Genes Genet. Syst. (2017) 92, p. 35–42 Phylogenomic analysis of Kinetoplastea 35 Global Kinetoplastea phylogeny inferred from a large-scale multigene alignment including parasitic species for better understanding transitions from a free-living to a parasitic lifestyle Euki Yazaki1, Sohta A. Ishikawa2*, Keitaro Kume1,3, Akira Kumagai4, Takashi Kamaishi5, Goro Tanifuji6, Tetsuo Hashimoto1,7 and Yuji Inagaki1,7† 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan 2Department of Biological Sciences, Graduate School of Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 3Graduate School of Systems and Information Engineering, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan 4Miyagi Prefecture Fisheries Technology Institute, 97-6 Sodenohama, Watanoha, Ishinomaki, Miyagi 986-2135, Japan 5National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, Minami-Ise, Mie 516-0913, Japan 6Department of Zoology, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan 7Center for Computational Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan (Received 12 October 2016, accepted 23 November 2016; J-STAGE Advance published date: 20 February 2017) All members of the order Trypanosomatida known to date are parasites that are most likely descendants of a free-living ancestor. Trypanosomatids are an excel- lent model to assess the transition from a free-living to a parasitic lifestyle, because a large amount of experimental data has been accumulated for well-studied mem- bers that are harmful to humans and livestock (Trypanosoma spp. and Leishmania spp.). However, recent advances in our understanding of the diversity of trypano- somatids and their close relatives (i.e., members of the class Kinetoplastea) have suggested that the change in lifestyle took place multiple times independently from that which gave rise to the extant trypanosomatid parasites. In the cur- rent study, transcriptomic data of two parasitic kinetoplastids belonging to orders other than Trypanosomatida, namely Azumiobodo hoyamushi (Neobodonida) and Trypanoplasma borreli (Parabodonida), were generated. We re-examined the transition from a free-living to a parasitic lifestyle in the evolution of kinetoplas- tids by combining (i) the relationship among the five orders in Kinetoplastea and (ii) that among free-living and parasitic species within the individual orders. The former relationship was inferred from a large-scale multigene alignment including the newly generated data from Azumiobodo and Trypanoplasma, as well as the data from another parasitic kinetoplastid, Perkinsela sp., deposited in GenBank; and the latter was inferred from a taxon-rich small subunit ribosomal DNA align- ment. Finally, we discuss the potential value of parasitic kinetoplastids identi- fied in Parabodonida and Neobodonida for studying the evolutionary process that turned a free-living species into a parasite. Key words: Azumiobodo hoyamushi, Kinetoplastea, multigene phylogeny, para- sites, Trypanoplasma borreli Edited by Kyoichi Sawamura INTRODUCTION * Corresponding author. E-mail: [email protected] † Corresponding author. E-mail: [email protected] Trypanosomatid flagellates have been studied exten- DOI: http://doi.org/10.1266/ggs.16-00056 sively, as some of them are causative agents of human 36 E. YAZAKI et al. African trypanosomiasis (sleeping sickness), Chagas dis- agent of ichthyobodosis, Ichthyobodo necator: Callahan ease, and leishmaniasis. Besides their clinical impor- et al., 2002) and intracellular parasites in the amoe- tance, these flagellates possess intriguing properties that bozoan Paramoeba pemaquidensis (i.e., Perkinsela sp. are shared by only a few or no other eukaryotes. Try- or Ichthyobodo-related organism: Dyková et al., 2003; panosomatids are known to possess a unique peroxisome- Caraguel et al., 2007; Dyková et al., 2008; Feehan et al., derived organelle, the glycosome, that encloses glycolytic 2013; Lukeš et al., 2014). Parabodonida includes fish enzymes (Opperdoes and Borst, 1977; Gualdrón-López parasites that cause cryptobiosis in salmonid and cypri- et al., 2012). Mitochondria of trypanosomatids contain nid fishes (e.g., Cryptobia salmositica and Trypanoplasma a complex network of two types of circular DNA mol- borreli: Woo and Poynton, 1995), as well as the snail ecules, maxicircles and minicircles, and their mitochon- parasite C. helicis (Leidy, 1846). Among the known neo- drial mRNAs undergo intricate and distinctive editing bodonids, there is a single parasitic member, Azumiobodo prior to translation (Lukeš et al., 2002, 2005). The 5′ hoyamushi, which infects ascidians and causes soft tunic termini of mRNAs from trypanosomatid nuclear genomes syndrome (Hirose et al., 2012). As the transition from a also undergo post-transcriptional modification (Campbell free-living to a parasitic lifestyle occurred after the diver- et al., 2003; Michaeli, 2011). Although the properties gence of the extant parabodonids/neobodonids, the para- described above are also observed in phylogenetic rela- sites in Parabodonida and Neobodonida are potentially tives of trypanosomatids (i.e., members of other orders in useful to retrace the evolutionary path from a free-living the class Kinetoplastea; see below), trypanosomatids, for to a parasitic lifestyle. which various experimental techniques in molecular and For a deeper understanding of the evolution of parasit- cell biology (e.g., genetic modification) are available, have ism in Kinetoplastea, a well-resolved, taxon-rich phylog- been the center of research on Kinetoplastea. eny is indispensable. Deschamps et al. (2011) analyzed Trypanosomatida, together with Eubodonida, Parabo- an alignment of 64 proteins and elucidated the relation- doida, Neobodonida and Prokinetoplastida, comprise the ship among Trypanosomatida, Eubodonida, Neobodonida class Kinetoplastea (Moreira et al., 2004; Simpson et al., and Parabodonida. However, their analyses contained 2006). All known members of Trypanosomatida and of two potential limitations. First, the alignment analyzed Prokinetoplastida are parasites (Simpson et al., 2006; in Deschamps et al. (2011) contained no prokinetoplastid Lukeš et al., 2014). However, the remaining three orders species. Second, each of Parabodonida and Neobodonida are dominated by free-living members, and only a few or was represented by only a single free-living species but none of the members are known to be parasitic. Pre- no parasitic member. In this study, we overcame these viously published phylogenies of small subunit ribo- limitations by analyzing a new alignment of 43 pro- somal DNA (SSU rDNA) sequences have constantly and teins (43-gene alignment), which covered all five orders robustly united Neobodonida, Parabodonida, Eubodon- in Kinetoplastea, and Parabodonida and Neobodonida ida and Trypanosomatida, excluding Prokinetoplas- were represented by both free-living and parasitic mem- tida (Simpson et al., 2002; Moreira et al., 2004; von der bers. Combining the global Kinetoplastea phylogeny, Heyden et al., 2004). This tree topology suggested that updated by analyzing the 43-gene alignment, with a Trypanosomatida and Prokinetoplastida acquired para- taxon-rich SSU rDNA phylogeny, we discuss the transi- sitic lifestyles separately (Moreira et al., 2004; Simpson tion from a free-living to a parasitic lifestyle in the evolu- et al., 2006; Lukeš et al., 2014). Owing to their impor- tion of Kinetoplastea. tance in public health, the origin of parasitism in the extant trypanosomatids is one of the major questions in MATERIALS AND METHODS the evolution of Kinetoplastea. To address this question, the precise relationship among Neobodonida, Parabodon- Cultures, RNA extraction and sequencing The lab- ida, Eubodonida and Trypanosomatida has been explored oratory culture of A. hoyamushi established by Hirose et mainly by analyzing SSU rRNA genes or genes encoding al. (2012) was grown and maintained in sea water con- highly conserved proteins, but has not been resolved with taining 2% heat-inactivated fetal bovine serum (Gibco, high statistical support (Dolezel et al., 2000; Simpson et Thermo Fisher Scientific, Waltham, Massachusetts, USA) al., 2002; Moreira et al., 2004; Simpson et al., 2004; von at 17 °C. Trypanoplasma borreli ATCC50836 was pur- der Heyden et al., 2004; Deschamps et al., 2011). A phy- chased from the American Type Culture Collection, and logenetic analysis of 64 genes encoding highly conserved grown in live-infusion-tryptose medium (Fernandes and proteins successfully designated Eubodonida as the clos- Castellani, 1966) at 17 °C. Total RNA was extracted est relative of Trypanosomatida (Deschamps et al., 2011). from the harvested cells using Trizol (Thermo Fisher Compared to pathogenic trypanosomatids, other Scientific, Waltham, Massachusetts, USA), following the parasitic members in Kinetoplastea have received less manufacturer’s protocol. Construction of a cDNA library research attention. Two types of parasites belong to and subsequent sequencing by the Illumina HiSeq2500 Prokinetoplastida: fish parasites (e.g., the causative system were performed at Hokkaido System Science Phylogenomic analysis of Kinetoplastea 37 (Sapporo, Hokkaido, Japan). We generated 401,725,240 anticipated that such sequences would probably group and 433,374,224 paired-end, 100-base reads from the with the Naegleria and Dictyostelium