Mitochondrial genomes (mtDNAs) of bilaterian animals are short, circular DNA molecules of 14-16 kb in length, typically characterized by the absence of introns and the presence of only short intergenic regions, with the exception of the control region (CR), a non-coding region assumed to contain the elements for the regulation of replication and transcription of the genome [1, 2]. To date, about 1,700 complete metazoan mitochondrial genomes have been sequenced and used in comparative mitogenomics and phylogenetic studies on different taxonomic scales [3–9].
The mitochondrial gene content is highly conserved across the different bilaterian phyla, with typically 37 genes [1, 2, 10]. Among them, 13 genes encode for proteins [ATP synthase subunits 6 and 8 (atp6 and atp8), cytochrome oxidase subunits (cox1, cox2, cox3), apocytochrome b (cytb), and dehydrogenase subunits (nd1, nd2, nd3, nd4, nd5, nd6 and nd4L)]. The remaining genes encode two ribosomal subunits (srRNA and lrRNA) and usually 22 tRNAs. However, cases of duplication and loss of tRNAs have been reported within bilaterians [11, 12]. Tunicate mitochondrial genomes illustrate such exceptions and typically encode 24 tRNAs, apart from two Phallusia species which lack the tRNA-Asp , and Halocynthia roretzi which encode two tRNA-phe . The two additional tRNAs present in tunicate mitochondrial genomes when compared to vertebrates are (i) the tRNA-Gly (for AGR codons), which is necessary for the translation due to the derived tunicate mitochondrial genetic code , and (ii) the tRNA-Met (for AUA codon), whose presence might reduce the conflict between translation initiation -- which requires a tRNA-Met (for AUG) -- and translation elongation that involves AUG codons .
The mitochondrial gene order is highly conserved within Deuterostomia [10, 17], and particularly in chordate genomes. Conversely, mitochondrial gene arrangement shows an important plasticity in some animal phyla, (e.g. molluscs and nematodes [1, 2]), and especially in tunicates [2, 13, 18–22]. Tunicates, or Urochordates, are marine deuterostomes characterized by markedly diversified developmental and life history traits, and traditionally encompass three major classes: Ascidiacea (sea squirts), Thaliacea (salps) and Appendicularia (larvaceans). Ascidiacea, commonly referred to as ascidians, is the most speciose and widespread group. Several ascidian species have been identified as invasive species, such as Styela clava and Pyura praeputialis [23–26], and have a strong ecological impact on the invaded marine ecosystems. Some species are also widely used as model organisms in evo-devo studies like Ciona intestinalis and Botryllus schlosseri [27–31]. According to the traditional classification, the class Ascidiacea is subdivided into three major orders: Phlebobranchia, Aplousobranchia, and Stolidobranchia. In contrast with this taxonomic view, 18S rRNA-based phylogenies have shown that ascidians are in fact paraphyletic [32–34]. According to the 18S rRNA phylogenetic framework, Aplousobranchia, Phlebobranchia, and Thaliacea are closely related, whereas Stolidobranchia forms a distinct and monophyletic group, which might be close to Appendicularia, although the position of the latter is still debated [32, 33].
To date, complete mitochondrial genomes of tunicates are mainly available for a single representative of Thaliacea (Doliolum nationalis) and five ascidians [13, 18–22], including four phlebobranchians (Ciona intestinalis type A and B, C. savignyi, Phallusia fumigata, P. mammillata) and one stolidobranchian (Halocynthia roretzi). The available mitochondrial data suggest that several unique features characterize mitogenomic evolution in tunicates relative to other chordate phyla. Two main peculiarities can be distinguished. The first refers to the highly variable gene order observed within the group, which implies that extensive gene rearrangements have occurred even at low taxonomic levels [13, 18, 19]. However, since most available tunicate complete mtDNA sequences belong to phylogenetically-related species (except H. roretzi) according to the 18S rRNA reference [32–34], it is not possible to evaluate whether mitochondrial gene rearrangements characterize the whole order or only the Aplousobranchia + Phlebobranchia + Thaliacea clade. The second specificity is that of an accelerated evolutionary rate of tunicates, as revealed by the long branches of the group in mitogenomic topologies [35–37] and the associated composition bias . However, whether this accelerated substitution rate is restricted to protein coding genes as in snakes  or is a more general feature of the whole mtDNA of Tunicates, has yet to be investigated.
These two peculiar evolutionary features of tunicate mitochondrial genome evolution have hampered their reliable phylogenetic placement within metazoans. Analyses of mitochondrial protein-coding genes have almost always systematically placed tunicates outside Bilateria [20, 35, 36, 39]. This is in sharp contrast with recent nuclear-based phylogenomic studies that identified tunicates as the closest living relatives of vertebrates within chordates [40–43]. Only two recent mitogenomic study have found marginal support for chordate monophyly. Bourlat et al.  grouped cephalochordates with vertebrates according to the traditional Euchordata hypothesis using a concatenation of the 13 protein coding genes under a site- and time-heterogeneous mixture model in Bayesian phylogenetic reconstructions. Alternatively, Zhong et al.  recovered the new chordate phylogeny under the maximum likelihood framework when removing the fastest evolving vertebrates species and when considering only the four most conserved mitochondrial proteins.
Here, we sequenced the complete mitochondrial genome of the solitary ascidian Herdmania momus (Ascidiacea: Stolidobranchia: Pyuridae), an Indo-Pacific species that was introduced into the Mediterranean Sea via the Suez Canal . We describe the structural and compositional features of H. momus mtDNA, discuss its evolutionary dynamics with respect to other tunicate and chordate mitochondrial genomes, and provide an updated metazoan phylogeny based on probabilistic analyses of the 13 mitochondrial proteins using site- and time-heterogeneous mixture models of amino acid substitutions.