Arthropod Phylogenetics in Light of Three Novel Millipede (Myriapoda: Diplopoda) Mitochondrial Genomes with Comments on the Appropriateness of Mitochondrial Genome Sequence Data for Inferring Deep Level Relationships

Background Arthropods are the most diverse group of eukaryotic organisms, but their phylogenetic relationships are poorly understood. Herein, we describe three mitochondrial genomes representing orders of millipedes for which complete genomes had not been characterized. Newly sequenced genomes are combined with existing data to characterize the protein coding regions of myriapods and to attempt to reconstruct the evolutionary relationships within the Myriapoda and Arthropoda. Results The newly sequenced genomes are similar to previously characterized millipede sequences in terms of synteny and length. Unique translocations occurred within the newly sequenced taxa, including one half of the Appalachioria falcifera genome, which is inverted with respect to other millipede genomes. Across myriapods, amino acid conservation levels are highly dependent on the gene region. Additionally, individual loci varied in the level of amino acid conservation. Overall, most gene regions showed low levels of conservation at many sites. Attempts to reconstruct the evolutionary relationships suffered from questionable relationships and low support values. Analyses of phylogenetic informativeness show the lack of signal deep in the trees (i.e., genes evolve too quickly). As a result, the myriapod tree resembles previously published results but lacks convincing support, and, within the arthropod tree, well established groups were recovered as polyphyletic. Conclusions The novel genome sequences described herein provide useful genomic information concerning millipede groups that had not been investigated. Taken together with existing sequences, the variety of compositions and evolution of myriapod mitochondrial genomes are shown to be more complex than previously thought. Unfortunately, the use of mitochondrial protein-coding regions in deep arthropod phylogenetics appears problematic, a result consistent with previously published studies. Lack of phylogenetic signal renders the resulting tree topologies as suspect. As such, these data are likely inappropriate for investigating such ancient relationships

PhyDesign results for all 13 protein-coding mitochondrial gene regions.

<p>The peaks for each gene region skewed toward the terminals of both trees. As a result, most signal deep in the trees is confounded by noise. A) Myriapod BI tree converted to ultrametric. B) Ecdysozoan BI tree converted to ultrametric. These results indicate that mitochondrial protein-coding sequences are not appropriate for reconstructing deep arthropod relationships, even when the data is encoded as amino acid residues. The color scheme follows <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068005#pone-0068005-g002" target="_blank">figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068005#pone-0068005-g003" target="_blank">3</a>.</p

Phylogenetic trees for the Myriapoda and Ecdysozoa based on mitochondrial protein-coding genes.

<p>The following phylogenies were reconstructed using maximum likelihood and Bayesian inference of amino acid sequences. The ML trees were obtained using RAxML with 1000 random addition searches followed by 1000 boostrap replicates. The BI trees were obtained from two phylobayes runs consisting of 10000 cycles. The first 2000 cycles were discarded as burn-in. A) Myriapod ML tree, B) myriapod BI tree, C) ecdysozoan ML tree, and D) ecdysozoan BI tree. In the myriapod trees, millipedes taxa are colored green, symphylans are blue, and centipedes are red. In the ecdysozoan trees, outgroup taxa (non-arthropods) are colored black, myriapods are green, chelicerates are red, “crustaceans” and non-insect hexapods are blue, and insects are yellow.</p

Mitochondrial genomes for the three taxa sequenced as part of this study.

<p>A. <i>Appalachioria falcifera</i>. B. <i>Abacion magnum</i>. C. <i>Brachycybe leconti</i>. The grey region corresponds to the A-T Rich Region (the origin of transcription and replication). The red sequences depict the ribosomal subunit DNA. The green regions represent protein-coding sequences. The pink regions correspond to transfer RNAs.</p

Amino acid conservation values based on identity for each of the 13 protein-coding regions of all currently available myriapod mitochondrial genomes.

<p>Amino acid conservation values based on identity for each of the 13 protein-coding regions of all currently available myriapod mitochondrial genomes.</p

Taxonomy and locality data for the specimens sequenced herein.

<p>Taxonomy and locality data for the specimens sequenced herein.</p

Myriapod mitochondrial genome syntenies depicted in a phylogenetic context.

<p>The phylogeny is adapted from Regier and Shultz <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068005#pone.0068005-Regier3" target="_blank">[18]</a> and Sierwald and Bond <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068005#pone.0068005-Sierwald1" target="_blank">[6]</a>. Grey regions are ribosomal subunit genes, white sequences code for transfer RNAs, and black region depicts major A-T Rich region in each genome. The other regions are protein coding; the color scheme is used in subsequent figures.</p