Internal Transcribed Spacer Secondary Structure Research Articles

Sphaerothrix gracilis gen. et sp. nov. (Nodosilineales, Cyanobacteria): a novel filamentous cyanobacterium isolated from tropical coastal microplastics

SUMMARYCyanobacteria are ancient organisms that contribute significantly to primary production in aquatic ecosystems. Although they are a group of microbes present in a wide range of environments, their diversity on anthropogenic substrates, namely microplastics, is still largely unknown. In the present study, we describe a new genus and species of cyanobacteria from the Nodosilineaceae family. Strains were isolated from foam microplastic particles obtained from beach sediments facing the Singapore Strait. This species is morphologically similar to the polyphyletic, cosmopolitan Leptolyngbya spp.; however, it forms an independent clade with 16S rRNA phylogeny and has a unique 16S–23S internal transcribed spacer secondary structure. To date, no cyanobacteria have been isolated and cultured from the surfaces of microplastic particles. Using a polyphasic approach combining morphological, phylogenetic and ecological traits, we describe a new genus and species Sphaerothrix gracilis. The presence of certain cyanobacteria rafting on microplastic surfaces can potentially indicate a long‐distance transport into new ecosystems with implications on toxicity and biodiversity.

Diversidade genética de mangostão cultivado e seus parentes silvestres (Garcinia spp.) com base na morfologia foliar e marcadores moleculares

ABSTRACT The mangosteen (Garcinia mangostana L.) germplasm still has limitations in fruit quality, drought tolerance and susceptibility to pests or diseases. This study investigated the genetic diversity and relationships of mangosteen with its wild relatives (Garcinia spp.) based on leaf morphology and the internal transcribed spacer (ITS) region, including its secondary structure. Based on leaf morphology, the mangosteen and its wild relatives generally showed a low genetic diversity. However, the leaf texture and pubescence had a high genetic diversity (0.71 and 0.77, respectively). Furthermore, based on the ITS markers, the genetic diversity of Garcinia at the interspecies level was much higher than that at the intraspecies one (0.043 and 0.005, respectively). The unweighted pair group method with the arithmetic average (UPGMA) revealed that mangosteen is grouped into four main clusters, with ‘Manggis Banjar’ and ‘Palembang’ in the same cluster. Similarly, the ITS positioned Garcinia into several clades, with ‘Manggis Banjar’, ‘Kandangan’ and ‘Palembang’ grouped into a similar clade. The biochemical reconstruction showed that Garcinia has unique ITS secondary structures, i.e., ring and four-helix models. Even though the cultivated mangosteen and its wild relatives had low diversity based on leaf morphology, the ITS markers showed a high genetic diversity. Furthermore, the reconstruction of the ITS secondary structure has supported this germplasm’s phylogenetic tree.

Structure-Based GC Investigation Sheds New Light on ITS2 Evolution in Corydalis Species.

Guanine and cytosine (GC) content is a fundamental component of genetic diversity and essential for phylogenetic analyses. However, the GC content of the ribosomal internal transcribed spacer 2 (ITS2) remains unknown, despite the fact that ITS2 is a widely used phylogenetic marker. Here, the ITS2 was high-throughput sequenced from 29 Corydalis species, and their GC contents were comparatively investigated in the context of ITS2's characteristic secondary structure and concerted evolution. Our results showed that the GC contents of ITS2 were 131% higher than those of their adjacent 5.8S regions, suggesting that ITS2 underwent GC-biased evolution. These GCs were distributed in a heterogeneous manner in the ITS2 secondary structure, with the paired regions being 130% larger than the unpaired regions, indicating that GC is chosen for thermodynamic stability. In addition, species with homogeneous ITS2 sequences were always GC-rich, supporting GC-biased gene conversion (gBGC), which occurred with ITS2's concerted evolution. The RNA substitution model inferred also showed a GC preference among base pair transformations, which again supports gBGC. Overall, structurally based GC investigation reveals that ITS2 evolves under structural stability and gBGC selection, significantly increasing its GC content.

The CIMS (Cyanobacterial ITS motif slicer) for molecular systematics

The 16S-23S rRNA Internal Transcribed Spacer (ITS) is a commonly employed taxonomic marker in cyanobacterial systematics. Due to numerous challenges in articulating phylogenetic relationships within this ubiquitous, ancient lineage, a polyphasic approach including 16S rRNA sequence data, ecology, morphology, and ITS secondary structure analysis has become the standard. In particular, the ITS motifs are being utilized in the erection of novel and cryptic taxa. However, this is challenging as researchers must manually mine and parse sequence data to visually find and identify ITS structures. This painstaking process deters researchers from using ITS motifs, may lead to inconsistencies, and is a rather dry, tedious enterprise. Thus, we present a simple, user-friendly web-based application for help in finding and preparing the most common cyanobacterial motifs (e.g., the Box-B, D1-D1´, tRNAs, etc.). After extensive testing, we note that the most common motifs are recovered at ca. 97%. These motifs can then be easily exported into Mfold or other similar folding packages. We hope that this will both provide a valuable tool for researchers but will also facilitate new discoveries and allow for greater consistency in publishing ITS comparisons. The tool can be accessed at www.phylo.dev.

Secondary Structure Form of ITS2 Region: A Significant Labeling Tool at all Taxonomic Levels

The general purpose of the molecular systematic studies is to illuminate the ITS2 structure of the target populations, to determine its phylogenetic boundaries, and to clarify intra-species and inter-species relationships. Particularly, the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA is used in molecular systematics because of availability of conserved regions with its highly repeated in number in plant genomes. Addition to the primary sequences of ITS2, also secondary structure form of the region became a valuable feature in species divergence and became to use like a morphological character. In the current study to indicate the secondary structure form of the ITS2 region as a useful tool in systematics, different taxa from 22 genera were used. The DNA samples were collected in the field studies in 2021 and sequences were aligned using ClustalW and Kimura-2 parameter to calculate the genetic distances. Phylogenetic tree was also constructed with Maximum Likelihood method with the best suitable model at MEGA X software. The secondary structure predictions of species and ΔG (Gibbs) free energy calculations, the tools of both the ITS2 database and mFOLD web server were used. The results indicated that ITS2 secondary structure estimations represented the genetic differences visibly with its helices and motifs like a morphological character. Consequently, even if primary structure of the ITS2 region revealed as valuable marker in molecular systematic studies, also, all tested secondary structure forms of the region will be used as an ideal marker for taxonomic and phylogenetic reconstructions at all taxonomic levels.

Compensatory Base Changes and Varying Phylogenetic Effects on Angiosperm ITS2 Genetic Distances.

A compensatory base change (CBC) that coevolves in the secondary structure of ribosomal internal transcribed spacer 2 (ITS2) influences the estimation of genetic distance and thus challenges the phylogenetic use of this most popular genetic marker. To date, however, the CBC effect on ITS2 genetic distance is still unclear. Here, ITS2 sequences of 46 more recent angiosperm lineages were screened from 5677 genera and phylogenetically analyzed in sequence-structure format, including secondary structure prediction, structure-based alignment and sequence partition of paired and unpaired regions. ITS2 genetic distances were estimated comparatively by using both conventional DNA substitution models and RNA-specific models, which were performed in the PHASE package. Our results showed that the existence of the CBC substitution inflated the ITS2 genetic distances to different extents, and the deviation could be 180% higher if the relative ratio of substitution rate in ITS2 secondary structure stems was threefold higher than that in the loops. However, the CBC effect was minor if that ratio was below two, indicating that the DNA model is still applicable in recent lineages in which few CBCs occur. We thus provide a general empirical threshold to take account of CBC before ITS2 phylogenetic analyses.

Amazonocrinis thailandica sp. nov. (Nostocales, Cyanobacteria), a novel species of the previously monotypic Amazonocrinis genus from Thailand

Cyanobacteria are distributed worldwide, and many new cyanobacterial species are discovered in tropical region. TheNostoc-like genus Amazonocrinis has been separated from the genus Nostoc based on polyphasic methods. However,species diversity within this genus remains poorly understood systematically because only one species (Amazonocrinisnigriterrae) has been described. In this study, two novel strains (NUACC02 and NUACC03) were isolated from moist ricefield soil in Thailand. These two strains were characterized using a polyphasic approach, based on morphology, 16S rRNAphylogenetic analysis, internal transcribed spacer secondary structure and ecology. Phylogenetic analyses based on 16SrRNA gene sequences confirmed that the two novel strains formed a monophyletic clade related to the genus Amazonocrinisand were distant from the type species A. nigriterrae. The 16S rRNA gene sequence similarity (<98.1%) betweennovel strains and all other closely related taxa including the Amazonocrinis members exceeded the cutoff for speciesdelimitation in bacteriology, reinforcing the presence of a new Amazonocrinis species. Furthermore, the novel strainspossessed unique phenotypic characteristics such as the presence of the sheath, necridia-like cells, larger cell dimensionand akinete cell arrangement in long-chains and the singularity of D1–D1′, Box-B, V2, and V3 secondary structures thatdistinguished them from other Amazonocrinis members. Considering all the results, we described our two strains as Amazonocrinisthailandica sp. nov. in accordance with the International Code of Nomenclature for Algae, Fungi and Plants.

Multi-Gene Phylogeny of the Ciliate Genus Trachelostyla (Ciliophora, Hypotrichia), With Integrative Description of Two Species, Trachelostyla multinucleata Spec. nov. and T. pediculiformis (Cohn, 1866).

Many hypotrich genera, including Trachelostyla, are taxonomically challenging and in a need of integrative revision. Using morphological data, molecular phylogenetic analyses, and internal transcribed spacer 2 (ITS2) secondary structures, we attempt to cast more light on species relationships within the genus Trachelostyla. The present multifaceted approach reveals that (1) a large-sized species with numerous macronuclear nodules, isolated from sandy littoral sediments in southern China, is new to science and is endowed here with a name, T. multinucleata spec. nov.; (2) two other Chinese populations previously identified as T. pediculiformis represent undescribed species; and (3) multigene phylogeny is more robust than single-gene trees, recovering the monophyly of the genus Trachelostyla with high bootstrap frequency. Additionally, ITS2 secondary structures and the presence of compensatory base changes in helices A and B indicate the presence of four distinct taxa within the molecularly studied members of the genus Trachelostyla. Molecular data are more suitable for delimitation of Trachelostyla species than morphological characters as interspecific pairwise genetic distances of small subunit (18S) rDNA, ITS1-5.8S-ITS2, and large subunit (28S) rDNA sequences do not overlap, whereas ranges of multiple morphometric features might transcend species boundaries.

18S and ITS2 rDNA sequence-structure phylogeny of Prototheca (Chlorophyta, Trebouxiophyceae)

Protothecosis is an infectious disease caused by organisms currently classified within the green algal genus Prototheca. The disease can manifest as cutaneous lesions, olecranon bursitis or disseminated or systemic infections in both immunocompetent and immunosuppressed patients. Concerning diagnostics, taxonomic validity is important. Prototheca, closely related to the Chlorella species complex, is known to be polyphyletic, branching with Auxenochlorella and Helicosporidium. The phylogeny of Prototheca was discussed and revisited several times in the last decade; new species have been described. Phylogenetic analyses were performed using ribosomal DNA (rDNA) and partial mitochondrial cytochrome b (cytb) sequence data. In this work we use Internal Transcribed Spacer 2 (ITS2) as well as 18S rDNA data. However, for the first time, we reconstruct phylogenetic relationships of Prototheca using primary sequence and RNA secondary structure information simultaneously, a concept shown to increase robustness and accuracy of phylogenetic tree estimation. Using encoded sequence-structure data, Neighbor-Joining, Maximum-Parsimony and Maximum-Likelihood methods yielded well-supported trees in agreement with other trees calculated on rDNA; but differ in several aspects from trees using cytb as a phylogenetic marker. ITS2 secondary structures of Prototheca sequences are in agreement with the well-known common core structure of eukaryotes but show unusual differences in their helix lengths. An elongation of the fourth helix of some species seems to have occurred independently in the course of evolution.

Molecular identification and phylogenetic analysis of Papaver based on ITS2 barcoding.

In forensic cases suspected to involve Papaver somniferum, species identification is key to the investigation. To accurately detect and identify P.somniferum as well as common adulterants of the same genus, 19 internal transcribed spacer 2 (ITS2) sequences of P.somniferum (256bp), Papaver canescens (254bp), Papaver nudicaule (254bp), Papaver pavoninum (250bp), Papaver radicatum (254bp), and Papaver rhoeas (256bp) were obtained. Based on the ITS2 sequence, similarity analysis via BLAST, the nearest Kimura-2-parameter (K2P) genetic distances were calculated, and a phylogenetic tree was constructed using MEGA X software for the identification of six species of Papaver. Finally, differences in the ITS2 secondary structure between species were analyzed. The best matches of the P.somniferum ITS2 sequence were of other P.somniferum from different sources. The nearest K2P genetic distances between P.somniferum and its counterparts from other sources were zero, which was the smallest pairwise genetic distance among distances from the other five Papaver species. Various sources of P.somniferum clustered into an independent branch in the phylogenetic tree. The secondary structures of P.somniferum and P.rhoeas were significantly different from those of the other four species of Papaver. In summary, P.somniferum can be effectively distinguished from five closely related plants of the same genus by using ITS2 as a DNA barcode.

Compensatory Base Changes Reveal Sexual Incompatibility among Members of the Anopheles subpictus Sensu Lato (Diptera: Culicidae) Species Complex in Sri Lanka.

The mosquito Anopheles (Cellia) subpictus sensu lato (s.l.) is a major secondary vector of malaria in Sri Lanka. The sibling species composition in this species complex in Sri Lanka remains debatable. Compensatory base changes (CBCs) in the secondary structures of internal transcribed spacer 2 (ITS2) are reliable sources to predict sexual incompatibility among closely related species. The objective of the present study was to investigate the An. subpictus s.l. populations in Sri Lanka using the CBC analysis. Mosquito DNA was amplified and sequenced for the ITS2 region. The sequences were annotated using ITS2 Database. ITS2 secondary structures were constructed and analyzed for CBCs using various bioinformatics tools. The ITS2 regions consisted of two different lengths, 575 bp and 480 bp. The two CBCs and three hemi CBCs identified in the present study suggest that there may be at least two sexually incompatible sibling species. In conclusion, it is likely that there may be only two reproductively isolated sibling species in the An. subpictus species complex in Sri Lanka. However, due to high divergence of ITS2 in these species, it is reasonable to assume that they may be undergoing a speciation event to separate as a distinct species.

Phylogenetic Utility of rRNA ITS2 Sequence-Structure under Functional Constraint.

The crucial function of the internal transcribed spacer 2 (ITS2) region in ribosome biogenesis depends on its secondary and tertiary structures. Despite rapidly evolving, ITS2 is under evolutionary constraints to maintain the specific secondary structures that provide functionality. A link between function, structure and evolution could contribute an understanding to each other and recently has created a growing point of sequence-structure phylogeny of ITS2. Here we briefly review the current knowledge of ITS2 processing in ribosome biogenesis, focusing on the conservative characteristics of ITS2 secondary structure, including structure form, structural motifs, cleavage sites, and base-pair interactions. We then review the phylogenetic implications and applications of this structure information, including structure-guiding sequence alignment, base-pair mutation model, and species distinguishing. We give the rationale for why incorporating structure information into tree construction could improve reliability and accuracy, and some perspectives of bioinformatics coding that allow for a meaningful evolutionary character to be extracted. In sum, this review of the integration of function, structure and evolution of ITS2 will expand the traditional sequence-based ITS2 phylogeny and thus contributes to the tree of life. The generality of ITS2 characteristics may also inspire phylogenetic use of other similar structural regions.

Phylogenetic analysis of 23 accessions of Indonesian banana cultivars based on Internal Transcribed Spacer 2 (ITS2) region

Pisang Kepok (Musa spp. [ABB ’Saba’ subgroup]) has several unique characteristics, such as tolerance to drought and Fusarium Foc (TR4) disease. Currently, the genetic diversity of Pisang Kepok in Indonesia is not well identified, although it is widely cultivated. Information on genetic diversity is essential for developing breeding strategies to achieve efficient cultivar improvement in the future. Aims of this research were to analyze the genetic variation of Pisang Kepok from some islands in Indonesia and to determine the genetic relationship between Pisang Kepok and other accessions banana cultivars based on ITS2 region, as a basis for future research in improving banana quality through molecular breeding. We have conducted the multiple sequence alignment and built the phylogenetic tree analysis using the Bayesian Inference Phylogeny method of one million generations (ngen = 1,000,000). The ITS2 region showed two clade ingroups: first clade consists of banana with B genome (balbisiana), while the second clade consists of banana with only A genome (acuminata). In general, all accessions of Pisang Kepok cultivars were clustered in the B genome of bananas cultivars. In addition, the ITS2 sequences and secondary structures among Pisang Kepok from various regions are identical, suggesting that there was no genetic variation in the ITS2 region of Pisang Kepok from multiple areas in Indonesia.

Alternative analyses of compensatory base changes in an ITS2 phylogeny of Corydalis (Papaveraceae).

Compensatory base changes (CBCs) that occur in stems of ribosomal internal transcribed spacer 2 (ITS2) can have important phylogenetic implications because they are not expected to occur within a single species and also affect selection of appropriate DNA substitution models. These effects have been demonstrated when studying ancient lineages. Here we examine these effects to quantify their importance within a more recent lineage by using both DNA- and RNA-specific models. We examined the phylogenetic implications of the CBC process by using a comprehensive sampling of ITS2 from ten closely related species of Corydalis. We predicted ITS2 secondary structures by using homology modelling, which was then used for a structure-based alignment. Paired and unpaired regions were analysed separately and in combination by using both RNA-specific substitution models and conventional DNA models. We mapped all base-pair states of CBCs on the phylogenetic tree to infer their evolution and relative timing. Our results indicate that selection acted to increase the thermodynamic stability of the secondary structure. Thus, the unpaired and paired regions did not evolve under a common substitution model. Only two CBCs occurred within the lineage sampled and no striking differences in topology or support for the shared clades were found between trees constructed using DNA- or RNA-specific substitution models. Although application of RNA-specific substitution models remains preferred over more conventional DNA models, we infer that application of conventional DNA models is unlikely to be problematic when conducting phylogenetic analyses of ITS2 within closely related lineages wherein few CBCs are observed. Each of the two CBCs was found within the same lineages but was not observed within a given species, which supports application of the CBC species concept.

Molecular Authentication of the Medicinal Species of Ligusticum (Ligustici Rhizoma et Radix, "Gao-ben") by Integrating Non-coding Internal Transcribed Spacer 2 (ITS2) and Its Secondary Structure.

Ligustici Rhizoma et Radix (LReR), an important Chinese medicine known as “Gao-ben,” refers to Ligusticum sinense Oliv. or Ligusticum jeholense Nakai et Kitag. However, a number of other species are commonly sold as “Gao-ben” in the herbal medicine market, which may result in a series of quality control problems and inconsistent therapeutic effects. The “Gao-ben” is commonly sold sliced and dried, making traditional identification methods difficult. Here, the mini barcode ITS2 region was examined on 68 samples representing LReR and 7 potential adulterant or substitute species. The results showed 100% success rates of PCR and sequencing and the existence of a barcoding gap. The neighbor-joining (NJ) tree indicated that all the tested samples could be exactly identified. The ITS2 secondary structure revealed a clear difference between true “Gao-ben” and three adulterant species. We therefore recommend the use of ITS2 as a mini barcode for distinguishing between closely or distantly related plant species that may be used in Chinese medicine.

Internal transcribed spacer 2 (ITS2) molecular morphometric analysis based species delimitation of foliar endophytic fungi from Aglaia elaeagnoidea, Flacourtia inermis and Premna serratifolia.

Molecular morphometrics is an emerging third dimensional aspect of fungal species delimitation. They have been demonstrated to be more informative than conventional barcoding methods. Hence in this study, foliar endophytic fungal (FEF) assemblages in three Magnoliopsida plants were delimited using nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence—secondary structural features based phylogenetic analysis, also known as molecular morphometrics. A total of 392 FEF isolates were obtained from the Aglaia elaeagnoidea, Flacourtia inermis, and Premna serratifolia leaves and grouped into 98 morphotypes. Among these host plants, P. serratifolia showed the maximum percentage of colonization frequency. Representatives of each morphotype was sequenced and subjected to further molecular characterization. The results revealed that morphotypes were belonged to the phylum of Ascomycota, distributed over two classes (Sordariomycetes (68.59%) and Dothideomycetes (31.41%)), 6 orders and 19 genera. Based on compensatory base changes (CBC) analysis and absolute identity of ITS2 structure, 21, 20 and 23 species were recognized from A. elaeagnoidea, F. inermis, and P. serratifolia respectively. Diversity indices were higher in A. elaeagnoidea, despite it accounted for a modest 16.8% of total isolates recorded in this study. The genus Colletotrichum was predominant in A. elaeagnoidea (39%) and P. serratifolia (48%). Similarly, Diaporthe (43%) was dominant in F. inermis. Several host-specific species were also observed. This study concludes that these plants host diverse species of Ascomycota. To the best of our knowledge, this is the first detailed report on FEF diversity from these plants. Also, the inclusion of ITS2 secondary structure information along with the sequence provides a further dimension to resolve the inherent problems in identification of fungal species.

A consensus secondary structure of ITS2 for the diatom Order Cymatosirales (Mediophyceae, Bacillariophyta) and reappraisal of the order based on DNA, morphology, and reproduction

In 1983, Hasle and colleagues removed cymatosiroid diatoms from the pennates, and erected a new centric diatom family, the Cymatosiraceae, mainly to accommodate for their newly discovered mode of sexual reproduction. The new family consisted of two subfamilies differing in frustule structure. The family was later elevated to the rank of Order Cymatosirales Round and Crawford. We revisited intra-ordinal relationships within Cymatosirales using combined genetic (DNA sequences), morphological (valve and frustule structure), and reproductive (auxospore type) characters. In total, 36 cymatosiroid strains from 19 species representing 13 genera (80% of the total number of extant genera; nine of them represented by their generitypes) were used in this study. Instead of only the commonly used loci (18S rRNA and plastidal genes) to infer diatom phylogeny, we developed a consensus secondary structure model of the Internal Transcribed Spacer 2 (ITS2) for this order and applied it to aid in sequence alignments for ITS2. This improved the alignment and thus the robustness of the phylogenetic framework. The compensatory base changes (CBCs) found in ITS2 secondary structures were mapped onto the multi-gene (18S rRNA + ITS2 + rbcL) phylogenetic tree topology. In all these trees, all species grouped into two morphologically and genetically distinct clades. Each clade was supported by multiple CBCs, as did all the clades representing genera. However, these clades did not correspond to the previously established subfamilies. Consequently, we amend the Order Cymatosirales and family Cymatosiraceae, and propose a new family, the Leyanellaceae. The structure of the auxospore was an additional synapomorphic character for Cymatosirales. Overall, we demonstrate a novel approach to study diatom phylogeny across a broader taxonomic range using ITS2 secondary structural information. Our results suggest that this approach might be useful in establishing higher taxonomic relationships in other groups of diatoms.

A new freshwater alga, Ulvella shanxiensis (Chlorophyta) described from China

A new freshwater alga species of Ulvella shanxiensis, is found in the circulating neutral water spring in China (Shanxi Province), growing epilithic and epiphytic on other substrates. This new species is characterized by olive-green, disc-shaped hollow thallus with irregularly vesicular morphology, which composed of 2–3 layers cells. Most characteristics were in agreement with Ulvella, but distinguished from the other freshwater members by thalli dimensions and cell diameter. In addition to describing the morphological structures of U. shanxiensis in detail, phylogenetic analysis based on sequences of the rbcL and 18S rDNA gene placed the algae in single clade with two samples, and demonstrated the separation between U. shanxiensis and other Ulvella species with a considerable sequence distance. Comparing with U. bullata, the internal transcribed spacer secondary structure of the new species has some compensatory base changes in 1 Helix and hemi-CBCs in 2–3 Helix, differed in several ways from that of other Ulvella algae. Consequently, these results of morphological observation and phylogenetic analysis suggest this alga as a new species, and bring a new record in the total number of recognized freshwater Ulvella species in China.

ITS2 sequence-structure phylogeny reveals diverse endophytic Pseudocercospora fungi on poplars.

For matching the new fungal nomenclature to abolish pleomorphic names for a fungus, a genus Pseudocercospora s. str. was suggested to host holomorphic Pseudocercosproa fungi. But the Pseudocercosproa fungi need extra phylogenetic loci to clarify their taxonomy and diversity for their existing and coming species. Internal transcribed spacer 2 (ITS2) secondary structures have been promising in charactering species phylogeny in plants, animals and fungi. In present study, a conserved model of ITS2 secondary structures was confirmed on fungi in Pseudocercospora s. str. genus using RNAshape program. The model has a typical eukaryotic four-helix ITS2 secondary structure. But a single U base occurred in conserved motif of U-U mismatch in Helix 2, and a UG emerged in UGGU motif in Helix 3 to Pseudocercospora fungi. The phylogeny analyses based on the ITS2 sequence-secondary structures with compensatory base change characterizations are able to delimit more species for Pseudocercospora s. str. than phylogenic inferences of traditional multi-loci alignments do. The model was employed to explore the diversity of endophytic Pseudocercospora fungi in poplar trees. The analysis results also showed that endophytic Pseudocercospora fungi were diverse in species and evolved a specific lineage in poplar trees. This work suggested that ITS2 sequence-structures could become as additionally significant loci for species phylogenetic and taxonomic studies on Pseudocerospora fungi, and that Pseudocercospora endophytes could be important roles to Pseudocercospora fungi's evolution and function in ecology.

A new subarctic strain of Tetradesmus obliquus—part I: identification and fatty acid profiling

We isolated a new subarctic strain Tetradesmus obliquus IPPAS S-2023 (Scenedesmaceae, Chlorophyceae) from rock baths in the White Sea. To verify its taxonomic assignment, internal transcribed spacer 2 (ITS2) of the strain was sequenced and its secondary structure was compared with predicted ITS2 secondary structures of Scenedesmaceae. The analysis of the ITS2 made it possible to assign the new strain IPPAS S-2023 to the species T. obliquus. The ultrastructural studies and energy-dispersive X-ray spectroscopy (EDX) analysis revealed a marked accumulation of vacuolar inclusions enriched in phosphorus and nitrogen (N) as well as cytoplasmiс oil bodies. Most of predicted properties of biodiesel derived from the fatty acids profile of the strain grown in the N-free medium complied with the requirements of European and American standards. The results suggest that the new subarctic strain T. obliquus IPPAS S-2023 is a promising candidate for nutrients biosequestration and for biodiesel production. In a companion paper, we assess its biomass production capability and suitability and demonstrated suitability of IPPAS S-2023 as a reference strain for studies on elevated CO2 stress effects selection of carbon dioxide-tolerant microalgae by comparison with a CO2-tolerant strain IPPAS S-2014.