Historical and Current Perspectives on the Systematics of the ‘Enigmatic’ Diatom Genus <i>Rhoicosphenia</i> (Bacillariophyta), with Single and Multi-Molecular Marker and Morphological Analyses and Discussion on the Monophyly of ‘Monoraphid’ Diatoms

<div><p>This study seeks to determine the phylogenetic position of the diatom genus <i>Rhoicosphenia</i>. Currently, four hypotheses based on the morphology of the siliceous valve and its various ultrastructural components, sexual reproduction, and chloroplasts have been proposed. Two previous morphological studies have tentatively placed <i>Rhoicosphenia</i> near members of the Achnanthidiaceae and Gomphonemataceae, and no molecular studies have been completed. The position of <i>Rhoicosphenia</i> as sister to ‘monoraphid’ diatoms is problematic due to the apparent non-monophyly of that group, so hypotheses of ‘monoraphid’ monophyly are also tested. Using an analysis of morphological and cytological features, as well as sequences from three genes, SSU, LSU, and <i>rbc</i>L, recovered from several freshwater <i>Rhoicosphenia</i> populations that have similar morphology to <i>Rhoicosphenia abbreviata</i> (Agardh) Lange-Bertalot, we have analyzed the phylogenetic position of <i>Rhoicosphenia</i> in the context of raphid diatoms. Further, we have used topology testing to determine the statistical likelihoods of these relationships. The hypothesis that <i>Rhoicosphenia</i> is a member of the Achnanthidiaceae cannot be rejected, while the hypothesis that it is a member of the Gomphonemataceae can be rejected. In our analyses, members of the Achnanthidiaceae are basal to <i>Rhoicosphenia</i>, and <i>Rhoicosphenia</i> is basal to the Cymbellales, or a basal member of the Cymbellales, which includes the Gomphonemataceae. Hypothesis testing rejects the monophyly of ‘monoraphid’ diatoms.</p></div

Maximum likelihood phylogram from three-marker concatenated alignment.

<p>Node support values are for maximum likelihood bootstrap values (500 bootstraps)/Bayesian posterior probability (as a percentage). “*” = 100, “-” = node incongruent between the two analyses.</p

Sampling location information <i>Rhoicosphenia</i> populations sequenced including species, ID, State, County, Site Name, Latitude, Longitude, Type, and Collection number.

<p>Sampling location information <i>Rhoicosphenia</i> populations sequenced including species, ID, State, County, Site Name, Latitude, Longitude, Type, and Collection number.</p

Primers used in amplification and sequencing of SSU, LSU, and <i>rbc</i>L.

<p>^a Forward PCR amplification primer, ^b Reverse PCR amplification primer.</p

Summary of Hypothesis Testing Results.

<p>The first column states the molecular markers for the phylogeny being tested, while the first row represents the hypothesis being tested. The values in the table are the p-values from the Approximately Unbiased (AU) test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152797#pone.0152797.ref073" target="_blank">73</a>], and hypotheses that can be rejected based on the AU test are indicated with a “*”.</p

<i>Rhoicosphenia</i> populations sequenced including name, ID, molecular marker sequences available, and GenBank accession numbers.

<p><i>Rhoicosphenia</i> populations sequenced including name, ID, molecular marker sequences available, and GenBank accession numbers.</p

Comparative analysis of the mitochondrial genomes of six newly sequenced diatoms reveals group II introns in the barcoding region of <i>cox1</i>

<p>Diatoms are the most diverse lineage of algae and at the base of most aquatic food webs, but only 11 of their mitochondrial genomes have been described. Herein, we present the mitochondrial genomes of six diatom species, including: <i>Melosira undulata</i>, <i>Nitzschia alba</i>, <i>Surirella</i> sp., <i>Entomoneis</i> sp., <i>Halamphora coffeaeformis</i>, and <i>Halamphora calidilacuna.</i> Comparison of these six genomes to the 11 currently published diatom mitochondrial genomes revealed a novel ubiquitous feature block consisting of <i>tatC</i>-<i>orf157</i>-<i>rps11.</i> The presence of intronic retrotransposable elements in the barcoding region of <i>cox1</i> in the <i>Halamphora</i> genomes may explain historic difficulty (especially PCR) with <i>cox1</i> as a universal barcode for diatoms. Our analysis suggests that high rates of variability in number and position of introns, in many commonly used coding sequences, prevent these from being universally viable as barcodes for diatoms. Therefore, we suggest researchers examine the chloroplast and/or nuclear genomes for universal barcoding markers.</p

SSU sequences Submission1762486.txt

SSU sequence

data for distributional analysis

Thallus and dispersal unit sizes of macroalgae used for analyses in the current manuscript

tufA sequences Submission1762279.txt

tufA sequence