribosomal DNA gene of 11 taxa of the genus Artemisia were sequenced and compared with other 14 species taken from GenBank. The aims of this study are to clarify phylogenetic relationships for 25 taxa within the genus Artemisia, and to highlight the phylogenetic position of some species of geobotanical interest from the Alps or from other European areas. The results support the monophyly of the genus Artemisia, and the presence of the five main clades, corresponding to the morphologically based sections, Absinthium, Artemisia, Seriphidium, Dracunculus and Tridentatae. Only A. annua and A. genipi are not classified in the section in which they were traditionally included: A. annua is assigned to Seriphidium and not Artemisia, and A. genipi to Absinthium and not Artemisia. The basal structure of the tree differed in the 45 equally parsimonious MP trees, and thus appeared as a polytomy in the consensus tree. This does not allow us to completely solve the relationships among the clades. The molecular data are complementary with the morphological and biogeographical information and all are essential to draw valid conclusions on the relative closeness of the various taxa.     

Systematic status and phylogenetic relationships of the enigmatic Tanacetum paradoxum Bornm. (Asteraceae, Anthemideae): evidences from nrDNA ITS, micromorphological, and cytological data

In order to evaluate the phylogenetic relationships and taxonomic classification of the Iranian endemic Tanacetum paradoxum Bornm. (Asteraceae, Anthemideae), a molecular analysis based on nrDNA ITS sequences of 16 species of Tanacetum together with representatives of 27 genera of subtribes Artemisiinae and Handeliinae as well as a micromorphological study of achenes and pollen grains was performed. The chromosome number of 2n?=?2x?=?16 and the genome size of T. paradoxum (2C?=?8.81?±?0.19?pg) are reported here for the first time. Due to the well-supported close phylogenetic relationship of T.?paradoxum to the species of the genus Artemisia (Bayesian inference, posterior probability PP?=?1), which is supported by cytological (x?=?8) and micromorphological features concerning the achene shape and pollen type, the combination Artemisia paradoxa (Bornm.) Sonboli is proposed.     

Chloroplast genome sequences of Artemisia maritima and Artemisia absinthium: Comparative analyses,mutational hotspots in genus Artemisia and phylogeny in family Asteraceae

Artemisia L. is a complex genus of medicinal importance. Publicly available chloroplast genomes of few Artemisia species are insufficient to resolve taxonomic discrepancies at species level. We report chloroplast genome sequences of two further Artemisia species: A. maritima (151,061 bp) and A. absinthium (151,193 bp). Both genomes possess typical quadripartite structure comprising of a large single copy, a small single copy and a pair of long inverted repeats. The two genomes exhibited high similarities in genome sizes, gene synteny, GC content, synonymous and non-synonymous substitutions, codon usage, amino acids frequencies, RNA editing sites, microsatellites, and oligonucleotide repeats. Transition to transversion ratio was <1. Maximum likelihood tree showed Artemisia a monophyletic genus, sister to genus Chrysanthemum. We also identified 20 highly polymorphic regions including rpoC2-rps2, trnR-UCU-trnG-UCC, rps18-rpl20, and trnL-UAG-rpl32 that could be used to develop authentic and cost-effective markers to resolve taxonomic discrepancies and infer phylogenetic relationships among Artemisia species.     

Multigene phylogenetic analysis reveals non-monophyly of Anisakis s.l. and Pseudoterranova (Nematoda: Anisakidae)

The nematode genera Anisakis s.l. and Pseudoterranova (Anisakidae) include causative agents of anisakiasis and pseudoterranovosis, parasitic diseases resulting from eating undercooked or raw fish or squid. Species in both genera have thus attracted considerable attention especially in public health and taxonomic studies. The phylogenetic relationships of these genera within the subfamily Anisakinae, however, remain to be investigated with dense taxonomic sampling. In this study, we collected an anisakid third-stage larva, and identified it morphologically and molecularly as Pseudoterranova ceticola. Phylogeny of 15 anisakine species, including the newly collected specimen of Ps. ceticola, was reconstructed based on sequences of three mitochondrial (cox1, cox2, and 12S rRNA) and two nuclear (ITS and 28S rRNA) regions. The obtained tree suggested the non-monophyly of Anisakis s.l. and Pseudoterranova. Anisakis s.l. was divided into two groups, which are distinguished from each other by the shape of the ventriculus. Based on phylogenetic relationships and morphology, three species with a shorter ventriculus (“A.” brevispiculata, “A.” paggiae, and “A.” physeteris) were assigned to the genus Skrjabinisakis, as recently proposed. Pseudoterranova ceticola was distantly related to the monophyletic Ps. decipiens species complex. Although the phylogenetic position of the type species Ps. kogiae has not been investigated due to a lack of sequence data, this species may morphologically and ecologically resemble Ps. ceticola, inferring a close kinship between the two 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.     

莪术基原植物DNA条形码序列的筛选与鉴定

为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件...     

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.     

DNA Barcode ITS Effectively Distinguishes the Medicinal Plant Boerhavia diffusa from Its Adulterants

Boerhavia diffusa (B. diffusa), also known as Punarnava, is an indigenous plant in India and an important component in traditional Indian medicine. The accurate identification and collection of this medicinal herb is vital to enhance the drug’s efficacy and biosafety. In this study, a DNA barcoding technique has been applied to identify and distinguish B. diffusa from its closely-related species. The phylogenetic analysis was carried out for the four species of Boerhavia using barcode candidates including nuclear ribosomal DNA regions ITS, ITS1, ITS2 and the chloroplast plastid gene psbA-trnH. Sequence alignment revealed 26% polymorphic sites in ITS, 30% in ITS1, 16% in ITS2 and 6% in psbA-trnH, respectively. Additionally, a phylogenetic tree was constructed for 15 species using ITS sequences which clearly distinguished B. diffusa from the other species. The ITS1 demonstrates a higher transition/transversion ratio, percentage of variation and pairwise distance which differentiate B. diffusa from other species of Boerhavia. Our study revealed that ITS and ITS1 could be used as potential candidate regions for identifying B. diffusa and for authenticating its herbal products.     

Strong plastid degradation is consistent within section Chondrophyllae,the most speciose lineage of Gentiana

Recovering phylogenetic relationships in lineages experiencing intense diversification has always been a persistent challenge in evolutionary studies, including in Gentiana section Chondrophyllae sensu lato (s.l.). Indeed, this subcosmopolitan taxon encompasses more than 180 mostly annual species distributed around the world. We sequenced and assembled 22 new plastomes representing 21 species in section Chondrophyllae s.l. In addition to previously released plastome data, our study includes all main lineages within the section. We reconstructed their phylogenetic relationships based on protein‐coding genes and recombinant DNA (rDNA) cistron sequences, and then investigated plastome structural evolution as well as divergence time. Despite an admittedly humble species cover overall, we recovered a well‐supported phylogenetic tree based on plastome data, and found significant discordance between phylogenetic relationships and taxonomic treatments. Our results show that G. capitata and G. leucomelaena diverged early within the section, which is then further divided into two clades. The divergence time estimation showed that section Chondrophyllae s.l. evolved in the second half of the Oligocene. We found that section Chondrophyllae s.l. had the smallest average plastome size (128 KB) in tribe Gentianeae (Gentianaceae), with frequent gene and sequence losses such as the ndh complex and its flanking regions. In addition, we detected both expansion and contraction of the inverted repeat (IR) regions. Our study suggests that plastome degradation parallels the diversification of this group, and illustrates the strong discordance between phylogenetic relationships and taxonomic treatments, which now need to be carefully revised.     

Beiträge zur Systematischen Anatomie derAsteraceae-Anthemideae: Die Trichome

Biseriate glandular trichomes and uniseriate flagellar covering hairs occur in all or most of the 20 genera examined and thus do not provide important taxonomical hints. In contrast, T- or Y-shaped hairs have been found inSantolina, Anthemis, Leucanthemopsis, Dendranthema, Tanacetum,Balsamita, Sphaeromeria (?),Artemisia, andAchillea clavenae only. Stellate hairs appear to be rare (inSantolina andArtemisia sect.Dracunculus). For details, additional hair types, and relationships see Tab.1 and Fig. 6.     

Phylogenetic reconstruction of endophytic fungal isolates using internal transcribed spacer 2 (ITS2) region

Endophytic fungi are inhabitants of plants, living most part of their lifecycle asymptomatically which mainly confer protection and ecological advantages to the host plant. In this present study, 48 endophytic fungi were isolated from the leaves of three medicinal plants and characterized based on ITS2 sequence – secondary structure analysis. ITS2 secondary structures were elucidated with minimum free energy method (MFOLD version 3.1) and consensus structure of each genus was generated by 4SALE. ProfDistS was used to generate ITS2 sequence structure based phylogenetic tree respectively. Our elucidated isolates were belonging to Ascomycetes family, representing 5 orders and 6 genera. Colletotrichum/Glomerella spp., Diaporthae/Phomopsis spp., and Alternaria spp., were predominantly observed while Cochliobolus sp., Cladosporium sp., and Emericella sp., were represented by singletons. The constructed phylogenetic tree has well resolved monophyletic groups with >50% bootstrap value support. Secondary structures based fungal systematics improves not only the stability; it also increases the precision of phylogenetic inference. Above ITS2 based phylogenetic analysis was performed for our 48 isolates along with sequences of known ex-types taken from GenBank which confirms the efficiency of the proposed method. Further, we propose it as superlative marker for reconstructing phylogenetic relationships at different taxonomic levels due to their lesser length.     

Sesquiterpene lactones and systematics of the genus Artemisia

The sesquiterpene lactones isolated from species in the genus Artemisia have been reviewed in an attempt to better understand the phylogeny and systematics of the four sections (subgenera), Abrotanum, Absinthium, Dracunculus and Seriphidium, proposed by Besser in 1829. The absence of hair on the receptacle is the only morphological characteristic separating species of Abrotanum from the species of Absinthium. There are no chemical characteristics segregating the species in these two subgenera since both produce eudesmanolides and guaianolides that are identical or biosynthetically similar. This suggests that the two subgenera could be combined into one (Artemisia) as proposed by Poljakov. The subgenus Seriphidium is composed of two geographical groups, one in the Old World and the other in the New World. The Old World species almost exclusively produce sesquiterpene lactones in the eudesmanolide class whereas the New World species (section Tridentatae) produce eudesmanolides and guaianolides, many of the latter being identical or structurally related to the sesquiterpene lactones in New World Abrotanum species. The chemical data in conjunction with geographic distributions suggest that the subgenus Seriphidium is polyphyletic and that the section Tridentatae originated from Abrotanum. Consequently, the Tridentate should be recognized as a subgenus separate and distinct from the Old World Seriphidium. There was insufficient information from the subgenus Dracunculus for interpretation.     

ITS sequence data resolve higher level relationships among the eucalypts.

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were obtained for 35 species of Eucalyptus s.s. and seven taxa representing five outgroup genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, and Stockwellia). The sequences were analyzed cladistically. The data distinguished clearly between the two major subgenera of Eucalyptus s.s. (Symphyomyrtus and Monocalyptus) but indicated that subgenus Eudesmia may be paraphyletic. ITS sequence data demonstrated the potential to resolve relationships between sections within subgenus Symphyomyrtus. Within sections, however, taxa were poorly differentiated. At the generic level, Corymbia appeared to be paraphyletic due to the exclusion of Angophora. The positions of Allosyncarpia and Arillastrum relative to the ingroup remain unresolved. ITS sequence data may prove valuable for resolving other phylogenetic relationships at higher taxonomic levels within Eucalyptus.     

Vertebrate evolution reflected in the evolution of nuclear ribosomal internal transcribed spacer 2

In eukaryotes, mature rRNA sequences are produced from single large (45S) precursor (pre-rRNA) as the result of successive removal of spacers through a series of rapid and intricate actions of endo- and exonucleases. The excision of internal transcribed spacer (ITS2), a eukaryotic-specific insertion, remains the most elusive processing step. ITS2 is the element mandatory for all eukaryotic pre-rRNAs that contain at least three processing cleavage sites for precise 5.8S and 28S formation. Conserved core sequences (cis-elements) binding to trans-factors provide for precise rRNA processing, whereas rapidly diverging regions between the core sequences preserve internal complementarity, which guarantees the spatial integrity of ITS2. Characteristic differences in the formation of such insertions during evolution should reflect the relationships between taxa. The phylogeny of the reptiles and the relationships between taxa proposed by scientists are controversial. To delineate the structural and functional features preserved among reptilian ITS2s, we cloned and sequenced 58 ITS2s belonging to four reptile orders: Squamata, Crocodilians, Aves, and Testudines. We studied the subsequent alignment and folding of variable regions. The sizes and packing of the loop–stems between conserved consensus segments in reptiles vary considerably between taxa. Our phylogenetic trees constructed on the basis of the reptile ITS2s primary structural alignments revealed a split between Iguania clade and all other taxa. True lizards (suborder Scleroglossa) and snakes (suborder Serpentes) show sister relationships, as well as the two other reptilian orders, Crocodilia + Aves and Testudines. In summary, our phylogenetic trees exhibit a mix of specific features deduced or, to the contrary, rejected earlier by other authors.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

The rpb2 gene represents a viable alternative molecular marker for the analysis of environmental fungal communities

Although the commonly used internal transcribed spacer region of rDNA (ITS) is well suited for taxonomic identification of fungi, the information on the relative abundance of taxa and diversity is negatively affected by the multicopy nature of rDNA and the existence of ITS paralogues. Moreover, due to high variability, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa. The part of single‐copy gene encoding the second largest subunit of RNA polymerase II (rpb2) was thus compared with first spacer of ITS as an alternative marker for the analysis of fungal communities in spruce forest topsoil, and their applicability was tested on a comprehensive mock community. In soil, rpb2 exhibited broad taxonomic coverage of the entire fungal tree of life including basal fungal lineages. The gene exhibited sufficient variation for the use in phylogenetic analyses and taxonomic assignments, although it amplifies also paralogues. The fungal taxon spectra obtained with rbp2 region and ITS1 corresponded, but sequence abundance differed widely, especially in the basal lineages. The proportions of OTU counts and read counts of major fungal groups were close to the reality when rpb2 was used as a molecular marker while they were strongly biased towards the Basidiomycota when using the ITS primers ITS1/ITS4. Although the taxonomic placement of rbp2 sequences is currently more difficult than that of the ITS sequences, its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses represent significant advantages.     

Chloroplastrps16 intron phylogeny of the tribeSileneae (Caryophyllaceae)

Intron sequences of the chloroplast generps16 from 46 species were used to examine phylogenetic relationships indicated by nrDNA ITS sequence variation in the tribeSileneae (Caryophyllaceae, Caryophylloideae). This region has previously not been utilized for phylogenetic purposes but the results presented here suggest that it is a consistent and valuable complement to the ITS sequences. Therps16 intron trees are largely congruent with the ITS trees. All the major hypotheses suggested by the ITS data are supported, often at similar bootstrap levels. The joint usage ofrps16 intron and ITS sequences provides a powerful tool for resolving many of the difficult taxonomic issues in the tribeSileneae. Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

基于ITS序列的东亚当归属植物的分类学研究

采用PCR直接测序法,测定了东亚地区狭义当归属Angelica s.s.及其近缘共7属40种代表植物的核糖体DNA ITS序列,并结合GenBank中相关植物的ITS序列(含外类群3种),应用遗传距离与系统树分析法对东亚地区狭义当归属植物内部以及当归属与其近缘属植物之间的亲缘关系进行了分析。结果表明:(1)广义当归属中的狭义当归属、柳叶芹属Czernaevia和高山芹属Coelopleurum之间的亲缘关系较近,山芹属Ostericum与它们的亲缘关系较远,这与果实形态、化学分析的结果一致,建议将山芹属作为一个相对独立的分类群处理。(2)ITS序列分析结果支持狭义当归属不是单起源的自然分类群,而应该被分成若干组的观点。(3)ITS序列以及化学成分分析结果表明,前胡属Peucedanum与狭义当归属之间的亲缘关系很近。(4)形态、化学成分以及ITS等多方面分析结果显示,当归A.sinensis与狭义当归属的多数植物之间均有一定的差距,其归属问题值得商榷。(5)ITS序列与化学成分的分析结果均显示藁本属Ligusticum不是一个自然类群。