Arafat et al. BMC Evolutionary Biology (2018) 18:65 https://doi.org/10.1186/s12862-018-1186-1 RESEARCH ARTICLE Open Access Extensive mitochondrial gene rearrangements in Ctenophora: insights from benthic Platyctenida Hanan Arafat1, Ada Alamaru1, Carmela Gissi2,3 and Dorothée Huchon1,4* Abstract Background: Complete mitochondrial (mt) genomes have been sequenced for thousands of animals and represent a molecule of choice for many evolutionary studies. Nevertheless, some animal groups have remained under-sampled. Ctenophora (comb jellies) is one such example, with only two complete mt sequences determined hitherto for this phylum, which encompasses ca. 150–200 described species. This lack of data derives from the extremely fast mt evolutionary rate in this lineage, complicating primer design and DNA amplification. Indeed, in the two ctenophore mt genomes sequenced to date, i.e. those of Mnemiopsis leidyi (order Lobata) and Pleurobrachia bachei (order Cydippida), both rRNA and protein coding genes exhibit an extraordinary size reduction and have highly derived sequences. Additionally, all tRNAs, and the atp6 and atp8 genes are absent. In order to determine whether these characteristics are shared by other ctenophores, we obtained the complete mt genomes of three benthic ctenophores belonging to the so far unsampled order of Platyctenida: Coeloplana loyai, Coeloplana yulianicorum and Vallicula multiformis. Results: The mt genomes of benthic ctenophores reveal the same peculiarities found in Mnemiopsis and Pleurobrachia, demonstrating that the fast evolutionary rate is a general trait of the ctenophore mt genomes. Our results also indicate that this high evolutionary rate not only affects the nucleotide substitution but also gene rearrangements. Indeed, gene order was highly rearranged among representatives of the different taxonomic orders in which it was close to random, but also quite variable within Platyctenida, in which the genera Coeloplana and Vallicula share only four conserved synteny blocks. However, the two congeneric Coeloplana species display exactly the same gene order. Because of the extreme evolutionary rate, our phylogenetic analyses were unable to resolve the phylogenetic position of ctenophores within metazoans or the relationships among the different Ctenophora orders. Comparative sequence-analyses allowed us to correct the annotation of the Pleurobrachia mt genome, confirming the absence of tRNAs, the presence of both rRNA genes, and the existence of a reassignment of codon TGA from tryptophan to serine for this species. Conclusions: Since Platyctenida is an early diverging lineage among Ctenophora, our findings suggest that the mt traits described above are ancestral characteristics of this phylum. Keywords: Coeloplana, Vallicula, mtDNA, Gene loss, Extreme evolutionary rate Background 1–6[nad1–6]), 22 transfer RNA (tRNAs) genes and 2 The typical metazoan mitochondrial (mt) genome is a ribosomal RNA (rRNA) genes (the small subunit riboso- small (~ 20 kb), circular DNA molecule containing 37 mal RNA [rns] and the large subunit ribosomal RNA genes – 13 protein coding genes (the cytochrome oxidase [rnl]) [1, 2]. This compact molecule possesses several 1–3[cox1–3]; the ATPase subunit 6 and 8 [atp6/8]; the characteristics that have made it a perfect sequencing cytochrome b [cob]; the NADH dehydrogenase subunit target. First, mt genomes are present in high copy num- bers [3], since eukaryotic cells usually contain several * Correspondence: [email protected] mitochondria and each mitochondrion possesses numer- 1School of Zoology, Tel-Aviv University, Tel-Aviv, Israel 4 ous copies of the mt genome. Second, mt genomes are The Steinhardt Museum of Natural History and National Research Center, – Tel-Aviv University, Tel-Aviv, Israel extremely compact most are intron-less and non-coding Full list of author information is available at the end of the article regions are extremely small [3]. Consequently, it is usually © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Arafat et al. BMC Evolutionary Biology (2018) 18:65 Page 2 of 11 easier to amplify and sequence mt genes than nuclear the “typical” mt genome described above (see Lavrov and ones. Finally, the same set of orthologous single-copy Pett [1]). Indeed, they are of very small size (10–11 kilo- genes is conserved in most animal taxa, thus facilitating bases [kb]); lack the atp8, atp6 and most/all tRNA genes; sequence comparisons and, above all, allowing the recon- demonstrate a length reduction; and a high sequence diver- struction of gene trees that correspond to species trees [4]. gence in both protein coding and rRNA genes. These mt The above-noted features have made the mt genome a genomes thus reveal an extremely high evolutionary rate. It molecule of choice for many fields of genomics and mo- is unclear, however, whether all these features are shared by lecular evolution. For example, complete mt genome se- all ctenophores or only by the few analysed lineages to date. quences have been used to resolve the phylogenetic Here, we report the sequencing and analysis of the relationships of many animal groups [5], to study the complete mt genomes of three benthic ctenophore spe- transfer of organelle genes to the nucleus [6] and to cies belonging to the order Platyctenida and the family study the evolution of gene rearrangement [7]. Addition- Coeloplanidae: Coeloplana loyai, Coeloplana yuliani- ally, the mt cox1 gene is used as a universal “DNA bar- corum and Vallicula multiformis. The analyses of these code” [8] for animals and, consequently, for a given new species have enabled us to amend the annotation of species, a complete mt genome is often sequenced from the Pleurobrachia mt genome and to confirm a modifi- one individual as a prerequisite of population studies [9]. cation of the genetic code in the latter. One direct consequence of the high interest in mt ge- nomes is the large and increasing number of complete mt genomes available in public databases (i.e., over 7300 Results complete mt genomes of animals are available at the Na- General features of the Coeloplanidae mt genomes tional Center for Biotechnology Information [NCBI] - The circular mt genomes of C. loyai, C. yulianicorum and https://www.ncbi.nlm.nih.gov/genome/browse/)[3], and V. multiformis are 11,574, 11,551, and 9961 base pairs (bp) numerous databases and web servers are dedicated to long, respectively. The three sequenced species contain the their annotations and comparisons [10, 11]. However, same set of genes: 11 protein coding genes (all those some animal phyla, such as Ctenophora, are under- present in typical animal mt genomes, except for atp6 and represented in these databases [12]. atp8) and 2 rRNAs (rnl and rns). In addition, from 1 to 4 Ctenophora (comb jellies) comprise a rather small unassigned ORFs (Unidentified Reading Frames, named phylum of marine animals that encompasses about 150– URFs 1–4), ranging from 102 to 687 bp, were identified in 200 species [13]. Most ctenophores are planktonic pred- each species (Fig. 1). All annotated URFs were predicted ators with a gelatinous body similar to jellyfish (Cnidaria, to encompass from 1 to 5 putative transmembrane do- Medusozoa), for which they are often mistaken. Unlike mains. All these genes are encoded on the same strand jellyfish and other cnidarians, ctenophores lack stinging (Fig. 1). cells and are characterized by the presence of comb rows No tRNAs were identified. Indeed, all putative tRNAs composed of cilia, which are used for swimming, and detected by the three tRNA annotation software programs tentacles, which possess a special cell type, the collo- (see Methods) were located within ORFs and possessed a blasts, used for grasping and feeding. Ctenophores are structure that did not fit the canonical mt tRNA structure traditionally divided into two classes, Tentaculata and [19]. They were thus considered to be false positive hits. Nuda, based on the presence or absence of tentacles While gene order is conserved between the mt se- throughout their lives, respectively [13]. However, mo- quences of the two Coeloplana species, only four synteny lecular analyses suggest that Tentaculata, which include blocks are conserved between the two genera Coeloplana eight orders, is paraphyletic, and that the single Nuda and Vallicula (cox1-cox2-nad4, nad4L-nad1, cob-cox3- order – Beroida – is nested within Tentaculata [14–17]. nad3,andrnl-rns)(Fig.2). The gene order is thus con- Among ctenophores, Platyctenida is the only order that served at the congeneric level but already quite variable at includes benthic species. Most members of the Platycte- the intra-family level. Considering the other ctenophore nida do not possess comb rows in the adult stage and taxonomic orders, only one synteny block is conserved externally resemble flat worms, except for the presence (cox3-nd3) between Platyctenida and the Lobata M. leidyi, of two long retractile tentacles. Interestingly, molecular and