<i>Lunachloris lukesovae</i> gen. et sp. nov. (Trebouxiophyceae, Chlorophyta), a novel coccoid green alga isolated from soil in South Bohemia, Czech Republic

<p>Culture collections of microorganisms can still hold undiscovered biodiversity; with molecular techniques, considerable progress has been made in characterizing microalgae which were isolated in the past and misidentified due to a lack of morphological features. However, many strains are still awaiting taxonomic reassessment. Here we analysed the phylogenetic position, morphology and ultrastructure of the strain CCALA 307 previously identified as <i>Coccomyxa</i> cf. <i>gloeobotrydiformis</i> Reysigl isolated in 1987 from field soil in South Bohemia, Czech Republic. Molecular phylogenetic analyses based on SSU rDNA and the plastid <i>rbc</i>L gene revealed that the strain CCALA 307 formed a distinct sister lineage to <i>Neocystis</i> and <i>Prasiola</i> clades within the Trebouxiophyceae. We describe this strain as a new genus and species, <i>Lunachloris lukesovae</i>. Multiple conserved nucleotide positions identified in the secondary structures of the highly variable ITS2 rDNA barcoding marker provide further evidence of the phylogenetic position of <i>Lunachloris</i>. Minute vegetative cells of this newly recognized species are spherical or ellipsoid, with a single parietal chloroplast without a pyrenoid. Asexually, it reproduces by the formation of 2–6 autospores. Since the majority of recent attention has been paid to algae from the tropics or extreme habitats, the biodiversity of terrestrial microalgae in temperate regions is still notably unexplored and even a ‘common’ habitat like agricultural soil can contain new, as yet unknown species. Moreover, this study emphasizes the importance of culture collections of microorganisms even in the era of culture-independent biodiversity research, because they may harbour novel and undescribed organisms as well as preserving strains for future studies.</p

ITS1 secondary structure model for <i>Ranunculus notabilis</i> 5613-1.

<p>Polymorphic sites detected in all studied taxa were highlighted in the secondary structure model for <i>R. notabilis</i> 5613-1. The DNA weblogo summarizes all ITS1 nucleotide polymorphisms detected by direct sequencing (red letters) and both direct sequencing and cloning (green letters). Bonds within the ITS1 secondary structure are shown for all positions which can be affected by either non-compensatory or compensatory ( =  hemi-CBCs) single-stranded nucleotide polymorphisms within helices. Italic letters mark non-compensatory base changes which are present only in a single clone.</p

ITS Polymorphisms Shed Light on Hybrid Evolution in Apomictic Plants: A Case Study on the <i>Ranunculus auricomus</i> Complex

<div><p>The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the <i>Ranunculus auricomus</i> complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict <i>R. variabilis</i> is an ancient hybrid of the diploid sexual species <i>R. notabilis</i> and the sexual species <i>R. cassubicifolius</i>. The additivity pattern shared by <i>R. variabilis</i> and the synthetic hybrids supports an evolutionary and biogeographical scenario that <i>R. variabilis</i> originated from ancient hybridization. Concerted evolution of ITS copies in <i>R. variabilis</i> is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.</p></div

NeighborNet analysis of interspecific ITS1+ITS2 variability within the <i>Ranunculus auricomus</i> complex.

<p>NeighborNet analysis of all ITS1+ITS2 sequences obtained by direct sequencing of the studied individuals. The spring leaf silhouettes illustrate the main phenotypic differences between the two morphotypes: the <i>auricomus</i>-morphotype (characteristic for <i>R. notabilis</i>, <i>R. variabilis</i> and the synthetic hybrid <i>R. cassubicifolius</i> × <i>notabilis</i>) and the <i>cassubicus</i>–morphotype (i.e., <i>R. carpaticola</i> and <i>R. cassubicifolius</i>). Individuals belonging to <i>R. carpaticola</i> are marked as “P”, <i>R. cassubicifolius</i> as “S”, <i>R. notabilis</i> as “N” and <i>R. variabilis</i> as “V”, respectively. Identical sequences representing the same ribotype are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103003#pone-0103003-g003" target="_blank">Figure 3</a>. Bootstrap values are given for the main clusters.</p

NeighborNet analysis of cloned ITS1+ITS2 variants.

<p>Particular clones belong to: <i>Ranunculus cassubicifolius</i> (“S”-clones), <i>R. notabilis</i> (“N”-clones), their synthetic hybrid (“X”-clones) and the putative hybrid, <i>R. variabilis</i> (“V”-clones). The spring leaf silhouettes illustrate the main phenotypic difference between the <i>auricomus</i> and the <i>cassubicus</i> morphotypes. Italic letters mark clones which exhibit non-compensatory base changes in the ITS1 or ITS2 secondary structures. Bootstrap values are given for the main clusters.</p

5.8S secondary structure model for <i>Ranunculus notabilis</i> 5613-1.

<p>(A) The partial 5.8S secondary structure model comprising conserved sequence regions (M1-M3), conserved helices (B5-B8) and highlighted sites affected by nucleotide substitutions (for the whole 5.8S secondary structure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103003#pone.0103003.s001" target="_blank">Figure S1</a>). (B) A summary Table of 5.8S nucleotide polymorphisms detected in clones.</p

List of directly sequenced individuals.

a)<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103003#pone.0103003-Hrandl14" target="_blank">[96]</a>.</p>b)<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103003#pone.0103003-Hrandl2" target="_blank">[46]</a>.</p>c)<p>functional sexual seed, but with aposporous initials (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103003#pone.0103003-Hojsgaard1" target="_blank">[58]</a>).</p>d)<p>functional sexual seed, but with low rates of aposporous seeds (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103003#pone.0103003-Hojsgaard1" target="_blank">[58]</a>).</p

Ribotype network representing all cloned ITS1+ITS2 variants.

<p>Particular clones belong to: <i>R. cassubicifolius</i> (“S”-clones), <i>R. notabilis</i> (“N”-clones), <i>R. cassubicifolius</i> × <i>notabilis</i> synthetic hybrid (“X”-clones) and <i>R. variabilis</i> (“V”-clones). The spring leaf silhouettes illustrate the main phenotypic difference between the <i>auricomus and cassubicus</i> morphotypes. Italic letters mark clones which exhibit non-compensatory base changes in the ITS1 or ITS2 secondary structures.</p

Distribution of the analyzed populations of the <i>Ranunculus auricomus</i> complex.

<p>Dots represent sampled populations, the areas with the same color show the entire distribution of the respective taxon within Central Europe (after <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103003#pone.0103003-Paun1" target="_blank">[49]</a>).</p

Geometric morphometric analysis of individuals exhibiting the characteristic “<i>auricomus</i>” morphology.

<p>(A) An example of the sampled fresh plant material for DNA sequencing and geometric morphometric analyses. (B) 2D landmarks digitalization on the leaf outline. (C) Principal components analysis of the shape variables (i.e., Relative warps analysis, RWA) extracted from <i>R. notabilis</i> (blue squares), <i>R. cassubicifolius</i> × <i>notabilis</i> (red crosses) and <i>R. variabilis</i> (violet triangles) 2D landmark data. (D) Mean shapes of <i>R. notabilis</i>, <i>R. cassubicifolius</i> and <i>R. variabilis</i> reconstructed from each centroid (visualized as symbols surrounded by black-lined circles) of the three scatter clusters shown in (C). The between-group differences were tested by permutation tests (lower left triangle, ***  =  p<0.001) and the distances between the mean shapes are expressed as Procrustes distances (upper right triangle).</p