Neighbour-joining tree of <i>Firmicutes</i>-like bacterial clones isolated from different instars of <i>H. parallela</i>.

<p>Only 1 representative clone from each OTU was included in the phylogenetic tree. Clones obtained in this study and closest relative bacterial clones that obtained previously form other scarab beetles were shown in bold. Bar charts represent the relative number of clones obtained from each larval stage. The bar represents 0.05 substitutions per site. For more information, see figure legends of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057169#pone-0057169-g004" target="_blank">Fig. 4</a>.</p

supplemental data

supplemental dat

Relative abundance of different bacterial groups found in the hindgut of <i>H. parallela</i> at different instars.

<p>Relative abundance of different bacterial groups found in the hindgut of <i>H. parallela</i> at different instars.</p

Collection sites of <i>H. parallela</i> larvae.

<p>The abbreviations of the province or municipality used are as follows: LN, Liaoning; TJ, Tianjin; SD, Shandong, HB, Hubei; ZJ, Zhejiang; FJ, Fujian; GD, Guangdong; GX,Guangxi; SC, Sichuan; NX, Ningxia. In this study, the ten natural populations of the <i>H. parallela</i> were named according to the abbreviation of the province or municipality where they were collected (for example, if the larvae were collected at Hubei province, the population was named HB).</p

Relative abundance of different bacterial groups found in the hindgut of <i>H. parallela</i> from the 10 distinct natural populations.

<p>Relative abundance of different bacterial groups found in the hindgut of <i>H. parallela</i> from the 10 distinct natural populations.</p

Neighbour-joining tree representing the Firmicutes-like bacterial clones isolated from <i>H. parallela</i> natural populations that besides <i>Clostridia</i>.

<p>Only 1 representative clone from each OTU was included in the phylogenetic tree. Bar charts represent the relative number of clones obtained from each natural population. The 16S rRNA gene sequence of <i>Cenarchaeum symbiosum</i> (U51469) was selected as an outgroup. Clones obtained in this study and closest relative bacterial clones that obtained previously from larvae of other scarab beetles, including <i>Pachnoda ephippiata</i> (<i>P. ephippiata</i>), <i>Dermolepida albohirtum</i> (<i>D. albohirtum</i>), and <i>Costelytra zealandica</i> (<i>C. zealandica</i>) were shown in bold. Bar charts represent the relative number of clones obtained from each natural population. Numbers at nodes are bootstrap support values ≥50%. The bar represents 0.05 substitutions per site respectively.</p

Neighbour-joining tree of <i>Bacteroidetes</i>, <i>Actinobacteria</i>, <i>Proteobacteria</i>bacterial clones isolated from different instars of <i>H. parallela</i>.

<p>Bar charts represent the relative number of clones obtained from each larval stage. Clones obtained in this study and closest relative bacterial clones that obtained previously form other scarab beetles were shown in bold. For other details, see figure legends of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057169#pone-0057169-g004" target="_blank">Fig. 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057169#pone-0057169-g009" target="_blank">Fig. 9</a>.</p

RDA biplots of selected (forward selection) environmental factors and the relative abundance of major bacterial groups in the hindgut of scarab larvae.

<p>The smaller the angle between the vectors (or a vector and an axis) and the longer the vectors, the more correlated are the variables represented by the vectors. The eigenvalues of the two axes are given in parentheses. Abbreviations used are as follows: Annu, Mean annual temperature; Ave, monthly temperature range; N, total nitrogen in the soil; C, total carbon in the soil; Rumin, <i>Ruminococcaceae</i>; Veillone, <i>Veillonellaceae</i>; Rhodo, <i>Rhodocyclaceae</i>, Clostri, <i>Clostridiaceae</i>; Klebsi, <i>Klebsiella</i>; Desulfo, <i>Desulfovibrio</i>; Eubac, <i>Eubacteriaceae</i>; Entero, <i>Enterobacter</i>; Raoult, <i>Raoultella</i>; Porphy, <i>Porphyromonadaceae</i>; Lachn, <i>Lachnospiraceae</i>, Chris, <i>Christensenellaceae</i>.</p

Neighbour-joining tree of all clones isolated from 10 <i>H. parallela</i> natural populations that affiliated to <i>Clostridia</i>.

<p>After construction, the tree was edited using the Interactive Tree of Life website (iTOL) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057169#pone.0057169-Letunic1" target="_blank">[77]</a>. The 16S rRNA gene sequence of <i>Thermotoga maritime</i> (AJ401021) was selected as an outgroup. Branches in red are clones obtained from other scarab larvae in previous studies. For clarity, clones were grouped into OTUs, with only 1 representative clone from each OTU included in the phylogenetic tree, and the last 4 numbers of their GenBANK accession numbers (JF964265–JF964858) were also included. Bar charts represent the relative number of clones obtained from each natural population. Bar in the center of the circle represents 0.01 substitutions per site. The abbreviations of the bacterial species were used are as follows: <i>R. gauvreauii</i>, <i>Ruminococcus gauvreauii</i> (EF529620); <i>C. jejuense</i>, <i>Clostridium jejuense</i> (AY494606); <i>E. brachy</i>, <i>Eubacterium brachy</i> (U13038); <i>D. auripigmenti</i>, <i>Desulfosporosinus auripigmenti</i> (AJ493051); <i>O. valericigenes</i>, <i>Oscillibacter valericigenes</i> (AP012044). <i>C. hongkongensis</i>, <i>Catabacter hongkongensis</i>(AB671763); <i>C. asparagiforme</i>, <i>Clostridium. asparagiforme</i> (ACCJ01000522); <i>E. fissicatena</i>, <i>Eubacterium fissicatena</i> (FR749937).</p

An image of representative gel showing DGGE analysis of PCR products amplified from individual clones.

<p>Lanes labeled 1 to 21 give examples of individual clones that were randomly picked out from the clone library of SC population. Clones with the same electrophoretic mobility in the gel were temporarily grouped into one group. The marker lanes (M) contained PCR-amplified 16S rRNA gene fragments of nine bacterial clones that showed different electrophoretic mobility in DGGE gel.</p