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GBE Gene Content Evolution in Discobid Mitochondria Deduced from the Phylogenetic Position and Complete Mitochondrial Genome of Tsukubamonas globosa Ryoma Kamikawa1,*, Martin Kolisko2,8, Yuki Nishimura3,4, Akinori Yabuki5, Matthew W. Brown6, Sohta A. Ishikawa3,4, Ken-ichiro Ishida3, Andrew J. Roger2,TetsuoHashimoto3,7,andYujiInagaki3,7 1Graduate School of Global Environmental Studies and Graduate School of Human and Environmental Studies, Kyoto University, Japan 2Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan 4Graduate School of Systems and Information Engineering, University of Tsukuba, Ibaraki, Japan 5Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan 6Department of Biological Sciences, Mississippi State University 7Center for Computational Sciences, University of Tsukuba, Ibaraki, Japan 8Present address: Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada *Corresponding author: E-mail: [email protected]. Accepted: January 14, 2014 Data deposition: This project has been deposited at GenBank/EMBL/DDBJ under the accession DRR014073 (pyrosequencing data) and AB854048 (complete mitochondrial genome sequence of Tsukubamonas). Abstract The unicellular eukaryotic assemblage Discoba (Excavata) comprises four lineages: the Heterolobosea, Euglenozoa, Jakobida, and Tsukubamonadida. Discoba has been considered as a key assemblage for understanding the early evolution of mitochondrial (mt) genomes, as jakobids retain the most gene-rich (i.e., primitive) genomes compared with any other eukaryotes determined to date. However, to date, mt genome sequences have been completed for only a few groups within Discoba, including jakobids, two closely related heteroloboseans, and kinetoplastid euglenozoans. The Tsukubamonadida is the least studied lineage, as the order was only recently established with the description of a sole representative species, Tsukubamonas globosa. The evolutionary relationship between T. globosa and other discobids has yet to be resolved, and no mt genome data are available for this particular organism. Here, we use a “phylogenomic” approach to resolve the relationship between T. globosa, heteroloboseans, euglenozoans, and jakobids. In addition, we have characterized the mt genome of T. globosa (48,463 bp in length), which encodes 52 putative protein-coding and 29 RNA genes. By mapping the gene repertoires of discobid mt genomes onto the well-resolved Discoba tree, we model gene loss events during the evolution of discobid mt genomes. Key words: gene loss, genome reduction, organelles, phylogenomics. Introduction 1999). Mitochondrial (mt) genomes are therefore descen- The transformation of bacterial endosymbionts into perma- dants of an a-proteobacterial genome, although all of the nent organelles is a major evolutionary process that has fun- mt genomes sequenced to date are highly reduced relative damentally shaped modern eukaryotic cells and their to any extant bacterial genomes, particularly those of free- genomes. One such organelle, the mitochondrion, was estab- living species (Gray et al. 2004). The ancestral mt genome lished via an endosymbiosis between an a-proteobacterium must therefore have been streamlined by massive “gene and a common ancestor of all living eukaryotes (Gray et al. loss”; these genes were either transferred from the ß The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 306 Genome Biol. Evol. 6(2):306–315. doi:10.1093/gbe/evu015 Advance Access publication January 21, 2014 Evolution of Discobid mt Genomes GBE mitochondrial to host genomes (endosymbiotic gene transfer) gracilis (Euglenida) are subsets of kinetoplast-encoded genes, or deleted if they were no longer necessary for the host– but the precise gene repertoires of the two mt genomes have endosymbiont partnership (Adams and Palmer 2003). This not been delineated due to their complex mt genome archi- reductive trend in mt genome evolution has continued even tectures (Marande et al. 2005; Spencer and Gray 2010; Vlcek after the diversification of the major eukaryotic lineages, yield- et al. 2011). It is noteworthy that Euglenozoa has also been ing a considerable variation in gene content among mt suggested to be a deeply branching eukaryotic lineage based genomes of closely related species (e.g., red algae; Hancock on supposedly “primitive” characteristics in mitochondrial et al. 2010). The patterns and frequencies of gene loss in mt protein import and nuclear DNA preparation machineries genome evolution are not simple, as homologous genes have (Cavalier-Smith 2010). Tsukubamonadida, which was estab- repeatedly been lost from mt genomes in separate branches lished by Yabuki et al. (2011), is represented by a single of the tree of eukaryotes (Gray et al. 1998; Lang, Seif et al. member Tsukubamonas globosa. As both morphological 1999; Adams and Palmer 2003). In order to retrace the evo- and phylogenetic analyses indicated T. globosa is a novel lutionary history of mt genomes in a particular clade, it is member of Discoba (Yabuki et al. 2011), its mt genome size necessary to have mt genome data from taxa representing and gene content are important for understanding the the diversity of the clade of interest and an accurate organis- dynamics of mt genomes in this evolutionarily important mal phylogeny for the corresponding taxa. protist group. Members of Jakobida, one of the subgroups of supergroup Here, we describe a 454-pyrosequencing-based transcrip- Excavata, possess the most gene-rich mt genomes known out tomic analysis of T. globosa that allowed us to include this of all eukaryotes. The most gene-rich mt genome determined organism into a “phylogenomic” data set comprised of 157 so far is of the jakobid Andalucia godoyi carrying 100 func- proteins. This data set was then subjected to both maximum- tionally assignable genes (66 and 34 genes encoding proteins likelihood (ML) and Bayesian phylogenetic analyses resulting in and structural RNAs, respectively; Burger et al. 2013). Other well-resolved tree of discobids. We also determined the com- jakobids also possess gene-rich mt genomes encoding 91–97 plete mt genome sequence of T. globosa. By combining the functionally assignable genes (Burger et al. 2013). The gene- phylogenetic position inferred from the phylogenomic analy- rich ancestral (i.e., bacteria-like) nature of jakobid mt genomes ses and the complete mt genome data, we are able to clarify has been suggested to support an early-branching position of the evolutionary dynamics of gene content in discobid mt the jakobid lineage among eukaryotes (Lang et al. 1997; genomes. Palmer 1997). More recently, the mt genomes of diverse jako- bids has been intensively sequenced to elucidate the evolu- tionary change in mt genomes within the jakobid lineage Materials and Methods (Burger et al. 2013). Although it remains unclear whether Transcriptomic Analysis jakobids are truly early branching, these organisms and their Tsukubamonas globosa was grown in URO-YT medium phylogenetic relatives are important for understanding the (http://mcc.nies.go.jp/medium/en/suy.pdf, last accessed evolutionary history of mt genomes. February 3, 2014) as described in Yabuki et al. (2011). Jakobida, together with Heterolobosea, Euglenozoa, and 8 0.864 mg of total RNA was extracted from 3 Â 10 cells Tsukubamonadida, form a well-supported clade, Discoba using Trizol (Life Technologies) following the manufacturer’s (Excavata; Rodrı´guez-Ezpeleta et al. 2007; Hampl et al. instructions. Construction of the cDNA library was performed 2009; Yabuki et al. 2011). For Heterolobosea, complete mt by Vertis Biotechnology AG (Freising, Germany), and 454 genome sequences are available for two closely related Titanium sequencing (454 Sequencing, Roche) was performed species Naegleria gruberi and N. fowleri, which commonly by Ge´nome Que´bec Innovation Centre at McGill University. encode 42 proteins and 23 structural RNA genes (Gray et al. We obtained 236,871 reads, which were assembled into 2004; Fritz-Laylin et al. 2011; Herman et al. 2013). 12,694 large unique contigs. The T. globosa sequences used Kinetoplastida, Diplonemida, and Euglenida comprise in this study were deposited to GenBank/EMBL/DDBJ (acces- Euglenozoa, but complete mt genome data are available sion no. DRR014073). only for several species belonging to Kinetoplastida (e.g., Trypanosoma cruzi; Westenberger et al. 2006). Kinetoplasts (mitochondria in kineoplastids) are known to contain Phylogenomic Analyses circular DNA in two forms, maxicircles and minicircles. The Our data sets used in this study were based on the gene sets of “maxicircle” encodes a reduced gene set (e.g., 18 protein- Brown et al. (2012) with some modifications of the taxa in- coding genes and 2 rRNA genes in T. cruzi), while numerous cluded (see supplementary table S1, Supplementary Material “minicircles” encode small RNA molecules that are essential online, for the details). We added the homologous sequences for