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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 Mg 2+ or Ca 2+ , 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., (© 2018 Phycological Society of America.)

Background: Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta., Methodology/principal Findings: Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses., Conclusions/significance: Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated, data analysis approach with demonstrated power to reconstruct evolutionary patterns for highly divergent lineages.

An enormous body of research is focused on finding ways to commercialize carotenoids produced by the unicellular green alga, Haematococcus, often without the benefit of a sound phylogenetic assessment. Evidence of cryptic diversity in the genus means that comparing results of pigment studies may be confounded by the absence of a phylogenetic framework. Moreover, previous work has identified unnamed strains that are likely candidates for species status. We reconstructed the phylogeny of an expanded sampling of Haematococcus isolates utilizing data from nuclear ribosomal markers (18S rRNA gene, 26S rRNA gene, internal transcribed spacer [ITS]-1, 5.8S rRNA gene, and ITS-2) and the rbcL gene. In addition, we gathered morphological, ultrastructural and pigment data from key isolates of Haematococcus. Our expanded data and taxon sampling support the concept of a new species, H. privus, found exclusively in North America. Despite overlap in numerous morphological traits, results indicate that ratios of protoplast length to width and akinete diameter may be useful for discriminating Haematococcus lineages. High growth rate and robust astaxanthin yield indicate that H. rubicundus (SAG 34-1c) is worthy of additional scrutiny as a pigment source. With the description of H. privus, the evidence supports the existence of at least five, species-level lineages in the genus. Our phylogenetic assessment provides the tools to frame future pigment investigations of Haematococcus in an updated evolutionary context. In addition, our investigation highlighted open questions regarding polyploidy and sexuality in Haematococcus which demonstrate that much remains to be discovered about this green flagellate. [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]

We analyzed the ITS2 primary and secondary structure (including Compensatory Base Changes ( CBCs)) of 17 new Dunaliella strains (11 D. viridis, two D. tertiolecta, and four Dunaliella sp.), and compared these with other Dunaliella sequences available from the ITS2 database to circumscribe their taxonomic position. The ITS2 primary and secondary structure analysis positioned the majority of D. viridis strains in four main clades, showing that D. viridis is polyphyletic. The detection of at least one CBC among these clades strongly suggests that they could correspond to different biological species. Unexpectedly, while D. viridis var. euchlora ( CCAP19/21) was positioned within the subgenus Dunaliella, D. viridis var. palmelloides ( CCAP11/34) was positioned clearly outside this subgenus, suggesting that this taxon may not be properly placed in Dunaliella. Furthermore, the detection of at least three compensatory base changes ( CBCs) between D. viridis var. palmelloides ( CCAP11/34) and the other strains analyzed, confirm that this strain is a different species. For these reasons we propose re-naming D. viridis var. palmelloides ( CCAP11/34) to incertae sedis, and D. viridis var. euchlora ( CCAP19/21) to Dunaliella sp. Therefore, the ITS2 primary and secondary structure data suggest a taxonomic re-structuring of D. viridis. [ABSTRACT FROM AUTHOR]

Abstract: Studies of alpha-2-macroglobulin (α2M), a universal protease inhibitor, have indicated that it plays a unique and critical role in the innate immune system of vertebrate and invertebrate animals. The distinctive mechanism of pathogen inhibition – through physical entrapment of the pathogen-derived protease – makes α2M an ideal candidate for molecular evolutionary analysis. Furthermore, recent studies revealed that the Osteichthyes are characterized by levels of α2M diversification that exceed those recorded in other animal groups. Our study of Danio rerio (zebrafish) indicated that (1) two distinct lineages of α2M and α2M-like isoforms exist and (2) at least some codons in the functional domains of α2M have been subjected to positive Darwinian selection. The findings of several hot-spots for nonsynonymous substitutions in the two functional domains such as bait region and receptor binding domain, suggest that host-immune selection have played a dominant role in these two genomic regions of α2M. The presence of two, non-monophyletic α2M lineages in zebrafish provides compelling evidence of an ancient gene duplication event. The accelerated rate of nucleotide substitution in the functional domains of α2M is consistent with similar observations of other immune system components. [Copyright &y& Elsevier]

The Great Salt Plains (GSP) in north-central Oklahoma, USA is an expansive salt flat (∼65 km2) that is part of the federally protected Salt Plains National Wildlife Refuge. The GSP serves as an ideal environment to study the microbial diversity of a terrestrial, hypersaline system that experiences wide fluctuations in freshwater influx and diel temperature. Our study assessed cyanobacterial diversity at the GSP by focusing on the taxonomic and physiological diversity of GSP isolates, and the 16S rRNA phylogenetic diversity of isolates and environmental clones from three sites (north, central, and south). Taxonomic diversity of isolates was limited to a few genera (mostly Phormidium and Geitlerinema), but physiological diversity based on halotolerance ranges was strikingly more diverse, even between strains of the same phylotype. The phylogenetic tree revealed diversity that spanned a number of cyanobacterial lineages, although diversity at each site was dominated by only a few phylotypes. Unlike other hypersaline systems, a number of environmental clones from the GSP were members of the heterocystous lineage. Although a number of cyanobacterial isolates were close matches with prevalent environmental clones, it is not certain if these clones reflect the same halotolerance ranges of their matching isolates. This caveat is based on the notable disparities we found between strains of the same phylotype and their inherent halotolerance. Our findings support the hypothesis that variable or poikilotrophic environments promote diversification, and in particular, select for variation in ecotype more than phylotype. [ABSTRACT FROM AUTHOR]

The hydrodictyacean green algal lineage has been the focus of much research due to the fossil record of at least some members, their ornamented cell walls, and their distinctive reproductive strategies. The phylogeny of the family was, until recently, exclusively morphology based. This investigation examines hydrodictyacean isolates from several culture collections, focusing on sequences from ribosomal data: 18S rDNA, 26S rDNA (partial), and internal transcribed spacer (ITS)-2 data. Results from phylogenetic analyses of independent and combined data matrices support the Hydrodictyaceae as a monophyletic lineage that includes isolates of Chlorotetraedron, Hydrodictyon, Pediastrum, Sorastrum, and Tetraedron. Phylogenetic analyses of rDNA data indicate that the three-dimensional coenobium of Hydrodictyon is evolutionarily distinct from the three-dimensional coenobium of Sorastrum. The more robust aspects of the ITS-2 data corroborate the 18S+26S rDNA topology and provide a structural autapomorphy for the Hydrodictyaceae and Neochloridaceae, that is, an abridgment of helix IV in the secondary structure. The rDNA data do not support monophyly of Pediastrum but rather suggest the existence of four additional hydrodictyacean genera: Monactinus, Parapediastrum, Pseudopediastrum, and Stauridium. [ABSTRACT FROM AUTHOR]

The phylogenetic affinities of Lobocharacium coloradoense were investigated by analysis of combined 18S and 26S rDNA data. Results from both parsimony and likelihood methods supported a close alliance among Lobocharacium , Characiosiphon , and Characiochloris. These three taxa formed a clade near the base of the “Dunaliella ” group within the chlamydomonad lineage. Protosiphon , which exhibits a siphonous habit similar to Characiosiphon and Lobocharacium , was not resolved as a close ally of the latter two taxa. The Lobocharacium alliance was characterized by the presence of an attachment pad associated with the nonmotile vegetative stage and pyrenoids that possess cytoplasmic invaginations. The pyrenoid feature is an ultrastructural trait that has now been observed in five different chlorophycean lineages. The Lobocharacium–Characiosiphon–Characiochloris clade is not predicted by any classifications of green algae. Additional taxon and data sampling need to be completed to resolve inconsistencies between the molecular phylogenetic evidence and at least some of the current family-level taxa. [ABSTRACT FROM AUTHOR]

The phylogenetic affinities of Lobocharacium coloradoense were investigated by analysis of combined 18S and 26S rDNA data. Results from both parsimony and likelihood methods supported a close alliance among Lobocharacium , Characiosiphon , and Characiochloris. These three taxa formed a clade near the base of the “Dunaliella ” group within the chlamydomonad lineage. Protosiphon , which exhibits a siphonous habit similar to Characiosiphon and Lobocharacium , was not resolved as a close ally of the latter two taxa. The Lobocharacium alliance was characterized by the presence of an attachment pad associated with the nonmotile vegetative stage and pyrenoids that possess cytoplasmic invaginations. The pyrenoid feature is an ultrastructural trait that has now been observed in five different chlorophycean lineages. The Lobocharacium–Characiosiphon–Characiochloris clade is not predicted by any classifications of green algae. Additional taxon and data sampling need to be completed to resolve inconsistencies between the molecular phylogenetic evidence and at least some of the current family-level taxa. [ABSTRACT FROM AUTHOR]

Ultrastructural analyses of the flagellar apparatus suggested that Sphaeroplea, Atractomorpha, the Hydrodictyaceae, and the Neochloridaceae, all of which produce biflagellate motile cells with directly opposed (DO) basal bodies, are allied in an order Sphaeropleales. Recent studies of 18S rDNA sequence data supported an alliance of the DO group, but no data from Sphaeroplea and its allies were included. This investigation presented a test of the phylogenetic hypothesis suggested by the flagellar apparatus evidence using sequence data from the nuclear-encoded small-subunit rDNA (18S) and large subunit rDNA (26S) genes, combined with additional taxon sampling. Results from phylogenetic analyses weakly supported monophyly of biflagellate DO taxa and indicated that pyrenoids with cytoplasmic invaginations are present in numerous distinct lineages. Analysis of both molecular data sets supported a class Chlorophyceae comprised of at least six major groups that generally correspond to currently recognized orders or families: Chaetophorales, Chae- topeltidales, Chlamydomonadales, Sphaeropleales, Sphaeropleaceae, and Oedogoniales. In addition, Cylindrocapsa, Elakatothrix, Treubaria, and Trochiscia formed a seventh chlorophycean clade that is new to science. This investigation demonstrated that the 26S rDNA gene provides more phylogenetic signal, per unit sequence, than the 18S rDNA gene and that combined analysis yields topologies with more robust support than independent analysis of either data set. [ABSTRACT FROM AUTHOR]

Comparative specificity of sporangial wall autolysins (i.e. vegetative lyric enzymes [VIE]) derived from sporulating cultures has been used to group Chlamydomonas taxa into 15 different VLE types. The VLE-14 group including isolates of C. geitleri, C. noctigama, C. monoica, C. pinicola, C. terricola, and C. hindakii, is one of the largest of these VLE groups. Genetic studies have shown that a number of the VLE-14 taxa are interfertile, albeit with little on reduced viability of progeny. A reevaluation of the VLE-14 group suggested that all members should be regarded a distinct isolates of C. noctigama. The present investigation tests the phylogenetic implications of the VLE evidence and examines the validity of the taxonomic reevaluation in a phylogenetic context by analysis of 18S rRNA gene sequence data. Results from analysis of the sequence data are consistent with an interpretation of the VLE evidence as indicative of monophyletic taxa. Phylogenetic analysis of the sequence data are also consistent with the taxonomic reevaluation and reidentification of the group. However, at least some of the VLE-14 isolates studied in this investigation fit criteria for distinct biologic or phylogenetic species. It is concluded that the VLE-14 taxa represent a very closely allied group that includes some isolates that are in the early stages of speciation by reproductive isolation. [ABSTRACT FROM AUTHOR]

The distribution of loroxanthin in chlorophycean flagellates is consistent with their phylogeny as inferred from molecular and organismal characters. Although polarity of this character cannot yet be determined, if the absence of loroxanthin is plasiomorphic for chlorophycean flagellates, the presence of loroxanthin defines a clade that includes the Euchlamydomonas morphological groups, two other species of Chlamydomonas, C. asymmetrica and C. oblonga, and Gonium pectorale. A subsequent reversal to loroxanthin absence is apomorphic for the more complex volvocacean taxa. These results suggest that loroxanthin distribution will be useful for resolving phylogenetic questions in green algae. [ABSTRACT FROM AUTHOR]

Comparative ultrastructural data have shown that atleast two distinct groups exist within Carteria. Similarly, interpretations of variation in gross morphological features have led to the discovery of morphologically distinct groups within the genus. Partial sequences from the nuclear-encoded small and large subunit ribosomal RNA molecules of selected Carteria taxa were studied as a means of 1)testing hypotheses that distinct groups of species exist within the genus and 2)assessing monophyly of the genus. Parsimony analysis of the sequence data suggests that three Carteria species, C.lunzenis, C. crucifera, and C.oliveri, form a monophyletic group that is the basal sister group to all other ingroup flagellate taxa (including species of Chlamydomonas, Haematococcus, Stephanosphaera, Volvox and Eudorina). Two other Carteria taxa, C.radiosa and Carteria sp. (UTEX isolate LB 762), form a clade that is the sister group to a clade that includes Haematococcus spp., Chlamydomonas spp., and Stephanosphaera. Thus, the sequence data support the interpretation of ultrastructural evidence that described two distinct Carteria lineages. Moreover, the sequence data suggest that these two Carteria groups do not form a monophyletic assemblage. Parsimony analysis of a suite of organismal (morphologica, ultrastructural, life history and biochemical) character data also suggest two distinct lineages among the five Carteria taxa; however, the organismal data are ambiguous regarding monopophyly of these Carteria taxa. When the two independent data sets are pooled, monophyly of Carteria is not supported; therefore, the weight of available evidence, both molecular and organismal, fails to support the concept of Carteria as a natural genus. [ABSTRACT FROM AUTHOR]

The genus Chlamydomonas Ehrenberg may contain as many as 450 described species. Morphological, physiological and molecular data show that variation among some Chlamydomonas species can be great, leading to speculation that multiple, generic-level lineages exist within this genus. The most recent systematic studies of Chlamydomonas have led to proposals of nine distinct morphological and 15 distinct sporangial autolysin groups. Partial sequences from the nuclear small subunit rRNAs from 14 Chlamydomonas species representing 12 autolysin and four morphological groups, and from three flagellates thought to be related to Chlamydomonas were determined in a phylogenetic study of relationships among these algae. Sequence comparisons among some Chlamydomonas species revealed differences comparable to the sequence divergence between soybeans and cycads. Cladistic analysis of the sequence data suggests that multiple lineages exist among species of Chlamydomonas. Some of these lineages represent alliances of both Chlamydomonas and non-Chlamydomonas taxa; thus, the current taxonomy does not reflect natural, or monophyletic, groups. Collectively, these lineages may represent distinct families or even orders. [ABSTRACT FROM AUTHOR]