Appendix S1

Average dimensions and ratios of ventral valve of Eohadrotreta zhenbaensis from the Cambrian (Series 2) Shuijingtuo Formation of the Three Gorges area, South China, demonstrating the three ontogenetic development stages respectively

Appendix S4

Average dimensions and ratios of ventral and dorsal valves of Eohadrotreta? zhujiahensis from the Cambrian (Series 2) Shuijingtuo Formation of the Three Gorges area, South China, including the whole ontogenetic development stages

The oldest known digestive system consisting of both paired digestive glands and a crop from exceptionally preserved trilobites of the Guanshan Biota (Early Cambrian, China)

<div><p>The early Cambrian Guanshan biota of eastern Yunnan, China, contains exceptionally preserved animals and algae. Most diverse and abundant are the arthropods, of which there are at least 11 species of trilobites represented by numerous specimens. Many trilobite specimens show soft-body preservation via iron oxide pseudomorphs of pyrite replacement. Here we describe digestive structures from two species of trilobite, <i>Palaeolenus lantenoisi</i> and <i>Redlichia mansuyi</i>. Multiple specimens of both species contain the preserved remains of an expanded stomach region (a “crop”) under the glabella, a structure which has not been observed in trilobites this old, despite numerous examples of trilobite gut traces from other Cambrian Lagerstätten. In addition, at least one specimen of <i>Palaeolenus lantenoisi</i> shows the preservation of an unusual combination of digestive structures: a crop and paired digestive glands along the alimentary tract. This combination of digestive structures has also never been observed in trilobites this old, and is rare in general, with prior evidence of it from one juvenile trilobite specimen from the late Cambrian Orsten fauna of Sweden and possibly one adult trilobite specimen from the Early Ordovician Fezouata Lagerstätte. The variation in the fidelity of preservation of digestive structures within and across different Lagerstätten may be due to variation in the type, quality, and point of digestion of food among specimens in addition to differences in mode of preservation. The presence and combination of these digestive features in the Guanshan trilobites contradicts current models of how the trilobite digestive system was structured and evolved over time. Most notably, the crop is not a derived structure as previously proposed, although it is possible that the relative size of the crop increased over the evolutionary history of the clade.</p></div

Evidence for crop and digestive glands in Guanshan trilobites.

<p>(A) <i>Palaeolenus lantenoisi</i> showing evidence of crop, digestive glands, alimentary canal along thorax, and excreted waste posterior to pygidium, GLF WLQ 228A. (B) Line drawing of (A). Cr = crop; cd = cephalic digestive glands; td = thoracic digestive glands. (C) <i>Palaeolenus lantenoisi</i>, showing crop cavity and antennae but no alimentary canal. (D) <i>Palaeolenus lantenoisi</i>, showing antennae, crop and alimentary canal but not preserving obvious digestive glands. (E) Line drawing of (D). Cr = crop; ac = alimentary canal. (F) <i>Palaeolenus lantenoisi</i>, showing crop cavity and only diffuse iron staining on thorax, GLF WLQ 174. (G) <i>Palaeolenus lantenoisi</i>, showing crop and no additional iron staining, GLF WLQ 214A. (H) <i>Redlichia mansuyi</i> showing cavity where crop would be located, GLF WLQ 216A. (I) <i>Redlichia mansuyi</i> showing crop and some additional iron staining, GLF WLQ 245A. Scale bar for (A), (H-I) = 5 mm; scale bar for all other = 1 mm.</p

Trilobite taxa for which part of the digestive system has been described.

<p>Trilobite taxa for which part of the digestive system has been described.</p

Overview of soft-body preservation.

<p>(A) <i>Palaeolenus lantenoisi</i>, GLF WLQ 174. (B) Elemental maps of Si (cyan) and Fe (yellow) overlain on SEM image of iron oxide framboids in crop of specimen shown in 3A. (C) Elemental maps of O (green) and Fe (yellow) overlain on SEM image of iron oxide framboids in crop of specimen shown in 3A. (D) Elemental map showing Fe concentration (yellow) at crop of specimen shown in 3A. There is no indication of iron in the center of the image because the framboids at the back of the cavity are outside the width of the detection field (see also 3B, 3C). (E) Elemental map showing Fe concentration (yellow) under left-hand side of doublure of cephalon, articulating half-rings on thoracic segments, and at crop for specimen shown in 3F. (F) <i>Palaeolenus lantenoisi</i>, GLF WLQ 214A. (G) Elemental map showing Fe concentration (yellow) at crop, posterior part of glabella, and palpebral lobe of specimen shown in 3I. (H) Elemental map showing Fe concentration (yellow) at thoracic and pygidial pleural spines of specimen shown in 3I. (I) <i>Palaeolenus latenoisi</i>, GLF WLQ 212A. (J) <i>Redlichia mansuyi</i>, GLF WLQ 245A. (K) Elemental map showing Fe concentration (yellow) at crop of specimen shown in 3J. Scale bar = 1 mm except where indicated. See Figs H-L in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184982#pone.0184982.s001" target="_blank">S1 File</a> for additional elemental maps for these specimens. See Figs M-N in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184982#pone.0184982.s001" target="_blank">S1 File</a> for additional elemental maps of pleural areas in <i>Redlichia spp</i>.</p

Iron concentrations in digestive tract of <i>Palaeolenus lantenoisi</i>, GLF WLQ 228A.

<p>Scale bars = 1 mm. Top right and left panels show elemental maps of Fe (yellow), Si (pink), O (green), and Al (cyan) concentrations in the areas indicated by black rectangles in the central photography. All other panels show elemental maps of just Fe concentrations (yellow) in the indicated areas. Note that here and in elemental maps in other figures, some areas where iron concentrations are expected but not evident are in ‘shadow’ due to the orientation of the specimen or because that part of the specimen is outside of the width of detection, so no elemental composition is available (compare the top two panels showing Fe, Si, O, and Al with those just below showing only Fe). See Figs B-G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184982#pone.0184982.s001" target="_blank">S1 File</a> for additional elemental maps of different parts of this specimen.</p

Suppression of <i>Ts</i>-<i>pmy</i> mRNA expression by siRNA1743 caused defects in larval molting.

<p>A. The larvae were electroporated with 8 µM siRNA1743 (a) or control siRNA (b) and incubated for 18 hours. The untreated larvae (c) were used as a negative control. The larvae with cuticle sheath at the end(s) were counted as molting ones. B. The molting percentage for each treatment was calculated. All of the assays were performed in triplicate. *<i>p</i><0.05 compared with the control siRNA-treated group.</p

Tables 4-12

Localities and basic statistics for the described Associations (number of individual for each taxon estimated by methods outlined in the text

The siRNA oligos used in this study.

<p>The siRNA oligos used in this study.</p