Evaluating nuclear (18S, ITS, and 28S) and chloroplast (<i>psbB</i>, <i>psbC</i>, <i>rbc</i>L, and <i>tufA</i>) genes of the green algae Monoraphidium (Sphaeropleales, Chlorophyta) for DNA barcoding

Compensatory base changes (CBCs) in internal transcribed spacer 2 (ITS2) rDNA secondary structures correlate with Ernst Mayr’s biological species concept. This hypothesis also referred to as the CBC species concept recently was subjected to large-scale testing, indicating two distinct probabilities. (1) If there is a CBC then there are two different species with a probability of ∼0.93. (2) If there is no CBC then there is the same species with a probability of ∼0.76. In ITS2 research, however, the main problem is the multicopy nature of ITS2 sequences. Most recently, 454 pyrosequencing data have been used to characterize more than 5000 intragenomic variations of ITS2 regions from 178 plant species, demonstrating that mutation of ITS2 is frequent, with a mean of 35 variants per species, respectively per individual organism. In this study, using those 454 data, the CBC criterion is reconsidered in the light of intragenomic variability, a proof of concept, a necessary criterion, expecting no intragenomic CBCs in variant ITS2 copies. In accordance with the CBC species concept, we could demonstrate that the probability that there is no intragenomic CBC is ∼0.99.

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3′-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

The rRNA cistron (18S-ITS1-5.8S-ITS2-28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3-21 nuclei per cell), 1-3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1-5.8S-ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3.

Compensatory base changes illuminate morphologically difficult taxonomy

One of the aims of the XXXIV Italian Antarctic Expedition is the study of the photosynthetic biodiversity of the Ross Sea. To achieve this goal, sea-ice samples were collected from Inexpressible Island and a strain of a green microalga (IMA076A) was isolated for morphological and molecular investigations. Combining: (1) phylogenetic analyses of the small subunit rDNA (18S rDNA) and of the internal transcribed spacer 2 (ITS-2) sequences; (2) species delimitation methods; (3) comparative analyses of the secondary structures of ITS-2 and compensatory base changes; (4) morphological, ultrastructural and ecological features, we described the strain IMA076A and its relatives as the new species Microglena antarctica sp. nov. The discovery of a new species of Chlorophyceae highlights that the biological diversity of Antarctic microalgae is more extensive than previously thought and that molecular phylogeny together with compensatory base changes (CBCs) approach are pivotal in the identification of cryptic microalgae.

BackgroundCurrent molecular phylogenetic studies of Lepidoptera and most other arthropods are predominantly based on mitochondrial genes and a limited number of nuclear genes. The nuclear genes, however, generally do not provide sufficient information for young radiations. ITS2 , which has proven to be an excellent nuclear marker for similarly aged radiations in other organisms like fungi and plants, is only rarely used for phylogeny estimation in arthropods, although universal primers exist. This is partly due to difficulties in the alignment of ITS2 sequences in more distant taxa. The present study uses ITS2 secondary structure information to elucidate the phylogeny of a species-rich young radiation of arthropods, the butterfly subgenus Agrodiaetus. One aim is to evaluate the efficiency of ITS2 to resolve the phylogeny of the subgenus in comparison with COI , the most important mitochondrial marker in arthropods. Furthermore, we assess the use of compensatory base changes in ITS2 for the delimitation of species and discuss the prospects of ITS2 as a nuclear marker for barcoding studies.ResultsIn the butterfly family Lycaenidae, ITS2 secondary structure enabled us to successfully align sequences of different subtribes in Polyommatini and produce a Profile Neighbour Joining tree of this tribe, the resolution of which is comparable to phylogenetic trees obtained with COI+COII . The subgenus Agrodiaetus comprises 6 major clades which are in agreement with COI analyses. A dispersal-vicariance analysis (DIVA) traced the origin of most Agrodiaetus clades to separate biogeographical areas in the region encompassing Eastern Anatolia, Transcaucasia and Iran.ConclusionsWith the inclusion of secondary structure information, ITS2 appears to be a suitable nuclear marker to infer the phylogeny of young radiations, as well as more distantly related genera within a diverse arthropod family. Its phylogenetic signal is comparable to the mitochondrial marker COI . Compensatory base changes are very rare within Polyommatini and cannot be used for species delimitation. The implementation of secondary structure information into character-based phylogenetic methods is suggested to further improve the versatility of this marker in phylogenetic studies.

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.

Recently developed computational tools in ITS2 sequence-structure phylogenetics are improving tree robustness by exploitation of the added information content of the secondary structure. Despite this strength, however, their adoption for species-level clarifications in angiosperms has been slow. We investigate the utility of combining ITS2 sequence and secondary structure to separate species of southern African Strychnos, and assess correlation between compensatory base changes (CBCs) and currently recognised species boundaries. Combined phylogenetic analysis of sequence and secondary structure datasets performed better, in terms of robustness and species resolution, than analysis involving primary sequences only, achieving 100 and 88.2% taxa discriminations respectively. Further, the Strychnos madagascariensis complex is well-resolved by sequence-structure phylogenetic analysis. The 17 Strychnos species corresponded to 14 ITS2 CBC clades. Four of the five taxa in section Densiflorae belong to a single CBC clade, whose members tend to form natural hybrids. Our finding supports the application of ITS2 as a complementary barcoding marker for species identification. It also highlights the potential of comparative studies of ITS2 CBC features among prospective parental pairs in breeding experiments as a rapid proxy for cross compatibility assessment. This could save valuable time in crop improvement. Patterns of CBC evolution and species boundaries in Strychnos suggests a positive correlation. We conclude that the CBC pattern coupled with observed ITS2 sequence paraphyly in section Densiflorae points to a speciation work-in-progress.

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.

La energia solar en fotosintesis : Moss

Maize brown sheath spot (MBSS), a new disease of maize, was discovered while surveying for maize leaf and sheath blight diseases in the Indian states of Assam, Jharkhand, Meghalaya, Manipur, and Odisha. Maize is the third most important cereal after rice and wheat in India. Unlike banded leaf and sheath blight disease caused by Rhizoctonia solani, MBSS symptoms on maize were discrete and limited to sheaths only. Symptoms of MBSS in the field were initially water-soaked necrotic lesions of 1 to 2 cm in diameter on the lowermost leaf sheaths, which then progressed to the upper sheaths. Lesions coalesced and covered approximately 2 to 5% of the sheath area. Infected dried lower leaves were shed, whereas infected upper leaves remained on the stem. The pathogen was isolated, characterized morphologically, pathologically, and molecularly, and identified as Waitea circinata var. prodigus, a basidiomycete known to cause basal leaf blight of seashore paspalum. The internal transcribed spacer (ITS) sequence 2 (ITS2) of rDNA from MBSS isolates formed a well supported clade with known W. circinata var. prodigus isolates. Molecular morphometric analysis of the ITS2 regions of the five known varieties of W. circinata detected distinguishing variations in GC content, compensatory base changes (CBCs), hemi- CBCs, indels, and altered base-pairing of helices. Variation in these characteristics may indicate that varieties are distinct biological species within W. circinata sensu lato. The geographical distribution and potential impacts of MBSS on the maize crop in India necessitate further investigations of pathogen identification and disease management.

We used an ITS2 primary and secondary structure and Compensatory Base Changes (CBCs) analyses on new French and Spanish Dunallela salina strains to investigate their phylogenetic position and taxonomic status within the genus Dunaliella. Our analyses show a great diversity within D. salina (with only some clades not statistically supported) and reveal considerable genetic diversity and structure within Dunaliella, although the CBC analysis did not bolster the existence of different biological groups within this taxon. The ITS2 sequences of the new Spanish and French D. salina strains were very similar except for two of them: ITC5105 "Janubio" from Spain and ITC5119 from France. Although the Spanish one had a unique ITS2 sequence profile and the phylogenetic tree indicates that this strain can represent a new species, this hypothesis was not confirmed by CBCs, and clarification of its taxonomic status requires further investigation with new data. Overall, the use of CBCs to define species boundaries within Dunaliella was not conclusive in some cases, and the ITS2 region does not contain a geographical signal overall.

Evaluation of ITS2 molecular morphometrics effectiveness in species delimitation of Ascomycota – A pilot study

A Consensus Secondary Structure of ITS2 in the Chlorophyta Identified by Phylogenetic Reconstruction

:Haematococcus pluvialis has received much interest because it can accumulate large quantities of the pigment astaxanthin. While different isolates of H. pluvialis seem to differ considerably in their physiology, their phylogenetic diversity has not yet been thoroughly studied. We studied the species diversity in a set of temperate European strains of H. pluvialis based on internal transcribed spacer (ITS) rDNA and rbcL molecular phylogenies and determined their congruence with morphology and temperature preferences. The ITS rDNA phylogeny resolved three lineages with the European strains. Three additional lineages were not represented by our European strains. Both statistical parsimony network analysis and the general mixed Yule coalescent recovered these six lineages as different species. An ITS1 structure analysis revealed up to three compensatory base changes (CBCs) between each of the three European lineages, while one CBC was found between lineages in the ITS2 secondary structure. Yet the CBCs were not always present in all strains of each lineage. The rbcL was much less variable and separated only two of the three ITS lineages. We propose an epitype for H. pluvialis and describe the two lineages containing European strains as new species, Haematococcus rubicundus and Haematococcus rubens. Although there is broad morphological overlap, strains of H. pluvialis and H. rubens have, on average, more elongated cells, more noticeable cytoplasmic strands and a more (extreme) pear-shaped protoplast apex than H. rubicundus. Optimal temperatures for growth were similar for the three species and varied between 17°C and 23°C, which is lower than reported earlier from other strains. This study provides a robust phylogenetic framework for further ecophysiological study of Haematococcus.