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Abstract Aided by a host of bioinformatics tools, primary and secondary structural analyses of the internal transcribed spacer 2 (ITS2) from the eukaryotic ribosomal RNA repeat have a long and enviable record of service to diversity studies of fungi, plants and protists. Automation of annotation, secondary structure estimation and sequence alignment have become routine for the vast majority of ITS2 sequences. Challenges to the bioinformatics pipeline for ITS2 analysis generally arise in cases where the sequence length lies well outside the norm. These sequences generally defy protocols for annotation and secondary structure prediction. The long ITS2 sequences (ca. 600 nucleotides) from the green alga, Jenufa , offered an opportunity to explore this problem. Custom BLAST parameters revealed the presence of 4-helix structures (200–250 nucleotides) embedded in the 5′ portion of several long ITS2 sequences of Jenufa. Of special note is the ITS2 sequence of J. lobulosa where a 4-helix structure was obtained for both the embedded ITS2 and for the complete sequence. Phylogenetic analysis of these typically-sized sequences resolved Golenkinia longispicula as the sister to Jenufa. Our observations indicate that other long ITS2 sequences should be examined for evidence of expansion or duplication. In addition, if the embedded ITS2 sequences are functional, then ribogenesis is almost certainly more diverse than is already apparent from studies of humans and yeast. Highlights • Custom BLAST parameters revealed the presence of 4-helix structures embedded in the long ITS2 sequences of Jenufa • For J. lobulosa a 4-helix structure was obtained for both the embedded ITS2 and for the complete sequence • ITS2 sequence-structure phylogenetics affirmed an alliance between Jenufa and Golenkinia (Chlorophyta, Chlorophyceae) [ABSTRACT FROM AUTHOR]

Primary and secondary structural data from the internal transcribed spacer two (ITS2) have been used extensively for diversity studies of many different eukaryotic organisms, including the green algae. Ease of amplification is due, at least in part, to the fact that ITS2 is part of the tandemly-repeated rRNA array. The potential confounding influence of intragenomic variability has yet to be addressed except in a few organisms. Moreover, few of the assessments of intragenomic variation have taken advantage of the deep sequencing capacity of sequence-by-synthesis protocols. We present results from our adaptation of the 16S Metagenomics Sequencing Library Preparation/Illumina protocol for deep sequencing of the ITS2 genes in selected isolates of the green algal genus, Haematococcus. Deep sequencing yielded from just under 20,000 to more than 500,000 merged reads, outpacing results from recent pyrosequencing efforts. Furthermore, a conservative evaluation of these data revealed a range of three to six ITS2 sequence haplotypes (defined as unique sets of nucleotide polymorphisms) across the taxon sampling. The frequency of the dominant haplotype ranged from 0.35 to 0.98. In all but two cases, the haplotype with the greatest frequency corresponded to a sequence obtained by the Sanger method using PCR templates. Our data also show that results from the sequencing-by-synthesis approach are reproducible. In addition to advancing our understanding of ribosomal RNA variation, the results of this investigation will allow us to begin testing hypotheses regarding the maintenance of homogeneity across multi-copy genes. [ABSTRACT FROM AUTHOR]

The use of 18S rDNA sequences for inferring phylogenies, in particular for higher taxonomic level analysis, has a long tradition in phycology. Similar to ITS2, the 18S rDNA displays a conserved secondary structure that could be used simultaneously with the primary sequence to increase the amount of information used when inferring phylogenetic relationships. Sequence-structure phylogenetics is already established for ITS2 research. Secondary structures no longer simply guide alignments and trees but are used simultaneously by encoding the sequence-structure information into a 12-letter alphabet. We used the knowledge gathered from the extensive body of ITS2 research regarding sequence-structure phylogenetics and applied it to 18S rRNA data; we present THESES db, the Algae 18S rDNA Sequence-Structure Database (), which contains sequences and their individual secondary structures for three major groups of algae (Chlorophyta, Bacillariophyta and Rhodophyta). This database was designed to serve as the starting point for future 18S rDNA sequence-structure based phylogenetic analyses that will eventually extend beyond phycology. One hundred phylogenetic trees generated from 18S sequence-only datasets and from parallel 18S sequence-structure datasets were compared for each taxon analyzed in this study (diatoms, green algae and red algae). Half of the comparisons produced trees with different topologies that frequently related to the status of sister genera. Using the lineage information for each species as listed in GenBank, we determined that the sequence-structure approach resolved a genus as monophyletic, while the sequence-only approach failed to do so in comparisons that comprised 3% of the cases examined. The reverse was true for a total of 8.3% of the comparisons that we generated. Future work, both in our labs and among the broader phycological community, will provide additional data to test the accuracy and robustness of a sequence-structure approach at different taxonomic ranks. [ABSTRACT FROM AUTHOR]

Ettlia is a green algal genus comprised of several economically important species that have contributed to a taxonomic nightmare for phycologists - the recognition that coccoid green algae are polyphyletic on a grand scale. This taxonomic confusion is slowly being resolved with the aid of molecular evidence and, in some cases, new structural data. The most recent investigation using 18S ribosomal (r)DNA data corroborated an extraordinarily close alliance between Haematococcus pluvialis, a predominantly flagellate unicell, and the coccoid alga, Ettlia carotinosa. This putative evolutionary relationship between the green algal species H. pluvialis and E. carotinosa has, once again, placed coccoid algae at the center of systematic debate. The only apparent similarity between these two species is the ability to produce astaxanthin. Because the 18S rDNA results were not particularly robust, additional data from the internal transcribed spacer 2 rRNA and 26S rRNA genes were collected. Results from analyses of these data corroborated a close alliance between E. carotinosa and H. pluvialis. In addition, the 18S rDNA data set was expanded using new, published data from other species of Ettlia. Phylogenetic analyses of this updated matrix confirmed the existence of at least four distinct Ettlia lineages. These observations indicated that (1) additional taxonomic revision of the genus Ettlia is needed, (2) E. carotinosa need not be merged into the genus Haematococcus, (3) the case of H. pluvialis and E. carotinosa is an example of ciyptic similarity, and (4) the extraordinarily close alliance between E. carotinosa and H. pluvialis offers an ideal system to study patterns of gene expression and morphogenesis in unicellular eukaryotes. [ABSTRACT FROM AUTHOR]