Hemitergite development and <i>engrailed</i> expression in whole mount (A, C, C3) and flat mounted (all others) stage 8 embryos of <i>Strigamia maritima</i>.

<p>All images orientated with anterior to the left. Images with the same letter indicate same specimen; A, C, D, D2 and D4: fluorescent nuclear staining (Sytox Green) to show the morphology; A1, C1, D1, D3 and D5 respectively same view as A, C, D, D2 and D4, under transmitted light, showing pattern of <i>en</i> expression. <b>A–A1.</b> Lateral view of whole-mount embryo with approximately 60% hemitergite extension on the anterior region of the trunk; note the correlation between the hemitergite dorsal border (<i>htb</i>) and the extent of the <i>engrailed</i> stripe. <b>B.</b> Ventral view of posterior trunk of embryo under transmitted light; note the reduced continuous dorsoventral <i>engrailed</i> stripe corresponding to the last leg-bearing segment (<i>llb</i>) at the posterior end. <b>C–C1.</b> Oblique view of embryo showing anterior region with approximately 75% of hemitergite dorsal extension and undifferentiated dorsal epithelium (<i>ude</i>). The tracheal pits (<i>tp</i>) become more conspicuous at this stage (insert). <b>D–D1.</b> Lateral view of whole-mount embryo with 90% hemitergite extension on the anterior region; note the correlation with the extent of the <i>engrailed</i> expression stripe. The anteriormost part of the trunk lacks expression due to the presence of residual yolk during dissection (<i>dotted line</i>). <b>D2–D3.</b> Close up of developing hemitergites and limb buds (<i>lb</i>). The morphology of the intercalary furrow (<i>if</i>) has become more accentuated. <b>D4–D5.</b> Detail of hemitergites, showing the anterior and posterior tergite borders (<i>solid lines</i>) and the intercalary furrow (<i>dashed line</i>), and together creating a clear distinction between the pretergite (<i>pt</i>) and metatergite (<i>mt</i>); note that the <i>engrailed</i> expression stripe is confined to the posterior portion of the (meta)tergite. Other abbreviations: <i>an</i>, antennae; <i>mxp</i>, maxilliped; <i>pct,</i> proctodeum; <i>arrowhead</i>, undifferentiated epithelium. Scale bars: 400 µm for A, A1, D, D1; 200 µm for B, C, C1; 100 µm for D2, D3; 50 µm for D4, D5; 75 µm for insert in C.</p

Schematic models of comparative dorsoventral expression of <i>engrailed</i> (blue), and correspondence with the tergite borders in extant arthropods.

<p>Anterior facing left. <b>A.</b> The expression of <i>engrailed</i> in the trunk segments of <i>Strigamia maritima</i> is reflective of the plesiomorphic condition of the arthropod trunk, consisting of a continuous dorsoventral stripe adjacent to the intersegmental boundary, and that extends into the limbs (<i>dotted line</i>). The posterior limit of the dorsal <i>engrailed</i> stripe is directly correlated with the posterior (meta)tergite (<i>mt</i>) border and the intersegmental boundary, and does not overlap with the anterior edge of the following (pro)tergite (<i>pt</i>). <b>B.</b> The expression of <i>engrailed</i> in the segments of the insect abdomen is similar to that observed in <i>Strigamia</i>, but differs in the absence of limbs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Rogers1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Campbell1" target="_blank">[47]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Krzemien1" target="_blank">[50]</a>. <b>C.</b> In the haplosegments of <i>Glomeris marginata,</i> the dorsal <i>engrailed</i> stripe is not expressed adjacent to the ventral intersegmental boundary, but rather anteriorly, approximately above the limbs; nevertheless, the tergite borders maintain the correlation with the expression of <i>engrailed</i> observed in <i>Strigamia</i> (A) and insects (B). The ventral side shows the typical activity of <i>engrailed</i> in the posterior portion of each segment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Janssen1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Janssen2" target="_blank">[51]</a>. Other abbreviations: <i>Tn</i>, trunk tergite number <i>n</i>; <i>An</i>, abdominal tergite number <i>n</i>. Numbering in C follows the nomenclature used by Janssen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Janssen1" target="_blank">[43]</a>.</p

Major body components in stage 7–8 embryos of <i>Strigamia maritima.</i>

<p>Specimens photographed with fluorescent nuclear staining (Sytox Green) to show the morphology. <b>A.</b> Lateral view of whole-mount embryo corresponding to Fig. 4D, head on the upper left. The juxtaposed ventral surface (<i>arrow</i>) of the embryo is obscured and out of sight. <b>B.</b> Flat-mounted posterior region of embryo corresponding to Fig. 2C, anterior to the left. In both A and B dots indicate the approximate boundary between the prospective dorsal and lateral tissues as observed in the adult. Squares indicate the approximate boundary between the prospective lateral and ventral tissues as observed in the adult.</p

Hemitergite development and <i>engrailed</i> expression in flat mounted stage 7 embryos of <i>Strigamia maritima</i>.

<p>All images orientated with anterior to the left. Images with the same letter indicate same specimen; A, B, B2, C: fluorescent nuclear staining (Sytox Green) to show the morphology; A1, B1, B3 and C2 respectively same view as A, B, B2, C, under transmitted light, showing pattern of <i>en</i> expression. <b>A–A1.</b> Lateral view of most of the left half of a germ band, showing developing hemitergites extended along the dorsal side only for 30% in the anterior half of the trunk, and the presence of developing tracheal pits (<i>tp</i>). For most of the germ band only the laterodorsal part is visible; note the lack of gene expression in the undifferentiated dorsal epithelium (<i>ude</i>). <b>B–B1.</b> Ventral view of middle trunk region. <b>B2–B3.</b> Close-up of panel B, showing developing hemitergites with weakly formed intercalary furrow (<i>if</i>). <b>B3.</b> Shows correspondence between the extent of the laterodorsal <i>engrailed</i> stripe and the hemitergite extension. <b>C–C1.</b> Ventral view of posterior trunk of nuclear stained embryo. At this stage a thin undifferentiated epithelium, not expressing <i>engrailed</i>, is interposed between the medial and lateral parts of the germ band (<i>arrowhead</i>). <b>C1.</b> Shows posterior correspondence between <i>engrailed</i> expression and hemitergite coverage. The last leg-bearing segment (<i>llb</i>) features the only dorsoventrally uniform <i>engrailed</i> stripe. Other abbreviations: <i>htb,</i> semicircular dorsal border of the hemitergites; <i>mxp</i>, maxilliped. Scale bars: 200 µm for A, A1, B, B1, D, D1; 100 µm for insert in A, C, C1.</p

Phylogenetic relationships, trunk segmentation, and gut metamerism of trilobites and their close relatives.

<p><b>A.</b> Phylogenetic position trilobites relative to other various groups of trilobite-like arthropods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-OrtegaHernndez1" target="_blank">[68]</a>. The trilobite-like articulation is synapomorphic for a distinct clade that includes trilobites, helmetiids and nektaspidids; the presence of this condition in saperiids is uncertain due to preservation and rarity of specimens. Xandarellids do not feature this type of exoskeletal organization, but rather are distinguished by the presence of several pair of walking legs under the posterior tergites (i.e. polypody). <b>B.</b> Inferred dorsoventral expression of <i>engrailed</i> (blue) in the trunk of trilobites and nektaspidids. The ventral expression of <i>engrailed</i> was most likely typical for arthropods in general, restricted to the posterior part of the segment, adjacent to the intersegmental border, and extending into the posterior portion of the limbs. The anteriorly shifted tergites that conform the trilobite-like articulation, coupled with the ancestral correlation between <i>engrailed</i> and the position of the tergites, strongly suggest that the domain of this gene was also anteriorly shifted, and thus expressed dorsally relative to the limbs. In nektaspidids, it is only possible to make accurate inferences on the dorsal expression of <i>engrailed</i> on the cephalothoracic articulation; consequently, the dorsal activity of this gene on the rest of the trunk segments remains uncertain. <b>C.</b> Metamerism of the digestive tract in trilobites, trilobite-like arthropods and remipedes (modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-LeroseyAubril1" target="_blank">[72]</a>). The digestive tract of the ptychopariid <i>Meniscopia beebei</i> shows a metameric organization that consists on several paired gut diverticulae that are located exactly under the tergite-to-tergite articulation throughout the trunk. The remipede <i>Speleonectes gironensis</i> also features paired gut diverticulae; however, in this case the diverticulae are confined within each segment, indicating that they follow the plesiomorphic ventral pattern of arthropod segmentation. The diverticulae in the gut of the nektaspidid <i>Naraoia spinosa</i> is consistent with the organization observed in <i>Meniscopia</i>, even though the only functional articulation.</p

Laterodorsal sclerite differentiation in flat mounted peripatoid juveniles of <i>Strigamia maritima</i>.

<p>All images orientated with anterior to the left. Images with the same letter indicate same specimen. All specimens photographed with fluorescent nuclear staining (Sytox Green) to show the morphology. <b>A.</b> Laterodorsal view of juvenile, showing anterior trunk region with dorsal midline (<i>dm</i>) and well-developed longitudinal pleural furrows (<i>lpf</i>) and presumptive tracheal pits (<i>tp</i>). <b>A1.</b> Close-up of pleural region, showing pleural quadrants (<i>dotted line</i>) formed by the intersection of the tergite borders (<i>solid lines</i>), the intercalary furrow (<i>if/dashed line</i>) and the longitudinal pleural furrows. <b>B.</b> Laterodorsal view of same specimen, showing middle trunk region. Other abbreviations: <i>pt</i>, pretergite; <i>mt</i>, metatergite. Scale bars: 200 µm for A, A2; 100 µm for A1.</p

Laterodorsal sclerite differentiation in flat mounted post-hatching juveniles of <i>Strigamia maritima</i>.

<p>All images orientated with anterior to the left. Images with the same letter indicate same specimen. All specimens photographed with fluorescent nuclear staining (Sytox Green) to show the morphology. <b>A.</b> Laterodorsal view of juvenile showing anterior trunk region and completed dorsal closure indicated by dorsal midline (<i>dm</i>). <b>A1.</b> Close up showing incipient formation of the longitudinal pleural furrows (<i>lpf</i>) in the lateral region. <b>B.</b> Laterodorsal view of juvenile, showing middle trunk region. <b>B1.</b> Close up showing similar morphological features as A1 for the middle of the trunk. The intersection between the longitudinal furrows and the tergite borders (<i>solid line</i>) form quadrants that will become distinct sclerites. The position of the intercalary furrow (<i>if</i>/<i>dashed line</i>) indicates that the presumptive tracheal pits (<i>tp</i>) are aligned with the metatergite (<i>mt</i>). Scale bars: 200 µm for A, B; 100 µm for A1, B1.</p

Phylogenetic distribution of dorsoventral trunk segmental mismatch within total-group Euarthropoda; note that these represent peculiar cases, and thus are not necessarily reflective of the fundamental organization of each major arthropod group as a whole.

<p>Among extant representatives, dorsoventral segmental mismatch, acting at the genetic and/or phenetic levels, has been reported in the trunk of <i>Drosophila </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Struhl1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Lawrence1" target="_blank">[78]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Shen1" target="_blank">[79]</a>, <i>Triops </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Nulsen1" target="_blank">[80]</a>, and <i>Glomeris </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Janssen1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Janssen2" target="_blank">[51]</a>. In <i>Cupiennius</i>, the dorsoventral mismatch is observable only at the gene expression level <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Damen1" target="_blank">[42]</a>. In most cases, differences in dorsoventral expression of segmentation genes are correlated with morphological segmental mismatch. In extinct taxa, various types of dorsoventral segmental mismatch are present in fuxianhuiids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Hou1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Bergstrm4" target="_blank">[53]</a>, trilobites and trilobite-like arthropods, but the extent of a possible genetic mismatch is uncertain in most cases (see discussion in text).</p

Reconstructions of the dorsoventral morphology of trilobites and trilobite-like arthropods.

<p>Anterior to the left. <b>A.</b> Exsagittal longitudinal section of the phacopid <i>Phacops</i> showing typical trilobite exoskeletal organization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Hessler1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Whittington3" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Bruton1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Bruton2" target="_blank">[33]</a>. The biomineralized tergites (<i>Tn</i>) overlay the lightly sclerotized series of sternites (<i>stn</i>) that are connected by flexible tendinous bars (<i>tnb</i>). Each sternite bears a pair of laterally attached gnathobasic (<i>gnb</i>) walking legs. The reconstruction of the dorsal (<i>dlm</i>) and ventral (<i>vlm</i>) longitudinal muscles follow the functional requirements for typical arthropod locomotion, and are shown attached to specific regions of the visceral exoskeleton such as the articulating furrow (<i>af</i>) and the apodemes (<i>apm</i>); note that although the apodemes actually are in direct contact with the visceral side of the tergite at the level of the articulating furrow (see Fig. 8f), such connection is not shown in here due to the schematic nature of this representation. The trilobite-like articulation consists of the anteriorly shifted position of the tergite borders relative to the sternite borders; consequently, a pair of cephalic legs (<i>Cln</i>) is located under the cephalo-thoracic articulation, and the thoracic legs (<i>Tln</i>) under each tergite-to-tergite junction. <b>B.</b> Longitudinal section of the nektaspidid <i>Misszhouia longicaudata</i> showing exoskeletal organization in an unmineralized trilobite-like arthropod <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Edgecombe1" target="_blank">[13]</a>. The tergites of nektaspidids are fused into a thoracopygidial shield (<i>TS</i>), with a single articulation at the cephalo-thoracic junction; as diagnostic for the trilobite-like articulation, a pair of cephalic legs is positioned directly under this region. Aspects of the ventral morphology, such as the sternite series, tendinous bars and limb attachment sites, are very similar to those of trilobites; however, there is no clear indication for muscle attachment sites on the visceral side of the thoracopygidial shield. Other abbreviations: <i>OR</i>, occipital ring; <i>ahr</i>, articulating half ring; <i>dvm</i>, dorsoventral muscle; <i>CS</i>, cephalic shield.</p

Opposing views on the interpretation of trilobite trunk segmentation and alternative phylogenetic hypotheses for the group.

<p><b>A.</b> Direct correspondence between the segments and the dorsal exoskeletal plates (i.e. tergites) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Bergstrm1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Raw1" target="_blank">[14]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Bergstrm2" target="_blank">[16]</a>, illustrated in the corynexochid <i>Olenoides serratus</i>; this interpretation is reflective of the plesiomorphic trunk segmentation present in most arthropods, in which the segment and tergite borders are coincident. Every second segment is shaded. <b>A1.</b> Morphological features and articulating devices of a typical trunk tergite, dorsal view. <b>B.</b> Indirect correspondence between segments and tergites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Strmer1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Palmer1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Hessler1" target="_blank">[20]</a>; the intersegmental boundaries of the trunk occur within each tergite, being delimited by the articulating (<i>af</i>) and pleural (<i>pf</i>) furrows. <b>C.</b> Trilobites have been variously interpreted as stem-euarthropods, stem-chelicerates, and stem-mandibulates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052623#pone.0052623-Budd2" target="_blank">[24]</a>, although most recent studies favour the latter affinities. The position of trilobites within total-group Euarthropoda indicates that the segment polarity gene <i>engrailed</i> had a role in the segmentation of these extinct organisms. Other abbreviations: <i>ahr</i>, articulating half ring; <i>fp</i>, fulcral process.</p