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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]

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty‐nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg2+ or Ca2+, addition of lectin‐inhibiting monosaccharides, or water‐soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling. [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]

Background and Aims The green algal class Chlorophyceae comprises five orders (Chlamydomonadales, Sphaeropleales, Chaetophorales, Chaetopeltidales and Oedogoniales). Attempts to resolve the relationships among these groups have met with limited success. Studies of single genes (18S rRNA, 26S rRNA, rbcL or atpB) have largely failed to unambiguously resolve the relative positions of Oedogoniales, Chaetophorales and Chaetopeltidales (the OCC taxa). In contrast, recent genomics analyses of plastid data from OCC exemplars provided a robust phylogenetic analysis that supports a monophyletic OCC alliance. Methods An ITS2 data set was assembled to independently test the OCC hypothesis and to evaluate the performance of these data in assessing green algal phylogeny at the ordinal or class level. Sequence-structure analysis designed for use with ITS2 data was employed for phylogenetic reconstruction. Key Results Results of this study yielded trees that were, in general, topologically congruent with the results from the genomic analyses, including support for the monophyly of the OCC alliance. Conclusions Not all nodes from the ITS2 analyses exhibited robust support, but our investigation demonstrates that sequence-structure analyses of ITS2 provide a taxon-rich means of testing phylogenetic hypotheses at high taxonomic levels. Thus, the ITS2 data, in the context of sequence-structure analysis, provide an economical supplement or alternative to the single-marker approaches used in green algal phylogeny. [ABSTRACT FROM AUTHOR]