Four new <i>Licmophora</i> species (Licmophorales), with a review of valve characters and exploration of cingulum characters, including a new septum type

<p><i>Licmophora</i> species are common epiphytes, with new species being discovered in under-sampled areas, and a need for a larger morphological character set for all species. In the rich flora of Guam, there are several distinctive new species, four of which we describe here, with a reconsideration and expansion of their useful taxonomic characters. <i>Licmophora attenuata</i> n. sp. formed large colonies on long mucilage stalks; narrowly cuneate valves, 209–339 µm long, were notable for a large, possibly hollow, spine, and the head-pole rimoportulae set back along the sternum. <i>Licmophora bulbosa</i> n. sp. was solitary or on short, branched mucilage stalks, with broadly cuneate valves, 95–122 µm long, with slightly inflated apices giving it a ‘turkey baster’ shape; valvocopulae bore a novel perforated silica ingrowth along the entire midrib, which we call a Tharngan strip. <i>Licmophora curvata</i> n. sp. attached by mucilage pads, the 87–170 µm long cells were distinctive by their curvature and 21–30 µm broad valves. <i>Licmophora labianatis</i> n. sp. was found in large colonies on long branched stalks, with 311–365 µm long valves that were distinctive for a delicate section mid-way along the valve where vimines were absent. <i>Licmophora</i> cells are heterovalvar and we define the ‘back’ frustule as the one with the basal rimoportula. Frustules are also heteropolar and thus have a left and right side. These terms do not correspond to epivalve/hypovalve and primary/secondary sides of valves and are useful for specifying locations of features. The <i>Licmophora</i> epitheca (i.e., the mature half of the frustule) has a cingulum comprising five copulae (a valvocopula + four pleurae) that differ from each other and between species; we describe their morphology in detail. Copular striae were often denser than valvar striae. Midribs were positioned symmetrically along the midline of the band, or asymmetrically, closer to one margin. Advalvar and abvalvar copular striae consisted of single slits or a series of pores (rimate vs porate) and the type often changed along the band. Midribs on valvocopulae transitioned in distinctive ways from the abvalvar side at the cell apex to the advalvar side. The 2nd and 4th pleurae (closed at the apex of the frustule, like the valvocopula) were sometimes very similar to one another but in other species the 4th pleura was reduced to an apical cap with a very narrow band. The 1st and 3rd pleurae, closed at the base, were usually distinct from one another in the size and shape of the open tips. The expanded set of valve + copula characters will be useful in resolving the taxonomy of the majority of short, straight <i>Licmophora</i> species, but studies of additional species are needed to narrow the list to the most useful copula characters.</p

Plastid genome maps of seven newly sequenced diatom species.

<p>Species that share the same circular map have the same gene order. Genes on the outside are transcribed clockwise; those on the inside counterclockwise. The ring of bar graphs on the inner circle display GC content in dark grey.</p

Location of the sampling site on the coast of Panama.

<p>Location of the sampling site on the coast of Panama.</p

Conserved Gene Order and Expanded Inverted Repeats Characterize Plastid Genomes of Thalassiosirales

<div><p>Diatoms are mostly photosynthetic eukaryotes within the heterokont lineage. Variable plastid genome sizes and extensive genome rearrangements have been observed across the diatom phylogeny, but little is known about plastid genome evolution within order- or family-level clades. The Thalassiosirales is one of the more comprehensively studied orders in terms of both genetics and morphology. Seven complete diatom plastid genomes are reported here including four Thalassiosirales: <i>Thalassiosira weissflogii</i>, <i>Roundia cardiophora</i>, <i>Cyclotella</i> sp. WC03_2, <i>Cyclotella</i> sp. L04_2, and three additional non-Thalassiosirales species <i>Chaetoceros simplex</i>, <i>Cerataulina daemon</i>, and <i>Rhizosolenia imbricata</i>. The sizes of the seven genomes vary from 116,459 to 129,498 bp, and their genomes are compact and lack introns. The larger size of the plastid genomes of Thalassiosirales compared to other diatoms is due primarily to expansion of the inverted repeat. Gene content within Thalassiosirales is more conserved compared to other diatom lineages. Gene order within Thalassiosirales is highly conserved except for the extensive genome rearrangement in <i>Thalassiosira oceanica</i>. <i>Cyclotella nana</i>, <i>Thalassiosira weissflogii</i> and <i>Roundia cardiophora</i> share an identical gene order, which is inferred to be the ancestral order for the Thalassiosirales, differing from that of the other two <i>Cyclotella</i> species by a single inversion. The genes <i>ilvB</i> and <i>ilvH</i> are missing in all six diatom plastid genomes except for <i>Cerataulina daemon</i>, suggesting an independent gain of these genes in this species. The <i>acpP1</i> gene is missing in all Thalassiosirales, suggesting that its loss may be a synapomorphy for the order and this gene may have been functionally transferred to the nucleus. Three genes involved in photosynthesis, <i>psaE</i>, <i>psaI</i>, <i>psaM</i>, are missing in <i>Rhizosolenia imbricata,</i> which represents the first documented instance of the loss of photosynthetic genes in diatom plastid genomes.</p></div

Phylogeny of Thalassiosirales and other diatom species based on twenty plastid protein-coding genes with gene/intron loss and plastid genome rearrangement events mapped on the branches.

<p>Number of genome inversions within Thalassiosirales were estimated based on Thalassiosirales ancestral genome using GRIMM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107854#pone.0107854-Tesler1" target="_blank">[29]</a>. Taxa in bold are new genomes sequenced in this study.</p

Comparison of inverted repeat boundaries in the seven diatom species newly sequenced for this study plus the two previously sequenced Thalassiosirales.

<p>Tree is that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107854#pone-0107854-g002" target="_blank">Figure 2</a> with previously sequenced outgroup taxa pruned for visual simplicity. The numbers in brown indicate plastid genome size; the numbers in black below each genome fragment indicate the sizes of the LSC, IR and SSC, respectively. Protein coding genes at the IR boundaries are listed in blue. Three red gene blocks are <i>rrn5</i>, <i>rns</i> and <i>rnl</i>, respectively. Names in bold are Thalassiosirales. Underscored names are for taxa newly sequenced for this study.</p

Gene order comparison of the plastid genomes of seven diatoms sequenced for this study plus previously sequenced Thalassiosirales.

<p>Alignments were performed in Geneious R6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107854#pone.0107854-Drummond1" target="_blank">[24]</a>with mauveAligner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107854#pone.0107854-Darling1" target="_blank">[28]</a>. Taxon names in bold are members of the Thalassiosirales. Names underscored are those sequenced for this study.</p

<i>Biremis panamae</i> sp. nov., TEM. A.

<p>A whole valve in valve view. <b>B.</b> A whole specimen observed from the valve interior. <b>C, D.</b> Close ups of a specimen illustrated in Fig. 5A; note the finely porous areolae occlusions, arrowhead in Fig. 5D.</p

Vegetative cells and auxosporulation in <i>Biremis</i> sp. A, B.

<p>Two focuses of a vegetative cell in girdle view. Each cell contains two chloroplasts either side of the centre, each of which comprises two plates (one is shown for each chloroplast in Fig. 7A, the other being out of focus beneath, on the opposite side of the cell) connected by a narrow bridge containing the pyrenoid (e.g. p). <b>C, D.</b> Two paired gametangia, each containing two rounded, rearranged gametes. The gametangia were paired with their girdles adjacent, the cell shown in Fig. 7D lying immediately below that in Fig. 7C. <b>E</b>. Two paired gametangia, unusual in being in contact only via their valves. Each gametangium contains a single subspherical zygote. Two nuclei are visible in the left-hand cell (arrows) and two of the four chloroplasts in the right-hand cell. <b>F, G.</b> Two focuses of a gametangium containing a zygote on the point of transformation into an auxospore. Note the slight central inflection of the zygote's outline, marking the deposition of the primary transverse perizonial band (cf. Fig. 7H, arrowhead). The two rows of foramina on the valves can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114508#pone-0114508-g002" target="_blank">Fig. 2G</a> (arrowheads). <b>H</b>. Expanded auxospore containing the initial epivalve (in section at arrow). The auxospore is encased in a well developed perizonium, containing a primary transverse band flanked by several secondary bands (see in section: see also Figs 7I, J). <b>I.</b> Peripheral focus of an expanded auxospore containing the initial epivalve. The two rows of foramina on one of the gametangium valves can be seen (arrowheads). The end of the auxospore is covered by a siliceous cap (arrow). <b>J.</b> Expanded auxospore containing a completed initial cell. The initial hypovalve (in section at h) lies at a distance from the perizonium, as a result of a strong contraction of the protoplast immediately before its formation; the initial epivalve lies opposite, directly moulded by the interior of the perizonium. The auxospore casing can be seen to consist of a perizonium of transverse bands (e.g. at white arrows) and two silicified hemispherical caps (e.g. at black arrow). [Scale bar 10 µm].</p

<i>Biremis panamae</i> sp. nov., SEM: external valve views.

<p><b>A.</b> Frustule with detached valves and a part of a copula with several rows of pores (arrowhead). <b>B.</b> Valve face showing occlusions of the marginal row of small areolae (arrowhead). <b>C, D.</b> External valve view showing variation in the valve face morphology. Note the depressed surface of the valve face areola occlusions (arrowhead).</p