ITS2 Secondary Structure Research Articles

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models.

Among ciliates, Paramecium has become a privileged model for the study of "species problem" particularly in the case of the "Paramecium aurelia complex" that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5'LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.

<p><strong>Morphological and molecular investigation of freshwater <em>Hildenbrandia</em> (Hildenbrandiales, Rhodophyta) with a new species reported from Japan</strong></p>

The morphological characterization and phylogenetic analysis based on molecular methods were executed on the freshwater Hildenbrandia specimens collected in this study. The cell size and filament height of the specimen collected from Pipa Lake, Japan were larger than the widely recognized species Hildenbrandia angolensis and Hildenbrandia rivularis, whereas overlapped with Hildenbrandia jigongshanensis. Based on molecular evidences of rbcL phylogenetic analysis and comparison of ITS1 secondary structure, specimen collected from Shiga Prefecture, Japan was proposed as a new species Hildenbrandia japananense. It brings the freshwater Hildenbrandia species in Japan to two. The specimens collected from Niangziguan, Shanxi province, China represent two new records of H. jigongshanensis. It enriches the species diversity of genus Hildenbrandia and increases the distribution diversity of this freshwater red algal taxon.

Application of ITS2 secondary structure phylogenetic information in DNA barcode identification of Chrysanthemum indicum and its related plants

By exploring additional phylogenetic information hidden in ITS2 secondary structure,the possibility of identifying Chrysanthemum indicum and its related species with DNA barcode of ITS2 nucleic acid sequence and its structure information were discussed.The genomic DNA was extracted from 12 samples. The ITS2 fragments were amplified by PCR and sequenced bidirectionally to obtain ITS2 sequence information. 28 sequences of related species for Ch. indicum were downloaded from Gen Bank. Until all 40 ITS2 sequences were aligned,ITS2 secondary structure prediction and structure comparison were finished. Then ITS2 secondary structure information was coded. After comparing ITS2 structure information and nucleic acid information,MP phylogenetic trees were built. The results showed that the secondary structures of ITS2 shared the same structure model--a four-fingered hand. They not only have the common characteristics of ITS2 secondary structures in plants,but also have many other conservative sequences,and their overall conservativeness is high. Among all species used in this study,their ITS2 secondary structures had obvious difference. In addition,the number of mutation sites in the joint matrix compared with the nucleic acid sequences increased by nearly 90%,which greatly enriched the number of mutation sites. This method of information analysis distinguished Ch. indicum from its related species. At the same time,the support rate of the branches of evolutionary trees and the identification rate of species were significantly improved. Although there was no distinction between Ch. zawadskii and Ch. morifolium,it effectively distinguished the three species,namely,Ch. hypargyrum,Ch.oreastrum,and Ch. dichrum. Therefore,the authors suggest that the ITS2 sequence combined with its structural data information should be applied to the identification of Ch. indicum and its related species,and be widely applied to DNA barcode research.

Evaluating High-Throughput Sequencing Data of Microalgae Living in Melting Snow: Improvements and Limitations1.

Melting snow fields are an extremophilic habitat dominated by closely related Chlamydomonadaceae (Chlorophyta). Microscopy-based classification of these cryophilic microalgae is challenging and may not reveal the true diversity. High-throughput sequencing (HTS) allows for a more comprehensive evaluation of the community. However, HTS approaches have been rarely used in such ecosystems and the output of their application has not been evaluated. Furthermore, there is no consensus on the choice for a suitable DNA marker or data processing workflow. We found that the correct placement of taxonomic strings onto OTUs strongly depends on the quality of the reference databases. We improved the assignments of the HST data by generating additional reference sequences of the locally abundant taxa, guided by light microscopy. Furthermore, a manual inspection of all automated OTU assignments, oligotyping of the most abundant 18S OTUs, as well as ITS2 secondary structure analyses were necessary for accurate species assignments. Moreover, the sole use of one marker can cause misleading results, either because of insufficient variability within the locus (18S) or the scarcity of reference sequences (ITS2). Our evaluation reveals that HTS output needs to be thoroughly checked when the studied habitats or organisms are poorly represented in publicly available databases. We recommend an optimized workflow for an improved biodiversity evaluation of not only snow algal communities, but generally 'exotic' ecosystems where similar problems arise. A consistent sampling strategy, two- molecular marker approach, light microscopy-based guidance, generation of appropriate reference sequences and final manual verification of all taxonomic assignments are highly recommended.

Phylogenetic complexities of the members of Rivulariaceae with the re-creation of the family Calotrichaceae and description of Dulcicalothrix necridiiformans gen nov., sp nov., and reclassification of Calothrix desertica.

A freshwater dwelling, tapering, heterocytous cyanobacterium (strain V13) was isolated from an oligotrophic pond in the Shrirampur taluka, Ahmednagar district of Maharashtra in India. Initial morphological examination indicated that strain V13 belonged to the genus Calothrix. Subsequent molecular and phylogenetic assessment based on 16S rRNA gene, led us to describe the freshwater/terrestrial clade of Calothrix strains without terminal hairs as a new genus Dulcicalothrix gen. nov., with the type species Dulcicalothrix necridiiformans sp. nov. (Strain V13) on the basis of the necridia forming ability of the strain. Also, the 16S-23S ITS secondary structure analysis clearly differentiated strain V13 from the other members of the clade. Past studies and the current state of knowledge makes it imperative to separate the groups Calothrix (marine/freshwater Calothrix), Macrochaete and Dulcicalothrix (freshwater/terrestrial Calothrix) into separate genera in accordance with the International Code of Nomenclature for Algae, Fungi and Plants. Robust phylogenetic evidence and previous reports strongly support the re-erection of the family Calotrichaceae distinct from the existing family Rivulariaceae.

Coccomyxa cimbrica sp. nov., a green microalga found in association with carnivorous plants of the genus Drosera L.

ABSTRACTCoccomyxa Schmidle (Trebouxiophyceae, Chlorophyta) is a genus of green microalgae that has worldwide distribution, with representatives found in a broad range of terrestrial and aquatic ecosystems. Members of this taxon can be free-living and/or associated with other organisms, establishing different types of relationships (e.g. parasitic, symbiotic). The most frequent organisms forming associations with Coccomyxa are fungi and marine animals, most importantly mussels. Moreover, there is a well-documented association between a Coccomyxa strain and Gingko biloba and some Coccomyxa-like microalgae have been found in the bark of other trees. Here we describe a green algal strain found in association with carnivorous plants of the genus Drosera L. The systematics of Coccomyxa based only on classical methods is problematic, due to the very simple morphology of the genus and its high phenotypic plasticity. Therefore, the microalga associated with Drosera plants was isolated, maintained in culture and subjected to an integrative taxonomy approach, including morphological, ultrastructural and molecular analyses, along with ecological considerations. Phylogenetic reconstructions based on the rbcL and 18S rRNA genes and molecular data obtained from the ITS2 spacer, including both sequence and secondary structure analyses, showed that the investigated microorganism belonged to a lineage distinct from those sequenced so far. An accurate taxonomic comparison between the microalga associated with Drosera and each of the species already described for the genus Coccomyxa was also carried out, considering morphology, ultrastructure and ecology. The results as a whole allowed us to erect a new species inside the genus Coccomyxa: Coccomyxa cimbrica K.Sciuto, B.Baldan & I.Moro

A bridge too far in naming species: a total evidence approach does not support recognition of four species in Desertifilum (Cyanobacteria).

A population of Desertifilum (Cyanobacteria, Oscillatoriales) from an oligotrophic desertic biotope was isolated and characterized using a polyphasic approach including molecular, morphological, and ecological information. The population was initially assumed to be a new species based on ecological and biogeographic separation from other existing species, however, phylogenetic analyses based on sequences of the 16S rRNA gene and 16S-23S ITS region, placed this strain clearly within the type species, Desertifilum tharense. Comparative analysis of morphology, 16S rRNA gene similarity, 16S-23S ITS secondary structure, and percent dissimilarity of the ITS regions for all characterized strains supports placing the six Desertifilum strains (designated as PD2001/TDC17, UAM-C/S02, CHAB7200, NapGTcm17, IPPAS B-1220, and PMC 872.14) into D.tharense. The recognition of Desertifilum salkalinema and Desertifilum dzianense is not supported, although our analysis does support continued recognition of Desertifilum fontinale. Pragmatic criteria for recognition of closely related species are proposed based on this study and others, and more rigorous review of future taxonomic papers is recommended.

Untangling a mess of worms: Species delimitations reveal morphological crypsis and variability in Southeast Asian semi-aquatic earthworms (Almidae, Glyphidrilus)

Semi-aquatic freshwater earthworms in the genus Glyphidrilus from Southeast Asia are characterized by both an extreme morphological crypsis among divergent phylogenetic lineages and a high morphological variability within the same phylogenetic lineages. The present study provides a new taxonomic framework for this problematic genus in SE Asia by integrating DNA sequence and morphological data. When single-locus and multilocus multispecies coalescent-based (MSC) species delimitation methods were applied to DNA sequence data, they usually yielded highly incongruent results compared to morphology-based species identifications. This suggested the presence of several cryptic species and high levels of intraspecific morphological variation. Applying reciprocal monophyly to the cytochrome c oxidase subunit 1 (COI) gene tree allowed us to propose the existence of 33 monophyletic species. Yet, often substantially more molecular operational taxonomic units (MOTUs) were obtained when species delimitation was based on COI and 16S rRNA sequences. In contrast, the ITS1 and ITS2 sequences suggested fewer MOTUs and did not recover most of the monophyletic species from the Mekong basin. However, several of these latter taxa were better supported when MSC species delimitation methods were applied to the combined mtDNA and ITS datasets. The ITS2 secondary structure retrieved one unnamed Mekong basin species that was not uncovered by the other methods when applied to ITS2 sequences. In conclusion, based on an integrative taxonomic workflow, 26 Glyphidrilus candidate species were retained and two remained to be confirmed. As such, this study provides evidence to suggest nine species new to science and to synonymize 12 nominal morphospecies. It also illustrates that the uncritical use of COI as a universal DNA barcode may overestimate species diversity because COI may be unable to distinguish between divergent conspecific lineages and different candidate species.

Identification of flower herbs in Chinese pharmacopoeia based on DNA barcoding

ObjectiveFlower herbs are an important category of traditional Chinese medicinal materials, some of which are used as healthcare tea in folk. However, the increasing adulteration of medicinal herbs is threatening consumer safety. The adulteration of flower herbs and their healthcare tea products in the market were investigated. MethodsA total of 33 flower herb samples from several retail pharmacies in China were randomly collected and 27 flower healthcare tea samples were purchased online. They were identified using ITS2-based Traditional Chinese Medicine Database (TCMD). Additionally, Lonicerae Japonicae Flos and the adulterants were compared in the ITS2 secondary structures. ResultsThere were one adulterant (Inulae Flos) in flower herb materials and eight adulterants in healthcare tea samples. Inula linariifolia was an adulterate species of Inulae Flos, Robinia pseudoacacia was of Sophorae Flos, and Lonicera macranthoides was of Lonicerae Japonicae Flos. Sophorae Flos and Lonicerae Japonicae Flos were two healthcare tea products with high adulteration rates. ConclusionThe TCMD is powerful tool to identify flower herbs and the adulterants that frequently occurred in the flower herb market, especially online shops.

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological, molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.

Identification and Phylogeny of Cryptic Species of the Daphnia Longispina Complex (Anomopoda, Daphniidae) Using ITS2 Secondary Structure

Cladocerans of the genus Daphnia O.F. Muller (Crustacea: Cladocera) are typical members of different types of water bodies and are used as model organisms in various biological disciplines. Within the genus, a considerable number of closely related and cryptic species are noted, the identification of which by morphological traits often causes difficulties. In the present study, species identification in the Daphnia longispina complex, namely, D. longispina O.F. Muller, D. hyalina Leydig, D. umbra Taylor, Hebert et Colbourne, and D. cf. longispina, was performed using data on the predicted secondary structure of the second internal transcribed spacer (ITS2) of the nuclear rDNA. Phylogenetic analysis carried out using the Profile Neighbor-Joining algorithm and the analysis of compensatory base changes supported the species status of D. hyalina and D. cf. longispina. It was demonstrated that the data on the ITS2 secondary structure can be used to resolve complex taxonomic problems in the study of cladocerans in general and the species complexes in particular.

Molecular authentication and differentiation of Dendrobium species by rDNA ITS region sequence analysis

Owing to their significant medicinal and edible values, the natural Dendrobium species have underdone over-collection and habitat destruction, and cultivated species emerged for candidates. However, these Dendrobium plants are similar in shape to be easily confused, leading to extreme difficulties for identification based on their morphological and chemical features. In this study, the rDNA ITS region sequence analysis was developed for rapid and accurate identification of thirteen wild and cultivated Dendrobium species belonging to two sections Formosae and Chrysotoxae. By cloning and sequencing the rDNA ITS region genes from 13 Dendrobium species, the phylogenetic relationships among them were analyzed. Results showed that the variation of the ITS region, together with the lengths and Guanine and Cytosine contents of ITS, 5.8s rDNA, ITS1 and ITS2 sequences occurred in the tested Dendrobium species, and which from section Chrysotoxae was higher than that from section Formsae. Phylogenetic analysis based on neighbor-joining and maximum p-arsimony trees indicated that the Dendrobium species of sections Formosae and Chrysotoxae could be well divided into two groups. A majority of Dendrobium species exhibited distinctive ITS2 secondary structures, while for those with close genetic relationships were similar. Therefore, the ITS2 region sequence analysis is simple, quick, and highly reliable that can be used as an effective tool for molecular identification and classification, as well as the reconstruction of the phylogeny of wild and cultivated Dendrobium species belonging to different sections.

Biogeography and phylogenetic relationships of Hyrcanian wild apple using cpDNA and ITS noncoding sequences

The Hyrcanian forest of northern Iran is considered one of the potential centres for the evolution and domestication of the genus Malus (Rosaceae). However, the biogeography, phylogenetic position, and taxonomic status of the Hyrcanian wild apples have never been evaluated. In our study, the nucleotide sequences of the internal transcribed spacer (ITS) and the trnH-psbA intergenic spacer region from 14 natural populations were analysed. Phylogenetic analysis based on the ITS and the Maximum-likelihood (ML) tree showed that all Hyrcanian samples were closely related to M. orientalis and M. asiatica and can be placed within section Malus and series Malus. Furthermore, based on a comparison of ITS2 secondary structures, the Hyrcanian samples were identical to M. orientalis and M. sieversii. Biogeographic scenarios constructed using Statistical Dispersal-Vicariance Analysis (S-DIVA) and the Bayesian Binary Method (BBM) indicated that the ancestor of Malus originated during the Eocene, ∼53 million years ago (Ma), and that China played a vital role in the expansion of the range of the genus. The members of Malus colonized the Hyrcanian region from China during the Miocene, ∼22-10 Ma.

Desmodesmus tropicus (Chlorophyta) in the Danube Delta – reassessing the phylogeny of the series Maximi

ABSTRACT Desmodesmus maximus (West & G.S.West) Hegewald, D. perforatus (Lemmermann) E.Hegewald and D. tropicus (W.B.Crow) E.Hegewald form the series Maximi within the genus Desmodesmus. Desmodesmus tropicus is a rare species although it is widely distributed in tropical regions including South-East Asia, South America and the USA. It is less common in temperate fresh waters, known only from Ukraine. In this study, we report for the first time its presence in the Danube Delta (Puiu Lake), Romania. The morphology and molecular phylogeny of two newly isolated strains were studied in relation to other strains of the genus Desmodesmus, series Maximi. Cell wall structures (rosettes, spines, tubes, honeycomb-shaped mesh with hexagonal units) which are important in the taxonomy of these algae were investigated by light-, scanning- and transmission electron microscopy. The phylogenetic position of the strains in the Algae and Cyanobacteria Culture Collection of the Institute of Biological Research was assessed using 18S rDNA sequences, ITS2 primary and secondary structures and by analysis of Compensatory Base Changes. Morphological traits and measurements of cells, coenobia and spines matched the description of D. tropicus, although some differences from the established morphological key (ridge-like structures resembling fine ribs) were also noticed. Identification as D. tropicus var. longiclathratus (Tell) Jeon & Hegewald was based on the presence of inner spines on four-celled and especially on eight-celled coenobia. The ITS2 sequence and secondary structure phylogeny supported the assignment of both AICB strains to D. tropicus, with no divergence from other D. tropicus sequences to support the identification of var. longiclathratus. Nevertheless, the morphological measurements and the ITS2 primary and secondary structure data indicated two distinct subclusters within D. tropicus, and a new possible taxon within the series Maximi.

Phylogenetically distant clade of Nostoc-like taxa with the description of Minunostoc gen. nov. and Minunostoc cylindricum sp. nov

Two cyanobacterial strains morphologically identified to the genus Nostoc were isolated from a wet rocky wall in a mid-subtropical region in China, and they were taxonomically and phylogenetically characterized based on the polyphasic approach combining morphological and genetic characteristics. 16S rRNA gene sequence analysis showed that the two strains containing six clones were all >99.6% similar to each other, but had < 94.3% similarities to the existing cyanobacterial genera. The phylogenies based on 16S rRNA and rpoC1 gene sequences indicated that their sequences grouped into a unique and robust cluster with high bootstrap values. This unique cluster was separated from the clade of the 'Nostoc sensu stricto' and the respective clades formed by the morphologically similar genera Mojavia, Desmonostoc, Aliinostoc, Komarekiella and Halotia. The 16S-23S rRNA ITS secondary structure of the both strains exhibited the unique pattern of D1-D1´, Box-B and V3 helix, distinguishing it from the other heterocytous genera. Such a clear cluster leads to the establishment of Minunostoc gen. nov., with the type species as Minunostoc cylindricum sp. nov.

Species composition and toxicity of the genus Pseudo-nitzschia in Taiwan Strait, including P. chiniana sp. nov. and P. qiana sp. nov.

In a field survey in the Taiwan Strait during April 2016, the species composition and the domoic acid production of the diatom genus Pseudo-nitzschia were investigated. A total of 80 strains of Pseudo-nitzschia were established, and species identification was determined based on a combination of morphological and molecular data. Fourteen taxa were recognized, i.e., P. americana, P. brasiliana, P. calliantha, P. cuspidata, P. galaxiae, P. lundholmiae, P. multiseries, P. multistriata, P. pseudodelicatissima, P. pungens var. aveirensis, P. pungenus var. pungens and P. sabit, as well as two novel species P. chiniana C.X. Huang & Yang Li and P. qiana C.X. Huang & Yang Li. Morphologically, P. chiniana is characterized by striae comprising one or two rows of poroids, and valve ends that are normally dominated by two rows of poroids within each stria. Whereas P. qiana is unique by having a narrow valve width (1.3–1.5 μm) and sharply pointed valve ends. Both taxa constitute their own monophyletic lineage in the phylogenetic analyses inferred from LSU and ITS2 rDNA, and are well differentiated from other Pseudo-nitzschia species. Pseudo-nitzschia chiniana forms a group with P. abrensis and P. batesiana in LSU and ITS trees, whereas P. qiana is sister to P. lineola. When comparing ITS2 secondary structure, five CBCs and seven HCBCs are recognized between P. chiniana and P. abrensis, and four CBCs and ten HCBCs between P. chiniana and P. batesiana. Two CBCs and eight HCBCs are found between P. qiana with P. lineola. The ability of the strains to produce domoic acid was assessed, including a potential toxin induction by the presence of brine shrimps. Results revealed production of domoic acid in six strains belonging to three species. Without presence of brine shrimps, cellular DA (pDA) was detected in four P. multiseries strains (1.6 ± 0.3, 26.6 ± 2.7, 68.3 ± 4.2 and 56.9 ± 4.7 fg cell−1, separately), one strain of P. pseudodelicatissima (0.8 ± 0.2 fg cell−1) and one strain of P. lundholmiae (2.5 ± 0.4 fg cell−1). In the presence of brine shrimps, pDA contents increased significantly (p < 0.05) in P. lundholmiae (strain MC4218) and P. multiseries (strain MC4177), from 2.5 ± 0.4 to 8.9 ± 0.7 and 1.6 ± 0.3 to 37.2 ± 2.5 fg cell−1 respectively.

Secondary structure of the internal transcribed rDNA (ITS) regions of Cosmopolites sordidus (Germar) and Odoiporus longicollis (Olivier): a first report in family Curculionidae

In the present work the consensus secondary structure of the internal transcribed rDNA spacers ITS1 and ITS2 of two weevils i.e. Cosmopolites sordidus (Germar) and Odoiporus longicollis (Olivier) has been described. The secondary structures of the ITS1 region of C.sordidus and O.longicollis have ten and five helices respectively, emerging from a central core. Two and five helices emerge from a central core in the ITS2 secondary structures of C.sordidus and O.longicollis respectively, and do not conform to the `paneukaryotic’ four domain model. The ITS2 sequences of these two weevils share three regions with maximum sequence identity. Of these, two are present at the 5’ and 3’ end and share structural similarity, suggesting their possible role in the processing of the rRNA. This is the first report of the secondary structure of the ITS region in family Curculionidae which can serve as a valuable resource for homology modelling and phylogenetic studies.

Identification of Chrysanthemum indicum and its adulterants based on ITS2 barcode

DNA barcode technology was used to establish a rapid identification method of Chrysanthemum indicum based on ITS2 sequences. The total DNA was extracted from 22 collected samples,and the ITS2 sequence was amplified by PCR and sequenced,and the information of ITS2 sequence was obtained. Another 14 items of the same family or the same genus were downloaded from Gen Bank.We aligned all 36 sequences,calculated the intraspecific and interspecific distances,and constructed Neighbor Joining( NJ) phylogenetic tree,using MEGA 7. 0. The difference of the secondary structure between the ITS2 sequences was compared. The results showed that the genetic distance of Ch. indicum and Ch. morifolium was overlapped,but the maximum intraspecific distance was far less than the minimum interspecific distance between and among Ch. indicum and other species,with an obvious barcoding gap. The NJ tree showed that Ch. indicum and Ch. morifolium shared a clade,and most of Ch. morifolium with some Ch. indicum were shared a subclade,while Inula lineariifolia,Sinosenecio oldhamianus and Senecio scandens belonged to one clade separately. ITS2 secondary structures for I. lineariifolia,S. oldhamianus and S. scandens were significantly different enough to identify completely but Ch. indicum and Ch. morifolium shared two secondary structures of A and B. It was proved that Ch. indicum was one of the evolutionary sources of Ch.morifolium. Therefore ITS2 sequence as DNA barcode can identify Ch. indicum and its adulterants accurately and quickly. The study provides an important basis for Ch. indicum for the identification of germplasm resources and the safety of clinical medication.

The genus Jaagichlorella Reisigl (Trebouxiophyceae, Chlorophyta) and its close relatives: an evolutionary puzzle

The genus Chlorella (in its traditional sense) is polyphyletic and belongs to at least twelve independent lineages of the Trebouxiophyceae and Chlorophyceae. Most of the aquatic species belong to the Chlorella and Parachlorella clades (within the so-called Chlorella-lineage of the Trebouxiophyceae), or to the genera Scenedesmus and Mychonastes (within the DO-group of the Chlorophyceae) according to phylogenetic analyses of the SSU and ITS rDNA sequences. In contrast to the aquatic species, the terrestrial strains investigated so far form a monophyletic lineage (Watanabea-clade) within the Trebouxia-lineage of the Trebouxiophyceae. Several genera with Chlorella-like morphology (Chloroidium, Heterochlorella, Watanabea, Kalinella, Viridiella and others) belong to the Watanabea clade. We studied 22 strains isolated from soil, bark, and artificial hard substrates, which have been traditionally identified as Chlorella luteoviridis or as unidentified Chlorella. To clarify the taxonomical status and intrageneric diversity of this group, we used an integrated approach (molecular phylogeny of SSU and ITS rDNA sequences, secondary structures, DNA barcoding, and morphology) including the ecological distribution. All investigated strains showed a low phenotypic plasticity, but a high genetic diversity, which could be only resolved in complex phylogenetic analyses based on the secondary structures of the investigated genes. Considering these results, we reestablished the genus Jaagichlorella for Heterochlorella and Heveochlorella, and proposed new combinations (J. luteoviridis, J. hainangensis, J. roystonensis, and J. sphaerica) as well as the new species, J. africana.

Characterization of Clinostomum metacercariae using microscopic and molecular approaches

One of the fundamental aspects in understanding the biology, diversity and epidemiology of a parasite lies in its proper identification. In the present study, morphological and molecular characterization of Clinostomum metacercariae recovered from an ornamental fish, Trichogaster fasciata, was carried out in order to ascertain its identity. To serve the purpose, scanning electron micrographs and gene sequences for two commonly used molecular markers, i.e., nuclear ribosomal internal transcribed spacer 2 (rDNA-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) were obtained. The sequences were further used for generating similarity index matrix as well as inferring phylogenies. Light and electron microscopic observations on metacercariae of the parasite revealed that it belongs to the genus Clinostomum. Identification of the same up to the level of species was made possible through sequence and phylogenetic analyses. The ITS2 sequence analyses of our species (KX758630) showed similarity to unidentified Clinostomum sp. reported from Nigeria (KY865625) and China (KP110579), and C. tilapiae recorded from South Africa (KX034048) and Nigeria (KY649353). However, the CO1 gene analyses suggested it to be highly identical to C. philippinense and the same was also corroborated in the phylogenetic analysis. Thus, morphological and molecular characterization revealed that the recovered metacercariae belong to the species C. philippinense. Additionally, a brief description of secondary structures of ITS2 of various species of Clinostomum has also been discussed.