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Arginine deiminase pathway enzymes: evolutionary history in metamonads and other eukaryotes Novák, Lukáš; Zubáčová, Zuzana; Karnkowska, Anna; Kolisko, Martin; Hroudová, Miluše; Stairs, Courtney W; Simpson, Alastair G B; Keeling, Patrick J; Roger, Andrew J; Čepička, Ivan; Hampl, Vladimír Oct 6, 2016

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RESEARCH ARTICLE Open AccessArginine deiminase pathway enzymes:evolutionary history in metamonads andother eukaryotesLukáš Novák1, Zuzana Zubáčová1, Anna Karnkowska1,5, Martin Kolisko2,5, Miluše Hroudová3, Courtney W. Stairs2,Alastair G. B. Simpson4, Patrick J. Keeling5, Andrew J. Roger2, Ivan Čepička6 and Vladimír Hampl1*AbstractBackground: Multiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy productionby arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, withonly two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess thecomplete set of all three enzymes. We have performed an extensive survey of available sequence data in order to mapthe distribution of these enzymes among eukaryotes and to reconstruct their phylogenies.Results: We have found genes for the complete pathway in almost all examined representatives of Metamonada, theanaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence ofthe complete pathway in the last common ancestor of metamonads and heterologous transformation experimentssuggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occursrarely, nevertheless, it was found in representatives of most major eukaryotic clades.Conclusions: Phylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remainpossible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may berelated to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Singleprotein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent genelosses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.Keywords: Arginine deiminase, Ornithine transcarbamylase, Carbamate kinase, Phylogeny, Metamonada,Preaxostyla, ProtistsBackgroundThe arginine deiminase pathway (ADI pathway, syn.: argin-ine dihydrolase pathway) catalyzes a conversion of argin-ine to ornithine, ammonium, and carbon dioxide, whilegenerating ATP from ADP and phosphate. The enzymesinvolved in the three steps of the pathway are arginine dei-minase (ADI, EC 3.5.3.6), ornithine transcarbamylase(OTC, EC 2.1.3.3), and carbamate kinase (CK, EC 2.7.2.2).The first reaction, catalyzed by ADI, is the deamination ofarginine to yield citrulline and NH4+. OTC then catalyzesthe conversion of citrulline and inorganic phosphate intocarbamoyl-phosphate and ornithine. Finally, CK catalyzesthe hydrolysis of carbamoyl phosphate to form CO2 andNH4+, while the phosphate group is used to regenerateATP from ADP.The ADI pathway is widely distributed among bacteria,where it is often a major means of energy production [1].However, the ammonium produced by this pathway hasalso been implicated in protecting some bacteria from theharmful effects of acidic environments [2, 3]. The pathwayhas been also described in Archaea [4]. This pathway hasonly been characterized in a few species of anaerobic eu-karyotes namely the parabasalids Trichomonas vaginalis[5] and Tritrichomonas foetus [6], and the diplomonads* Correspondence: vlada@natur.cuni.cz1Department of Parasitology, Charles University, Faculty of Science, Prague,Czech RepublicFull list of author information is available at the end of the article© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe 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.Novák et al. BMC Evolutionary Biology  (2016) 16:197 DOI 10.1186/s12862-016-0771-4Giardia intestinalis [7], Hexamita inflata [8], and Spironu-cleus salmonicida [9]. All these species belong to Metamo-nada (Excavata), a clade of anaerobic protists withsubstantially modified mitochondria designated as hydro-genosomes or mitosomes. Metamonada consists of threelineages – Fornicata (e.g., Giardia and Spironucleus), Para-basalia (e.g., Trichomonas and Tritrichomonas), and finallyPreaxostyla (Trimastix, Paratrimastix [10], and oxymo-nads) [11]. Currently, there is no information about theADI pathway in Preaxostyla.In Trichomonas vaginalis the ADI pathway generates upto 10 % of the energy produced by glucose fermentation[12]. OTC and CK were shown to be cytosolic, while ADIwas described as membrane-associated in both Trichomo-nas vaginalis and Tritrichomonas foetus [6]. The ADI ofTrichomonas vaginalis was later shown to be localized inhydrogenosomes and an in situ pH buffering function hasbeen proposed [13]. The ADI pathway of Giardia intestina-lis is completely cytosolic and produces up to 8 times moreATP than sugar metabolism [7]. Besides this energy-producing function, it has been proposed that the enzymesplay an important role in the pathogenesis of Giardia intes-tinalis and Trichomonas vaginalis. The protists secrete ADIand OTC from their cells causing arginine depletion thusreducing the ability of the infected tissue to produce anti-microbial nitric oxide [14, 15]. Other known effects of theseparasite's ADI pathway enzymes include growth arrest ofintestinal epithelial cells [16], inhibition of T cell prolifera-tion [15], and alteration of the phenotype and cytokine pro-duction of dendritic cells [17]. Another diplomonad with acharacterized ADI pathway, the free-living Hexamitainflata, inhabits environments with varying levels of dis-solved oxygen. It has been suggested that the ADI pathwaymay contribute to the metabolic flexibility of this organism,producing a significant amount of ATP under oxygen-limited conditions, while glycolysis is the main energysource under oxic or microoxic conditions, however theoxygen relationship might be incidental or secondary [8].Of the three enzymes, only ADI itself is considered tobe specific to the ADI pathway. CK has an additional rolein purine and nitrogen metabolism and OTC may catalyzesynthesis of citrulline as a nitrogen reservoir in plants [18]or be a part of ornithine-urea cycle in animals, diatomsand dinoflagellates [19, 20]. Therefore, the presence ofADI in organisms where no ADI pathway is known isintriguing and deserves further investigation. For example,within the chlorophytes the ADI gene was found in threespecies of Chlorella [21, 22] and Chlamydomonas rein-hardtii [23] And ADI activity has been reported in mul-tiple species of Chlorodendrophyceae, Trebouxiophyceae,Chlorophyceae, and Ulvophyceae [24], that is, in all classesof the “crown group” of Chlorophyta [25].The first known sequence of a eukaryotic ADI, fromGiardia intestinalis, showed no specific relationship to anybacterial or archaeal clade [26]. Later analyses included se-quences from Trichomonas vaginalis, Spironucleus vortens,Sp. barkhanus, and Sp. salmonicida (Metamonada), Euglenagracilis and 'Seculamonas' sp. (Discoba), Chlamydomonasreinhardtii and Chlorella sp. (core Chlorophyta [27]), andMastigamoeba balamuthi and Dictyostelium discoideum(Amoebozoa). All the eukaryotic sequences formed a well-supported clade related to Archaea, consistent with a singleorigin of ADI in the eukaryotic domain [9, 13].Due to its involvement in other pathways, it is not sur-prising that OTC is more widespread among eukaryotescompared to the other ADI pathway enzymes. The phylo-genetic analysis of OTC by Zúñiga et al. [26] recoveredtwo distinct eukaryotic clades branching in different posi-tions among bacteria, one comprising sequences fromEmbryophyta and other composed of metazoan and fungalsequences. The only eukaryote outside these two cladeswas Giardia intestinalis, which was also the only one witha characterized ADI pathway. The sequence from Giardiaintestinalis branched among bacterial sequences withoutclose relationship to any other eukaryotic clade. Later ana-lyses demonstrated that Spironucleus salmonicida andTrichomonas vaginalis OTC sequences formed a well-supported clade with Giardia intestinalis [9], suggestingthe existence of a third independent group of eukaryoticOTCs present in Metamonada and potentially involved inthe ADI pathway. The same analysis also showed twostramenopile sequences branching clearly inside theMetazoa-Fungi group.Sequences of CK from Giardia intestinalis, Hexamitasp., and Trichomonas vaginalis formed a relatively well-supported clade not closely related to any bacterial or ar-chaeal sequences [26]. The monophyly of eukaryotic CKswas later questioned after adding sequences from Spironu-cleus salmonicida and Carpediemonas membranifera, withthe Trichomonas vaginalis sequence branching separatelyfrom other eukaryotes, although statistical support for thistopology was very low [9].In summary, the complete set of ADI pathway enzymeshas been found in representatives of two out of threemajor lineages of Metamonada: Parabasalia and Fornicata.All the metamonad enzymes appear to be closely relatedto each other. This raises several questions about the evo-lutionary history of the pathway among eukaryotes. Is itpresent also in the third and least investigated lineage ofmetamonads, Preaxostyla? Was it present in the commonancestor of the Metamonada? Do representatives of othereukaryotic lineages possess the ADI pathway as well? If so,do all the eukaryotic enzymes involved in the ADI pathwayoriginate from the same source or do they represent inde-pendent acquisitions?Here, we take advantage of the recent progress ingenome and transcriptome sequencing of less studiedprotists to perform an up-to-date survey andNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 2 of 14phylogenetic analysis of ADIs, OTCs, and CKs. Thissurvey focuses on elucidating the evolutionary historyof the arginine deiminase pathway in eukaryotes, withspecial emphasis on Metamonada. In addition tophylogenetic studies, we determine the subcellularlocalization of these enzymes in two members of Pre-axostyla, Paratrimastix pyriformis and oxymonadMonocercomonoides sp. PA203.ResultsDistribution of ADI, OTC, and CK across eukaryotesOur survey revealed the presence of ADI, OTC, and CKin the three main eukaryotic clades defined by Adl et al.,2012 [28] (Fig. 1). The first and presumably the mostspecific enzyme of the pathway, i.e. without any role out-side the ADI pathway reported so far, is ADI itself. Thiswas found in 40 taxa, as shown on the schematic tree inAmoebozoaADI OTC CKArchaeplastidaSarBacillariophyceae (19)Breviateaat avacxEMetamonadaDiscobaOpisthokontaStramenopilesRhizariaAlveolataHolozoaHolofungiChlorophytaParabasaliaFornicataPreaxostylasekcite ro hpaiDaehpromATrichomonas vaginalisLacusteria cypriacaunidentified free-living trichomonadGiardia intestinalisHexamita inflataSpironucleus salmonicidaCarpediemonas membraniferaErgobibamus cyprinoidesChilomastix cuspidataParatrimastix pyriformisTrimastix marinaMonocercomonoides sp.Pyrsonympha sp.Naegleria gruberiHarpagon schusteriEuglena gracilisEutreptiella gymnastica-likeHerpetomonas muscarum'Seculamonas' sp.Metazoa (11)Salpingoeca rosettaSphaeroforma arcticaCapsaspora owczarzakiFungi (16)Fonticula albaDictyosteliidae (3)Acanthamoeba castellaniiMastigamoeba balamuthiPygsuia biformaGloeochaete wittrockianaRhodophytaChlorella variabilisHelicosporidium sp.Coccomyxa subellipsoideaChlamydomonas reinhardtiiVolvox carteriDunaliella tertiolectaTetraselmis sp.Ostreococcus lucimarinusPyramimonas parkeaePicocystis salinariumCryptophyta (3)Haptophyta (7)Labyrinthulomycetes (4)Oomycetes (4)Gregarina niphandrodesChlorarachniophyta (2)Dinoflagellata (13)Reticulomyxa filosaEmbryophyta (8) (4)other Ochrophyta (6)Pseudopedinella elasticaAureococcus anophagefferensFig. 1 Distribution of enzymes of the arginine deiminase pathway across eukaryotic diversity. Arginine deiminase (ADI), ornithine transcarbamylase (OTC),carbamate kinase (CK). Taxon names in boldface indicate lineages containing at least one representative with a sequenced nuclear genome. Numbers inbrackets indicate number of sequences from the given taxon included in our analyses. Colored rectangles indicate major eukaryotic groups as follows: darkbrown – Metamonada; light brown – Discoba; violet – Opisthokonta; blue – Amoebozoa; cyan – Breviatea, green – Archaeplastida; orange – Cryptophyta;yellow – Haptophyta; red – SAR. Excavata, Amorphea, and Diaphoretickes are names of the 3 putative largest clades of eukaryotes as proposed in Adl et al.,2012; relationships between them are not resolvedNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 3 of 14Fig. 1, and these taxa represent most eukaryotic super-groups (highlighted by colored backgrounds). Of these,16 species (most metamonads, Harpagon, Mastiga-moeba, Pygsuia, Chlorella, and Coccomyxa) encoded allthree enzymes, while the other species encoded only oneor two enzymes. ADI was not detected in any represen-tative of the clades Metazoa, Fungi, Embryophyta, Cryp-tophyta, and Haptophyta, nor in Sar [28], with the singlequestionable exception of Gregarina niphandrodes (seebelow). OTC was the most widespread enzyme, beingfound in 131 taxa including the major multicellulargroups of Metazoa, Fungi, and Embryophyta. CK wasdetected in all the investigated metamonads, multipleBacillariophyceae, Dinoflagellata and 8 other species.Please note that the given numbers do not represent theactual quantity of eukaryotic species with the particulargene since several groups, e.g. Metazoa, Bacillariophy-ceae, are represented by only a limited number of ran-domly selected sequences.Phylogenetic analysesArginine deiminaseCompared to the previous analyses we present a more ro-bust analysis including 40 eukaryotic species (Fig. 1). Thephylogenetic tree (Fig. 2) shows two clearly separated(RAxML bootstrap support/IQ-TREE bootstrap support:100 %/100 %) clans of ADIs, one comprising all bacteriaand one isolated eukaryote, Gregarina niphandrodes, in ahighly nested but poorly resolved position, and the secondcomposed of clans of Archaea plus a few Bacteria (64 %/97 %) and Eukaryota (51 %/81 %). The topology within theeukaryotic branch is poorly resolved overall, however, a fewclades of lower-than-supergroup rank were recovered withstrong support (i.e. bootstrap support > 80 %). These areParabasalia, Diplomonadida, Oxymonadida, Chlorophyta,and Dictyosteliida.Ornithine transcarbamylaseOur analysis included sequences from selected representa-tives of Metazoa, Fungi, and Embryophyta and 110 se-quences from 103 other eukaryotes (Fig. 1). Severalbacterial sequences of aspartate transcarbamylase, a proteinclosely related to OTC, were included to provide an out-group for rooting the tree. Our analysis of OTC phylogeny(Additional file 1) supports the existence of three largegroups and two separately-branching eukaryotic OTCs.The first large clade is strongly supported (100 %/100 %)and contains Metazoa, Fungi, Oomycota, Bacillariophyceae(i.e. diatoms), a few lineages of other Stramenopiles(Nannochloropsis, Vaucheria, Ectocarpus, Heterosigma andOchromonas), the holozoan Capsaspora, and one singleexcavate, the trypanosomatid Herpetomonas muscarum,which branches among Fungi.The second eukaryotic group, already indicated in theanalysis by Andersson et al. [9], is well supported (83 %/100 %) and includes all Metamonada, Harpagon shusteri,Naegleria gruberi, Reticulomyxa filosa, Pygsuia biforma,Mastigamoeba balamuthi, and many representatives ofautotrophic groups, namely dinoflagellates, cryptophytes,euglenophytes, chlorarachniophytes, and stramenopiles likeAureococcus, Aureoumbra, Pelagococcus, Pelagomonas, andPseudopedinella. The monophyly of Parabasalia is well sup-ported. A sequence from a recently described archaeonLokiarchaeum sp. is also included in this group, however atan unsupported position.The third group is composed of euglenophytes, greenalgae with green plants, red algae, and haptophytes, withhaptophytes branching inside red algae. A clade ofmostly Desulfobacteraceae bacterial sequences branchesinside this group of eukaryotic sequences.The only two eukaryotes outside these three large cladesare the choanoflagellate Salpingoeca rosetta (sequence ob-tained from the genome), which branches as sister toMicrogenomates bacterium (78 %/100 %), and the rhizarianPaulinella chromatophora (red star in Additional file 1) in-side Cyanobacteria with good statistical support (100 %/100 %). Since the Paulinella sequence originates from thegenome of the chromatophore, not the Paulinella nucleus,it actually represents a cyanobacterial OTC.Carbamate kinaseWe have included 47 sequences from 44 eukaryotic speciesin our analysis (Fig. 1). Our tree (Additional file 2) showseukaryotes falling into several separate clusters. One of twosubstantial groups is an unsupported clan of Fornicata,Parabasalia, Harpagon schusteri, Pygsuia biforma, and Mas-tigamoeba balamuthi. A well-supported Preaxostyla clade(96 %/100 %) branches at a different place among bacteria,as a sister group to Hadesarchaea archaeon and Anaeroli-neae bacterium (96 %/100 %). The second large eukaryoticclan (100 %/100 %) is composed of all the dinoflagellate se-quences, as well as sequences from diatoms, Pedinella andAureococcus. Dinoflagellata form a well-supported groupwithin this clan. Three sequences from diatoms do notbranch together with other ochrophytes (the photosyntheticStramenopiles), and instead form a separate well-supportedclan (100 %/100 %) among bacteria. This may represent asecond form of the enzyme, since Thalassiosira pseudo-nana appears in both diatom groups. The only two knownCKs from green plants (Chlorella and Coccomyxa) branchtogether (97 %/99 %) but separated from other eukaryotes.The Reticulomyxa CK sequence is also isolated from therest of eukaryotes.ConcatenationWe also performed a phylogenetic analysis of a concat-enation of all three enzymes. In the first step, we haveNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 4 of 14prepared an alignment supermatrix in which we have in-cluded all eukaryotes and representatives of prokaryotesthat contain a complete set of the three enzymes, and mayuse the ADI pathway. In order to detect potential incon-gruities between gene partitions caused by lateral genetransfer we have performed a phylogenetic analyses of theindividual gene partitions from this supermatrix. Based onthese gene trees (Additional file 3) we removed taxon-gene sequences that branched with bootstrap supporthigher than 50 % within a clan of sequences outside itsown domain (e. g. eukaryotic sequence outside Eukaryota)from the concatenated alignment – namely CKs from0.5Eutreptiella gymnastica-like 1Desulfovibrio cuneatusTrichomonas vaginalis 1Labrenzia albaClostridium perfringensPolysphondylium pallidumAlphaproteobacteria (6)Harpagon schusteriTreponema denticolaThermoplasmata (4)Lacusteria cypriacaSpironucleus salmonicidaGaldieria sulphurariaGammaproteobacteria (2)Sulfolobales (3)Bacilli (2)Pyrsonympha sp.Enterococcus faecalisMastigamoeba balamuthiFusobacteria (2)Unidentified free-living trichomonadChilomastix cuspidataBurkholderia ambifariaMonocercomonoides sp.Fervidicoccus fontisTrichomonas vaginalis 2Helicosporidium sp.Tolumonas auensisTM6 bacterium (2)Fonticula albaSalinisphaera shabanensisChlamydia trachomatisEnterobacteriales (5)Ferroplasma acidarmanusMethanofollis liminatansEuglena gracilis 2Aerococcus sanguinicolaMicrovirga lotononidisCyanobacteria (4)Sulfolobus (3)Rhizobium phaseoliAzospirillum brasilenseEutreptiella gymnastica-like 2Thermotogae (3)Gloeochaete witrockianaPyramimonas parkeaeMethanoculleus (2)Cyanidioschyzon merolaePseudovibrio sp.Anaerophaga thermohalophilaGammaproteobacteria (13)Carpediemonas membraniferaLactobacillales (2)Neisseriales (5)Naegleria gruberiGiardia intestinalisSpironucleus vortensPropionibacterium acnesTrueperella pyogenesEuglena gracilis 1Bradyrhizobium diazoefficiensCoccomyxa subellipsoideaRhodella maculataMycoplasma (2)Lamprocystis purpureaAcanthamoeba castellaniiCandidatus Korarchaeum cryptofilum (2)Geothrix fermentansActinobacteria (3)'Seculamonas ecuadoriensis'Sphaeroforma arcticaChlamydomonas reinhardtiiTetraselmis sp.Volvox carteri f. nagariensisThiorhodovibrio sp.Ergobibamus cyprinoidesChondrus crispusSinorhizobium medicaePygsuia biformaCandidatus Hepatoplasma crinochetorumPsychrobacter sp.Picocystis salinarumAminobacter sp.Clostridium sp. L74Dictyostelium fasciculatumCaldivirga maquilingensisDictyostelium discoideumRhizobium etliAlistipes (3)Pseudomonas aeruginosaBordetella petriiLeptotrichia goodfellowiiSpiroplasma turonicumSpirochaetes (2)Borrelia afzeliiChlorella variabilisGregarina niphandrodesBurkholderia sp.Auxenochlorella protothecoidesDunaliella tertiolectaCandidatus Micrarchaeum acidiphilum (2)82/8289/9971/9664/9783/10073/10093/10053/-99/10050/9898/10085/10088/10099/10086/10096/10057/10082/10097/10082/9893/9964/8781/10058/9287/10092/10051/8158/9980/10080/10075/9951/8482/10054/6998/9990/10092/9769/9956/8655/9655/9370/9799/10055/9997/10071/10064/9789/10085/10093/100DiPaChDipOx-/100-/88-/100-/62-/98-/98-/87-/68-/99-/98-/60-/63-/70-/58-/58-/56-/58-/60-/68 -/92-/84-/100-/99-/65-/68-/96-/90-/93-/100-/82-/84-/78Fig. 2 Phylogenetic tree of ADI sequences. The tree based on a 257 positions long protein alignment of 152 sequences was constructed in RAxMLusing the LG4X + Γ model of substitution. Eukaryotic taxa are highlighted in different colors according to the major group they belong to. The colorcode is the same as in Fig. 1. The values at nodes represent RAxML bootstrap support/IQ-TREE bootstrap support. Only values above 50 % are shown.Black circles indicate support of 100 %/100 %. Vertical black bars indicate well-supported eukaryotic clades: Pa – Parabasalia; Di – Dictyosteliida; Ch –Chlorophyta; Ox – Oxymonadida; Dip – Diplomonadida. Species with multiple sequences included: Euglena gracilis 1 – GI 109790819; Euglena gracilis 2– GI 109784514; Eutreptiella gymnastica-like 1 – CAMPEP 0200414012; Eutreptiella gymnastica-like 2 – CAMPEP 0200422928; Trichomonas vaginalis 1 –TVAG 183850; Trichomonas vaginalis 2 – TVAG 344520. The tree is unrootedNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 5 of 14Monocercomonoides sp., Pyrsonympha sp., Chlorella var-iabilis, and Coccomyxa subelipsoidea. We also removedOTCs from Chlorella variabilis, and Coccomyxa subelipsoi-dea because in the large single gene tree (Additional file 1)they branch within a clade which is sister to ChlorobiBacteria with 100 % IQ-TREE bootstrap support.The analysis performed on the alignment after removalof these sequences (Additional file 4) revealed a strongbipartition (100 %/100 %) grouping Eukaryota and Ar-chaea to the exclusion of Bacteria and within this part ofthe tree the eukaryotes formed a well-supported (100 %/100 %) clan sister to the archaeon Candidatus Korarch-aeum cryptofilum. In order to recover the relationshipswithin the Eukaryota–Archaea group without the dis-turbing long branch of Bacteria we repeated the analysiswithout the bacterial sequences (Fig. 3). In this unrootedtree Eukaryota are grouped with Candidatus Korarch-aeum cryptofilum to the exclusion of the rest of Archaeawith high support (97 %/98 %). We also performed aEukaryota-only analysis of the concatenated dataset forthe purpose of hypotheses testing (Additional file 5).Hypotheses testingWe used approximately unbiased (AU) and expected like-lihood weight (ELW) tests to assess whether the inferredphylogenies are in a significant conflict with the mono-phyly of eukaryotes, metamonads and with the expectedeukaryotic species tree. The results are summarized inTable 1. The AU tests rejected monophyly of metamonadsin the OTC and CK trees and monophyly of expectedeukaryotic phylogeny in concatenation. The ELW testsrejected the monophyly of metamonads in the OTC tree,the monophyly of both clades in the CK and monophylyof metamonads and the expected eukaryotic phylogeny inthe concatenation tree.Localization of ADI pathway enzymes in PreaxostylaAnother aim of this work was to infer the subcellularlocalization of ADI pathway enzymes in members of thepoorly studied Preaxostyla clade. Genomic and transcrip-tomic projects have revealed the presence of all threeenzymes in Monocercomonoides sp. and Pyrsonympha sp.,while only OTC and CK enzymes were detected in Tri-mastix marina and Paratrimastix pyriformis. We havechosen Monocercomonoides sp. PA203 and Paratrimastixpyriformis for further study.We investigated the presence of mitochondrion-targetingsignals in the enzymes of interest (Additional file 6) usingthe signal prediction software TargetP 1.1 [29] and MitoprotII v1.101 [30]. TargetP did not predict any targeting signals.Mitoprot II predicted a single mitochondrion-targeting sig-nal, for the OTC sequence of Paratrimastix pyriformis.To validate the results of mitochondrion-targeting signalprediction we used the Trichomonas vaginalis T197/9872/8175/9099/10059/-99/10062/7298/10070/5878/950.2Spironucleus salmonicidaHarpagon schusteriGiardia intestinalisUnidentified free-living trichomonadCandidatus Micrarchaeum acidiphilumLacusteria cypriacaCandidatus Korarchaeum cryptofilumPicrophilus torridusPygsuia biformaChilomastix cuspidataCarpediemonas membraniferaCaldivirga maquilingensisFervidicoccus fontisFerroplasma acidarmanusMonocercomonoides sp.Ergobibamus cyprinoidesCoccomyxa subellipsoideaTrichomonas vaginalisMethanofollis liminatansThermoplasma acidophilumChlorella variabilisMastigamoeba balamuthiPyrsonympha sp.Fig. 3 Phylogenetic tree of concatenated ADI, OTC, and CK sequences with Bacteria removed. The tree based on a 750 positions long proteinalignment of 23 sequences was constructed in RAxML using LG4X + Γ model. Eukaryotic taxa are highlighted in different colors according to themajor group they belong to. The color code is the same as in Fig. 1. The values at nodes represent RAxML bootstrap support/IQ-TREE bootstrapsupport. Only values above 50 % are shown. Black circles indicate support 100 %/100 %. The tree is unrootedNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 6 of 14heterologous expression system, with the assumption thatan undetected mitochondrion-targeting signal may none-theless be recognized by the Trichomonas hydrogenosomalimport machinery. We transfected Trichomonas vaginaliscells with plasmids containing HA-tagged OTC and CKfrom Paratrimastix pyriformis and ADI, OTC, and CKfrom Monocercomonoides sp. In all cases fluorescence mi-croscopy showed that the heterologously expressed proteinsdo not co-localize with the signal from the hydrogenosomalmarker protein (malic enzyme), but instead formed adiffuse pattern all over the cell (Fig. 4). This demonstratesthat the inserted proteins are not recognized ashydrogenosomal-import targets in Trichomonas vaginalis.The results of these experiments are consistent with thefact that most ADI pathway enzymes in eukaryotes arelocalized in the cytosol.DiscussionADI pathway enzymes are widespread in eukaryotesBefore this study, only two closely related lineages of eu-karyotes had been conclusively shown to possess acomplete ADI pathway. These were Parabasalia andDiplomonadida, both being members of Metamonada, asubgroup of Excavata. Our survey has shown the presenceof all three enzymes in ten other eukaryotic species.Among these are other members of Metamonada – in-cluding free-living members of Fornicata related to thepredominantly parasitic diplomonads (Ergobibamus cypri-noides, Chilomastix cuspidata, Carpediemonas membrani-fera), and members of the third metamonad lineage,Preaxostyla (Monocercomonoides sp. and Pyrsonymphasp.). The ADI pathway was also identified in non-metamonads including the heterolobosean Harpagonschusteri, the amoebozoan Mastigamoeba balamuthi, thebreviate Pygsuia biforma, and the green algae Chlorellavariabilis and Coccomyxa subelipsoidea. Further func-tional studies are needed to determine whether these en-zymes function within an ADI pathway in these species. Itis possible that the possession of the complete pathway isconnected with their anaerobic lifestyle since most ofthese organisms are anaerobes, microaerophiles or aer-obes able to live for long periods under anaerobic condi-tions [31–37].Many investigated eukaryotes possessed incompletesets of ADI pathway enzymes. The presence of OTC orCK on their own is not surprising, as they are known tobe involved in other biochemical processes including theornithine-urea cycle or purine biosynthesis. The pres-ence of ADI on its own was unexpected, yet we identi-fied ADI in a broad spectrum of eukaryotic lineageswithout complete pathway. The apparent absence ofOTC or CK may be due to the incompleteness of tran-scriptome or genome data, nevertheless, our observationsuggests that ADI may also function outside the contextof the ADI pathway in some eukaryotes.Phylogenetic histories of enzymesNone of the enzyme phylogenies is completely consistentwith the expected species relationships. In single genetrees, eukaryotes are always dispersed in multiple clades,suggesting complicated evolutionary histories. The back-bone topologies were generally weakly supported, andmany of these incongruences are probably the result oflow phylogenetic signal. Nevertheless, some conflicts withspecies phylogeny are better supported and some wereconfirmed by phylogenetic tests. These can potentially beattributed to lateral gene transfers (LGTs, also known ashorizontal gene transfers – HGTs) or endosymbiotic genetransfers (EGTs). The sister relationship of Preaxostylaand Spirochaeta in the CK tree, and the position of Gre-garina within Bacteria in the ADI tree are two such exam-ples of potential LGT, albeit the latter may also representa contamination. Since haptophytes are known to harborsecondary plastids of potentially red algal origin [38], theposition of haptophytes within a red algal clade in theOTC analysis, might represent a potential EGT. Con-versely, many moderately and robustly supportedeukaryotic clades are taxonomically reasonable, indicatingthe important role of vertical inheritance.The taxon sampling in the concatenation analyses waslower, because the analyses included only those taxa thatmay utilize the arginine deiminase pathway and not thosethat use individual enzymes for other purposes. The reso-lution of the concatenation tree was higher than the indi-vidual gene trees and strongly supported the monophyly ofeukaryotes (99 %/100 %) and their close relationship to Ar-chaea. The clade of eukaryotes branched with Archaea(100 %/100 %) as a sister to Candidatus Korarcheum cryp-tophylum. Increased support of these nodes should partlybe ascribed to the lower number of taxa but it also suggeststhat the phylogenetic signal regarding these deep nodes forthis narrow set of taxa and after exclusion of obvious casesof LGT is largely congruent.Table 1 Results of approximately unbiased (AU) and expectedlikelihood weights (ELW) testsData set – hypothesis AU test ELW testADI – Metamonada monophyly. 0.64 0.10OTC – Eukaryota monophyly 0.64 0.5OTC – Metamonada monophyly 0 0CK – Eukaryota monophyly 0.12 0CK – Metamonada monophyly 0 0Euk. Conc. – Metamonada monophyly 0.17 0Euk. Conc. – expected euk. phylogeny 0 0The tests were performed for 4 sets of taxa – ADI: ADI dataset (as in Fig. 2),OTC: OTC dataset (as in Additional file 1), CK: CK dataset (as in Additional file2), Euk. Conc.: concatenation dataset without prokaryotic sequences (as inAdditional file 5)Novák et al. BMC Evolutionary Biology  (2016) 16:197 Page 7 of 14ADI pathway is ancestral in MetamonadaThe presence of the complete ADI pathway is wide-spread in metamonads, protists that specifically inhabitlow-oxygen environments. In most of the phylogenies,metamonad taxa branch close to each other, but they neverform an exclusive clade. In CK trees there is a well-supported relationship between Preaxostyla (Paratrimastix,Monocercomonoides, and Pyrsonympha) sequences and aprokaryotic clan of Hadesarchaea archaeon and Anaeroli-neae bacterium. Metamonada do not appear monophyleticeven in the concatenation trees and their monophyly wasrejected by ELW test. Taking together all this informationwe propose that the complete arginine deiminase pathwaywas present already in the common ancestor of Metamo-nada and was vertically inherited by the extant metamonadlineages, with a few exceptions. The exceptions are the pu-tative losses of ADI in Trimastix marina and Paratrimastixpyriformis and the putative replacement by a bacterial CKin the Preaxostyla. It is also possible that some enzymes orthe whole pathway were laterally transferred from metamo-nads to other eukaryotes, which would disrupt the mono-phyly of Metamonada on trees.Our localization experiments suggest that all en-zymes in both Paratrimastix pyriformis and Monocer-comonoides sp. are localized in the cytosol, like theenzymes in Giardia intestinalis but unlike the ADI inTrichomonas vaginalis. We therefore conclude thatcytosolic localization of the pathway is an ancestraltrait of all metamonads. It should be noted, however,that the cytosolic localization of ADI inPA203 ADIPA203 OTCPA203 CKPp OTCPp CKFig. 4 Localisation of Monocercomonoides and Paratrimastix enzymes in Trichomonas vaginalis cells. Immunofluorescence micrographs of Trichomonasvaginalis, in which the HA-tagged versions of enzymes were expressed. Green signal from anti-HA antibody does not localize to hydrogenosomes ofTrichomonas vaginalis, which are marked by red anti-malic enzyme antibody. Blue signal indicates DAPI-stained nuclei. Abbreviations: PA203– Monocercomonoides sp. PA203; Pp – Paratrimastix pyriformis; ADI – arginine deiminase; OTC – ornithine transcarbamylase; CK –carbamate kinaseNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 8 of 14Monocercomonoides sp. may not be informative, sincethis protist does not contain mitochondrion [39].Paratrimastix pyriformis harbors an organelle similarto the hydrogenosome, but no ADI-coding gene hasbeen found in its transcriptome to test itslocalization.ADI pathways in other eukaryotesBesides Metamonada, five other species contain a completeADI pathway and all branch within the eukaryotic clade onthe concatenation tree (Fig. 3). These represent four differ-ent supergroups of eukaryotes; only Harpagon is from thesame supergroup as Metamonada (supergroup Excavata),and even then it belongs to a different sub-branch (Dis-coba). Moreover, Excavata are likely not monophyletic at all[40] and the root of eukaryotes may be situated inside thegroup. Harpagon, Mastigamoeba, and Pygsuia ADI path-way enzymes branch close to Metamonada in all three genetrees, and so it is very probable that the pathway in thesethree species was derived from the same source as thepathway in Metamonada.The situation in Chlorella variabilis and Coccomyxa sub-elipsoidea is less clear. These organisms are the only greenalgae known to contain all three ADI pathway enzymes.Their ADIs and OTCs branch together with other greenalgae and plants in the individual gene trees (while no otherPlantae have CK), supporting the presence of the completeset in the last common ancestor of Chlorophyta. However,Chlorophyta did not branch in a common eukaryotic cladewith Metamonada, Harpagon, Mastigamoeba and Pygsuiain OTC and CK phylogenies, suggesting that these two en-zymes in Chlorophyta may have independent origins. In theconcatenation tree, Chlorella variabilis and Coccomyxasubelipsoidea represented only by ADI sequences branchtogether with other eukaryotes. Presence of the ADI path-way in the last common ancestor of Chlorophyta would beconsistent with the ADI enzymatic activity previously re-ported from members of Chlorodendrophyceae, Trebouxio-phyceae, Chlorophyceae, and Ulvophyceae [24]. However,the function of the other enzymes in a typical ADI pathwayis questionable, since the OTCs from Chlorella autotro-phica, Chlorella saccharophila (Trebouxiophyceae), andDunaliella tertiolecta (Chlorophyceae) were found to haveno measurable activity in the direction of the ADI pathway,i.e. conversion of citrulline to ornithine [24]. It is thereforepossible that the two Chlorophyta species with all three en-zymes nonetheless do not use the ADI pathway.Origin of the eukaryotic ADI pathwayThe simplest explanation of the fact that the complete setsof ADI pathway enzymes from several eukaryotic lineagesare related is that they are inherited from their commonancestors. The taxonomic composition of the eukaryoticclade in the concatenation tree is so broad that theircommon ancestor must have been either the lasteukaryotic common ancestor (LECA) or its close descend-ant. This assumption is reasonable even if we would notconsider Chlorella and Coccomyxa. An alternative explan-ation for the close relationships of ADI pathways wouldbe that the genes were acquired more recently by oneeukaryotic lineage (perhaps Metamonada, where it is mostcommon), and then spread from this lineage into othersvia eukaryote-to-eukaryote lateral gene transfers.Based on our data we are unable to decide which alter-native is more likely. Vertical inheritance of the ADI path-way from LECA would be consistent with the sisterhoodof the eukaryotic clade and the archaeon CandidatusKorarchaeum cryptofilum in the concatenation tree (Fig. 3,Additional file 4), since recent studies indicate Korarch-aeota are indeed closely related to the eukaryotes [41].Moreover, OTC sequences from Lokiarchaeum sp., whichis the closest known relative to eukaryotes [42], are relatedto the Metamonada-containing eukaryotic clade. CK se-quence of this archaeon branches outside eukaryotes sisterto a proteobacterium Desulphobacula toluolica (97 %/100 %), but nodes separating these two from Metamonadadid not receive strong support. ADI sequence fromLokiarcheum is not available, and so this organism wasnot included in the concatenation analysis. We must alsotake into account that the position of the root of theeukaryotic tree is still an unresolved question [40, 43, 44].If the root falls outside Amorphea + Discoba (position ofMetamonada relative to the root is not known), then ourresults do not necessarily uncover the condition of LECA.The later acquisition of the pathway by Metamonada andits spread to unrelated eukaryotes by eukaryote-to-eukaryote transfer is supported by the fact that theconcatenation trees are incongruent with the expectedrelationships of taxa.ConclusionsOur broad survey of the arginine deiminase pathway en-zymes has shown that they are present in representatives ofall major lineages of eukaryotes. Sixteen protists (mostmetamonads, Harpagon, Mastigamoeba, Pygsuia, Chlorella,and Coccomyxa) contain the complete set of the three en-zymes, while other organisms contain incomplete sets. Theenzyme ADI is present in several species without thecomplete arginine deiminase pathway, suggesting its in-volvement in other cellular processes. The topology of indi-vidual gene trees is generally not very well supported andparticularly in OTC and CK trees the eukaryotic enzymesform multiple clearly unrelated clades consisting of mix-tures of eukaryotic supergroups. This indicates that mul-tiple prokaryote-to-eukaryote and eukaryote-to-eukaryoteLGT events took place in the history of these enzymes. It ispossible that in some groups the enzyme acquisition wasNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 9 of 14connected with its involvement in novel biochemical pro-cesses like the ornithine-urea cycle.Based on the presence of the complete pathway inmost metamonads and based on the phylogenetic affinityof metamonad enzymes, we conclude that the ancestorof metamonads already possessed this pathway. Theconcatenation analyses suggest that eukaryotes with thecomplete ADI pathway, including Metamonads, Harpa-gon, Mastigamoeba and Pygsuia (and possibly Chlorellaand Coccomyxa), may have acquired the genes from asingle, archaeon-related source. One intriguing possibil-ity is that the acquisition of the pathway may date backas deep as to LECA, but other scenarios involving LGTevents are also plausible. To resolve the last issue, it willbe necessary to obtain data from a more diverse set ofprokaryotes and eukaryotes, especially those branchingclose to the root of the eukaryotic tree and close to theroot of major eukaryotic lineages.MethodsObtaining sequencesThe majority of eukaryotic sequences included in the sur-vey were obtained from the NCBI database (Release 68),JGI database [45], or Marine Microbial Eukaryote Tran-scriptome Sequencing Project [46]. Initial searches wereperformed using BLASTp and tBLASTn algorithms [47]with Giardia intestinalis, Trichomonas vaginalis, andseveral bacterial sequences as queries. The searches ofpublic databases were then repeated several times whilerestricted to a particular major eukaryotic lineage (e.g.Cryptophyta, Alveolata) and with a phylogenetically clos-est available sequence as a query. All the eukaryoticsequences with E-value lower than 10−3 were downloadedand used in subsequent analyses.The prokaryotic sequences included in the survey wereretrieved from the NCBI database using the same queryas in the search for eukaryotic sequences. The searchwas also repeated several times with varying taxonomicrestrictions to ensure that all the bacterial and archaealphyla containing the particular enzyme are representedin the analysis. We used all archaeal sequences with E-value lower than 10−3 and a limited number of bacterialsequences with E-value lower than 10−3 and annotatedas the protein of interest. It is important to note that theset of bacterial sequences used in our analyses is not ex-haustive and therefore we do not infer any evolutionaryhypotheses about Bacteria in this study.In order to mitigate the risk of missing prokaryotic datainfluencing the relationships between eukaryotic groupswe enriched our datasets with the closest prokaryotic ho-mologs to each of the eukaryotic sequences by searchingthe NCBI database using BLASTp with each eukaryoticsequence as a query and downloaded the prokaryotic se-quence with the lowest e-value from each search.We investigated whether those eukaryotic sequenceswhich were not branching within eukaryotic clades repre-sent bona fide eukaryotic sequences or contamination ofthe data sets. Nucleotide sequences obtained from tran-scriptomic data were checked for similarity with sequencesdeposited in NCBI and those that were identical or verysimilar to bacterial genomes (bit score higher than 200 andsimilarity along the entire length of the sequence) were ex-cluded. In the case of sequences obtained from genomes,the entire gene content of the contiguous sequence scaffoldwas used as a query for BLAST search of the NCBI data-base in order to identify any known sequences with high se-quence similarity along the entire length of the sequence,indicating possible contamination. Furthermore, candidatesequences and neighboring genes were investigated for thepresence of introns and the origin and annotation of sur-rounding genes. These steps should identify some se-quences originating from contamination, however, otherscould still remain due to the lack of data from the source ofthe contamination or incorrect assembly of genomic dataresulting in chimerical sequences.The sequences downloaded from public databases werecombined with sequences extracted from genomic andtranscriptomic projects performed in the laboratories ofco-authors. Brief information on the generation of thesedata sets is given below. Details of theMonocercomonoidessp. PA203 genome and transcriptome project are given inKarnkowska et al. [39]. Details of the Paratrimastix pyri-formis transcriptome project are given in Zubáčová et al.[48]. Partial cDNA sequences corresponding to Paratri-mastix pyriformis OTC and CK obtained in the transcrip-tome project were completed at their 5` ends by RACEusing FirstChoice RLM-RACE kit (Life Technologies,AM1700). Amplifications by PCR were carried out usingTakara Hot-Start ExTaq DNA Polymerase (Takara,RR006A) in 50 μl reactions. Outer 5` RLM-RACE PCRwas done using the 5` RACE outer primer supplied in thekit and the following 5` RACE gene-specific outerprimers: TpOTCout: CCAGCAGGAAGAGAAGGAGGand TpCKout: GCTTGCCGTAGTTGATGATG. Inner 5`RLM-RACE PCR was done using the 5` RACE inner pri-mer supplied in the kit and the following 5` RACE gene-specific inner primers: TpOTCinn: AAGAGCTCGT-GATCTGGAAG and TpCKinn: GCCAGAGGCGATGA-CAATGA (here and elsewhere, all primers reported in the5’ to 3’ direction). The following touchdown program wasused for each of the two PCRs: 95 °C (5 min), 15 cycles of95 °C (1 min), 60 °C to 45 °C (35 s.) and 72 °C (2 min),20 cycles of 95 °C (1 min), 45 °C (35 s.) and 72 °C (2 min),then a final polymerization step at 72 °C for 6 min. PCRproducts were cloned into pGEM-T Easy plasmid vector(Promega, A1360) and sequenced.Sequences from Lacusteria cypriaca (strain LAI), an un-identified free-living trichomonad (strain LAGOS2D) andNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 10 of 14Pyrsonympha sp. were mined from RNA-seq data setsgenerated using the llumina MiSeq sequencing platform.Sequences from Trimastix marina, Carpediemonas mem-branifera, Chilomastix cuspidata, Mastigamoeba bala-muthi, and Pygsuia biforma were mined from RNA-seqdata sets generated using the Illumina HiSeq sequencingplatform. Sequences from Harpagon schusteri were minedfrom RNA-seq data obtained using the 454 sequencingplatform and sequences of Ergobibamus cyprinoides weremined from data sets generated by combination of Sangerand 454 sequencing platforms. The assembled sequenceswere submitted to GenBank under accession numbersKT883858-KT883885.Phylogenetic analysesInferred amino acid sequences were aligned using MAFFTversion 7 [49] and the resulting alignments were manuallytrimmed. Highly variable misaligned sections of severaleukaryotic sequences, possibly results of sequencing er-rors, were removed from the alignment manually. Theconcatenated alignment was constructed from the singlegene alignments using SequenceMatrix [50]. The finalalignments can be downloaded from our web page: http://protistologie.cz/hampllab/NovakData.zip [51]. Phylogen-etic inference was performed using substitution modelssuggested by ProtTest 2.4 server [52] – LG4X + Γ model.Maximum Likelihood trees were inferred using RAxML-HPC2 version 8 available on CIPRES [53], with 10 startingtrees and also using IQ-TREE v1.4.2 [54] under the LG +C20 + F +G4 model for the single gene trees and LG +C40 + F +G4 model for concatenated datasets. The modelthat best fits the data was determined by IQ-TREE accord-ing to the Bayesian information criterion (BIC). The LGmatrix was combined to an amino acid class frequencymixture model with 20 (for single gene trees) and 40 (forconcatenated datasets) frequency component profiles.Statistical support for branches was assessed by multipara-metric bootstrapping (1000 replicates) in RAxML and bythe ultrafast bootstrap approximation (UFboot) with 1000replicates in IQ-TREE.Topology testsPhylogenetic hypotheses were tested by an approximatelyunbiased (AU) test [55] and expected likelihood weights(ELW) method [56] implemented in IQ-TREE 1.4.2 [54].For all datasets we tested whether their ML phylogeny is insignificant conflict with the monophyly of Metamonada. Inthe case of OTC and CK, we also tested whether the phyl-ogeny is in significant conflict with the monophyly of eu-karyotes. In the case of the concatenated dataset, we testedwhether their ML phylogeny is in significant conflict withthe monophyly of Metamonada as well as whether the rela-tionships within the eukaryotic clade (Additional file 5) sig-nificantly conflicts with a user-defined expected species tree(Additional file 7). For the latter two tests we used aconcatenated dataset without prokaryotic sequences toeliminate the influence of relationships among prokaryotesand position of the eukaryotic root. The ADI sequence at-tributed to Gregarina niphandrodes was excluded from alltests including ADI data, as this clearly represents contam-ination or very recent LGT.To perform AU and ELW tests, a set of 1003 topologieswas created, containing the unconstrained ML topology in-ferred by RAxML, 1000 topologies inferred by RAxML dur-ing bootstrapping, and the best trees inferred by RAxMLunder the selected constraints (eukaryotic monophyly,Metamonada monophyly or eukaryotic phylogeny). Sitelikelihoods for topologies were calculated by IQ-TREEusing the LG+C20 + F +G4 model. The sets of site likeli-hoods were then compared using the AU test in IQ-TREE,with 10 000 replicates.Cloning of ADI pathway genesADI pathway genes were amplified from Paratrimastixand Monocercomonoides cDNAs by PCR. Paratrimastixpyriformis and Monocercomonoides sp. PA203 cDNAswere prepared from 100 mL of bacterized culture and1000 mL of culture filtered according to Hampl et al. [57],respectively. Isolations of total RNA were performed usingTRI Reagent RNA Isolation Reagent (Sigma, T9424). Ex-tractions of eukaryotic mRNA from total RNA were doneusing a Dynabeads mRNA Purification Kit (Life Technolo-gies, 61006). The SMARTer PCR cDNA Synthesis Kit(Clontech, 634925) was used for cDNA synthesis follow-ing by cDNA amplification with an Advantage 2 PCR Kit(Clontech, 639207) using 21 cycles (Paratrimastix) and19 cycles (Monocercomonoides) of amplification.The following primers were used for amplifications offull-length cDNAs of the ADI pathway genes of Paratri-mastix and Monocercomonoides (restriction sites NdeI,VspI, and BamHI are in bold): Tp OTC-F (TAACA-TATGCCTCGCCACCTTACCAAGAT), Tp OTC-R (TAAGGATCCGTCAAGGAGGGGCTGGCCCA), Tp CK-F (TAACATATGCGTATCCTCATCGCTCTCG), Tp CK-R (TAAGGATCCGGCGACAATGTGGGTACCAG), PA203 ADI-F (TAACATATGATGCAAGATATTCACGTTCC), PA203 ADI-R (TAAGGATCCCTGATTTCCCAGA-GATGCTA), PA203 OTC-F (ATCATTAATATGTCCGCTCCCGTTAGACA), PA203 OTC-R (TAAGGATCCCT-CAATGGTCATTTTCTTGT), PA203 CK-F (CACTTCA-CATTACATATGGTGAGAATTTTAATTGCTC), PA203CK-R (CGTATGGGTAGGATCCTGGAACAATGTGAGTTCCTT). For transfection of Trichomonas vaginalis, thegenes were cloned into TagVag2 plasmid vector [58] usingrestriction digestion and ligation, or directly using the In-fusion HD Cloning Kit (Clontech, 639648) in the case ofMonocercomonoides CK. Lab-grown chemicallyNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 11 of 14competent Escherichia coli XL1 cells were used fortransformations with ligation mixtures, whereas Stellarcompetent cells (Clontech, 636763) were used fortransformation with the in-fusion reactions. Bacterialclones were checked by colony PCR for the presence ofthe plasmids followed by sequencing of isolatedplasmids.Selectable transfection of Trichomonas vaginalisDespite extensive efforts, we did not achieve either stableor transient transfection of Paratrimastix pyriformis andMonocercomonoides sp. PA203 with plasmid vectors spe-cifically prepared for those two organisms (data notshown). Therefore, the Trichomonas vaginalis heterol-ogous expression system was used to infer the subcellularlocalizations of the Paratrimastix and Monocercomonoidesenzymes. Versions of ADI pathway genes with a C-terminal 2xHA-tag were electroporated into Trichomonascells according to the protocol described by Sutak et al.[59]. Briefly, 250 mL of Trichomonas vaginalis T1 culture(strain kindly provided by Michaela Marcinčiková, Dept.of Parasitology, Charles University) was used for two elec-troporations performed for each of the genes. Cells wereelectroporated with 30 μg of TagVag2 plasmid isolatedusing the Wizard Plus Midipreps DNA Purification Sys-tem (Promega, A7640). The exponential protocol (350 V,975 μF, ∞ Ω, 4 mm cuvette) of the GenePulser Xcell Elec-troporation System (Biorad, 165–2660) was used for eachtransfection. Trichomonads were selected with 200 μg/mlof G418 (ZellBio, G-418-5) for at least five passages. Ex-pression of the proteins was analyzed by Western blottingof cell homogenates (data not shown) and immunofluor-escence microscopy with antibody.Immunofluorescence microscopyADI pathway proteins of Paratrimastix and Monocercomo-noides were identified in Trichomonas cells using an anti-HA rat monoclonal antibody (Roche, 11867423001). Anantibody raised against malic enzyme, a hydrogenosomalmarker in Trichomonas vaginalis [60], was used for double-labeling (antibody kindly provided by prof. Jan Tachezy,Dept. of Parasitology, Charles University). Alexa Fluor-488goat anti-rat (green) and Alexa Fluor-594 goat anti-rabbit(red) (Life Technologies, A-11006 and A-11037) were usedas secondary antibodies. Immunostaining was done accord-ing to Sagolla et al. [61] on superfrost microscopic slidescoated with poly-L-lysine (Sigma, P8920). Preparationswere counterstained with DAPI in Vectashield mountingmedium (Vector Laboratories, H – 1200) and observedusing a IX81 fluorescent microscope (Olympus) equippedwith an IX2-UCB camera. Images were processed usingCellR software (Olympus) and ImageJ 1.42q.Additional filesAdditional file 1: Phylogenetic tree of OTC sequences. The tree based on a242 positions long protein alignment of 444 sequences was constructed inRAxML using the LG4X + Γ model of substitution. Eukaryotic taxa arehighlighted in different colors according to the major group they belong to.The color code is the same as in Fig. 1. The values at nodes represent RAxMLbootstrap support/IQ-TREE bootstrap support. Only values above 50 % areshown. Black circles indicate support of 100 %/100 %. Species with multiplesequences included: Alexandrium tamarense 1 – CAMPEP 0186340278;Alexandrium tamarense 2 – CAMPEP 0186191854; Alexandrium tamarense 3 –CAMPEP 0186247540; Durinskia baltica 1 – CAMPEP 0200033980; Durinskiabaltica 2 – CAMPEP 0200081736; Euglena gracilis 1 – c20598 g1 i1; Euglenagracilis 2 – c34673 g1 i6; Eutreptiella gymnastica-like 1 – CAMPEP 0200420840;Eutreptiella gymnastica-like 2 – CAMPEP 0200409666; Karenia brevis 1 –CAMPEP 0188881430; Karenia brevis 2 – CAMPEP 0188950444; Karlodiniummicrum 1 – CAMPEP 0200795676; Karlodinium micrum 2 – CAMPEP0200767534. The tree is rooted with sequences of bacterial aspartatecarbamoyltransferase (ATC; EC 2.1.3.2). (PDF 506 kb)Additional file 2: Phylogenetic tree of CK sequences. The tree based on a251 positions long protein alignment of 256 sequences was constructed inRAxML using the LG4X+ Γ model of substitution. Eukaryotic taxa arehighlighted in different colors according to the major group they belong to.The color code is the same as in Fig. 1. The values at nodes represent RAxMLbootstrap support/IQ-TREE bootstrap support. Only values above 50 % areshown. Black circles indicate support of 100 %/100 %. Vertical black barsindicate well-supported eukaryotic clades: Ch – Chlorophyta; Din –Dinoflagellata; Dip – Diplomonadida; Pa – Parabasalia; Pr – Preaxostyla.Species with multiple sequences included: Giardia intestinalis 1 – GSB 16453;Giardia intestinalis 2 – GL50803 16453; Thalassiosira pseudonana 1 – GI223995860; Thalassiosira pseudonana 2 – GI 224000745; Trichomonas vaginalis1 – TVAG 420500; Trichomonas vaginalis 2 – TVAG 261970; Trichomonasvaginalis 3 – TVAG 420510. The tree is unrooted. (PDF 482 kb)Additional file 3: Phylogenetic trees of gene partitions used forconcatenation. The values at nodes represent maximum likelihood bootstrappercentages. Eukaryota highlighted in grey. Red taxon names indicateremoved sequences. Positions of the particular genes in the alignment: ADI 0–257, OTC 258–499, CK 500–750. The trees are unrooted. (PDF 270 kb)Additional file 4: Phylogenetic tree of concatenated ADI, OTC, and CKsequences. The tree based on a 750 positions long protein alignment of 67sequences was constructed in RAxML using LG4X + Γ model. Eukaryotic taxaare highlighted in different colors according to the major group theybelong to. The color code is the same as in Fig. 1. The values at nodesrepresent RAxML bootstrap support/IQ-TREE bootstrap support. Only valuesabove 50 % are shown. Black circles indicate support 100 %/100 %. The treeis unrooted. (PDF 272 kb)Additional file 5: Phylogenetic tree of concatenated ADI, OTC, and CKsequences with Bacteria and Archaea removed. The tree based on a 750positions long protein alignment of 15 sequences was constructed inRAxML using LG4X + Γ model. The tree is unrooted. (PDF 2 kb)Additional file 6: The probability of mitochondrial localization of ADIpathway enzymes in Monocercomonoides sp. PA203 and Paratrimastixpyriformis as predicted by TargetP and MitoProt II. (DOCX 12 kb)Additional file 7: Topology of the expected species tree of eukaryotes.The tree is unrooted. (PDF 2 kb)AcknowledgementsAuthors would like to thank to Tomáš Pánek, Vít Céza, and Vojtěch Vacek, whoprovided RNA or cDNA for sequencing of Lacusteria cypriaca, the unidentifiedfree-living trichomonad, Harpagon shusteri, and Monocercomonoides sp. PA203,as well as Qianqian Zhang and Aaron Heiss, who prepared the Trimastix marinaRNAseq data.FundingZ. Z. and localization experiments were funded from Czech Science foundationproject 13-22333P. A. K. and V. H. were supported by the project BIOCEV –Biotechnology and Biomedicine Centre of the Academy of Sciences and CharlesUniversity (CZ.1.05/1.1.00/02.0109) from the European Regional DevelopmentNovák et al. BMC Evolutionary Biology  (2016) 16:197 Page 12 of 14Fund. I. Č., V. H. and sequencing was supported by Czech Science foundationprojects P506-12-1010 and GAP506/11/1317. M. K. and P. J. K. were supportedby a grant from the Tula Foundation to the Centre for Microbial Diversity andEvolution. C. W. S. was supported by Natural sciences and engineering researchcouncil of Canada - Alexander Graham Bell Canadian graduate scholarship. P. J.K., A. J. R., and A. G. B. S. are Senior Fellows of the Canadian Institute for Ad-vanced Research. Funding bodies did not play any role in the design ofthe study, collection, analysis, interpretation of data and in writing themanuscript.Availability of data and materialsSequences were deposited in GenBank under accession numbers KT883858-KT883885.Authors’ contributionsVH and LN designed the research. ZZ conducted the experiments. AK, MK,MH, CWS, AGBS, PJK, AJR, and IC took part in obtaining the sequences. LNand AK conducted the phylogenetic analyses. AK conducted the topologytests. LN and VH wrote the manuscript. All authors read, edited, andapproved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable.Ethics approval and consent to participateNot applicable.Author details1Department of Parasitology, Charles University, Faculty of Science, Prague,Czech Republic. 2Department of Biochemistry and Molecular Biology,Dalhousie University, Halifax, Canada. 3Institute of Molecular Genetics,Academy of Sciences of the Czech Republic, Prague, Czech Republic.4Department of Biology, Dalhousie University, Halifax, Canada. 5Departmentof Botany, University of British Columbia, Vancouver, Canada. 6Department ofZoology, Charles University, Faculty of Science, Prague, Czech Republic.Received: 27 December 2015 Accepted: 28 September 2016References1. Cunin R, Glansdorff N, Piérard A, Stalon V. Biosynthesis and metabolism ofarginine in bacteria. 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