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BMC Biology. 2021 May 17;19(1):103
The Author(s)
Záhonová, Kristína
Lax, Gordon
Sinha, Savar D
Leonard, Guy
Richards, Thomas A
Lukeš, Julius
Wideman, Jeremy G
Background: The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. Results: We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. Conclusions: Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.
RESEARCH ARTICLE Open AccessSingle-cell genomics unveils a canonicalorigin of the diverse mitochondrialgenomes of euglenozoansKristína Záhonová1,2, Gordon Lax3, Savar D. Sinha4, Guy Leonard5, Thomas A. Richards5, Julius Lukeš1,6* andJeremy G. Wideman4*AbstractBackground: The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids,diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrialgenomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereasmitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids.In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicingto produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns)and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestralbacteria-like circular mitochondrial genome remains unanswered.Results: We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogeneticand phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) withineuglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched aseither a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes wereidentified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genesand one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoansincluding full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome.Conclusions: Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestralmitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoanmitochondria.Keywords: Single-cell amplified genome, Evolution, Mitochondrial ribosome, Phylogeny© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.* Correspondence: jula@paru.cas.cz; jeremy.wideman@asu.edu1Institute of Parasitology, Biology Centre, Czech Academy of Sciences, ÄeskéBudÄjovice (Budweis), Czech Republic4Center for Mechanisms of Evolution, Biodesign Institute, School of LifeSciences, Arizona State University, Tempe, USAFull list of author information is available at the end of the articleZáhonová et al. BMC Biology (2021) 19:103 https://doi.org/10.1186/s12915-021-01035-yBackgroundThe major components of the eukaryotic tree of life thatremain underexplored comprise heterotrophic flagellates(HFs) [1]. Investigation of HFs enables improved under-standing of both eukaryotic evolution and diversity.However, traditional methods for acquiring moleculardata from HFs necessitates the establishment of cultures,which can be difficult [1], especially as many of thesecellular forms depend on symbiotic interactions. Newapproaches have focused on leveraging single-cell ampli-fied genome (SAG) sequencing technologies to generatedata enabling placement of these mysterious groups intothe tree of life [2â4]. Single-cell and traditional ap-proaches have recently uncovered HFs that are not partof well-studied groups (e.g., Rhodelphis, SAG D1, Ancor-acysta) [2, 5, 6], and phylogenetic placement of orphantaxa has helped to resolve major branching relationships(e.g., Hemimastigophora, Picozoa, telonemids) [7â9].Further sampling of HFs is predicted to shed light onkey evolutionary transitions that led from ancestral todiverged states across the eukaryotes.Among HFs, the phylum Euglenozoa includes line-ages with some of the most divergent cellular charac-teristics. Euglenozoans comprise four well-definedclades: kinetoplastids, diplonemids, euglenids, andsymbiontids [10â12]. Most euglenozoans, includingdiplonemids [13, 14], most kinetoplastids [15], andmost euglenids are free-living HFs [16, 17]. Symbion-tids are anaerobic and covered in epibiotic bacteria[10, 11]. Investigations of the parasitic kinetoplastidTrypanosoma brucei have shown that although theirmitoribosomes, nuclear pore complexes, and mito-chondrial import machineries are extremely divergedfrom most other eukaryotes, they function in a similarmanner [18â20]. Increased sampling of rare eugle-nozoans that branch at the base of each major lineagewill provide insights into how these cellular character-istics diversified in structure and protein composition,without obvious functional divergence.Compared to lineages like vertebrates and fungi, inwhich relatively little mitochondrial genome (mtDNA)variation has occurred, euglenozoans have amassedmitochondrial genomic oddities like no other eukaryoticgroup [21]. Juxtaposed with their closest relatives (i.e.,jakobids, Tsukubamonas, and heteroloboseans), whichhave bacteria-like, circular-mapping, gene-rich mtDNAs,the transcripts of which do not require trans-splicing orRNA editing [22â25], euglenozoans exhibit diversemtDNA architectures and mitochondrial transcript pro-cessing mechanisms [26â29]. The mtDNA of the eugle-nid Euglena gracilis is composed of linear chromosomesbearing full-length genes, non-functional pseudogenes,and terminal repeats in diverse arrangements [28, 30].Kinetoplastid mtDNA, like that of T. brucei, is split intomutually interlocked maxi- and minicircles [31]. Maxi-circles contain unrecognizable mitochondrial genes, thetranscripts of which undergo extensive uridine insertion/deletion editing, facilitated by minicircle-encoded guideRNAs and complex protein machinery [27]. Diplonemidshave mitochondrial genes split into small fragments,often on separate molecules [32, 33], which require ex-tensive trans-splicing and four kinds of RNA editing toproduce mature transcripts [29, 32â34]. How this diver-sity evolved in euglenozoans is unclear.A recently sequenced SAG (called âD1â) branched sis-ter to all known kinetoplastids in phylogenetic analyses[2]. Its mitochondrial contigs contained genes that werefragmented in a similar fashion as diplonemids, requir-ing diplonemid-like trans-splicing to produce maturetranscripts, but lacked any RNA editing [2]. While thesedata allow us to infer that the ancestor of kinetoplastidsand diplonemids had a diplonemid-like mtDNA archi-tecture, we cannot infer how the various idiosyncrasiesof euglenozoan mtDNAs emerged from a canonicalarchitecture. Here, we provide a starting point for un-derstanding early transitions by identifying a deep-branching euglenozoan SAG with an ancestral-likemtDNA lacking any RNA editing and/or trans-splicing,which encodes full-length mitoribosomal genes, andmultiple mitochondrial genes not present in other eugle-nozoan mtDNAs.Results and discussionDiverse euglenozoans can be recovered in SAG analysesWe sequenced and assembled 20 SAGs previously amp-lified and reported to be euglenozoans via V9 18S map-ping [4]. Assemblies varied in size from 1.2Mb (EU20)to 57.6Mb (EU16) (Additional file 1: Table S1). Similarto previous reports [2, 35, 36], our SAGs had very lowBUSCO completion scores (0â26% including fragmentedgenes) with the eukaryota_odb9 dataset (Additional file1: Table S1). Although all SAGs contained predomin-antly eukaryotic sequences, six (EU3â5, EU7, EU15, andEU19) were judged to be heavily contaminated as theyhad relatively high (53â83%) BUSCO completion scoreswhen using the bacteria_odb9 dataset. These resultswere consistent with the BlobTools plots (Additional file2: Fig. S1). When the eukaryotic small subunit rDNA(18S) genes from all 20 SAGs were used as BLAST quer-ies against the NCBI nucleotide database, their topBLAST hits were all euglenozoan 18S rDNAs (Add-itional file 1: Table S1).18S rDNA and concatenated nuclear phylogenies reveal adeep-branching euglenozoanUsing a previously published 18S dataset [7], we per-formed phylogenetic analysis including 18S sequencesfrom a broad diversity of eukaryotes (Fig. 1; AdditionalZáhonová et al. BMC Biology (2021) 19:103 Page 2 of 14file 2: Fig. S2). All 20 SAGs branched within euglenozo-ans, which were recovered as a monophyletic clade withfull support. The majority of these euglenozoan SAGsbranched within major clades (3 kinetoplastids, 1 eugle-nid, and 14 diplonemids) with full support. Only SAGsEU17 and EU18 could not be confidently placed withinany known euglenozoan clade. To better place these twoSAGs, we conducted phylogenetic analyses with add-itional euglenozoan 18S (332 sequences) and 16 discobidsequences as an outgroup (Additional file 2: Fig. S3).Euglenozoan monophyly was obtained, and kinetoplas-tids, diplonemids, and symbiontids all formed monophy-letic groups. As in some previous investigations [37, 38],euglenids were not retrieved as a monophyletic clade.EU2 branched sister to Rapaza viridis, confirming itseuglenid identity (100% standard bootstrap [BS]). EU17and EU18 branched with very low support (35% BS) sis-ter to petalomonads, a basal euglenid lineage [39].To further investigate the position of EU17 and EU18within euglenozoans, we performed a multi-gene phylo-genetic analysis. Because their 18S sequences differed onlyin three nucleotides (not shown) and because of the lowcompleteness of their individual assemblies (4.0% and4.3% BUSCOs with eukaryota_odb9, respectively; Add-itional file 1: Table S1), we co-assembled EU17 and EU18(Additional file 1: Table S1), producing SAG EU17/18(6.9% eukaryota_odb9 BUSCOs). Of 20 nucleus-encodedproteins used in a recent euglenid study [39], only fourFig. 1 18S rDNA sequences from 20 SAGs branch with euglenozoans. 18S rDNA sequences were collected from 20 sequenced SAGs forphylogenetic analysis. The 18S rDNA alignment, based on a previously published dataset [7], contained 131 taxa and 1551 nucleotide positions.The maximum likelihood phylogenetic tree was estimated under GTR + Î with standard bootstrapping (BS) and ultrafast bootstrapping (UFB).Support values are shown if â¥ 50% and â¥ 75% for BS and UFB, respectively. Fully supported nodes are shown as black circles. For clarity, themajority of eukaryotes are collapsed (for full tree see Additional file 2: Fig. S2)Záhonová et al. BMC Biology (2021) 19:103 Page 3 of 14Fig. 2 Concatenated nuclear phylogenetic analysis indicates EU17/18 represent a deep-branching euglenozoan. Assemblies of EU17/18 and EU2were searched for a set of marker proteins based on a previously published study including 20 nucleus-encoded proteins [39] for phylogeneticanalysis. The final alignment contained 62 taxa and 10,366 amino acid positions. EU17/18 and EU2 had 82% and 90% missing data, respectively.The maximum likelihood tree was estimated under two models, LG + C60 + F + Î (C60) and LG4X, with standard bootstrapping (BS) and ultrafastbootstrapping (UFB). The tree topology shown is from the C60 analysis. Support values for < 50% BS and < 75% UFB are denoted by a dash (-),whereas an asterisk (*) marks a topology that does not exist in a particular analysis. Fully supported nodes are shown as black circlesZáhonová et al. BMC Biology (2021) 19:103 Page 4 of 14and two of these genes were identified in EU17/18 andEU2, resulting in only 18% and 10% completeconcatenated alignments, respectively. While EU2branched again sister to R. viridis with 90% BS and 99%ultrafast bootstrap (UFB) support (LG + C60 + F + Î), and85/97% BS/UFB (LG4X), EU17/18 consistently formed asister clade to petalomonads with moderate, but uncon-vincing support values given the extent of the missing data(64/94% BS/UFB using LG + C60 + F + Î model, and 42/81% BS/UFB using LG4X model) (Fig. 2). Taken together,Fig. 3 EU17/18 (a) and EU2 (b) mtDNA structure and gene content are unique among known euglenozoans. The reassembly of EU17/18 wasobtained using the SAGAWE pipeline (see âMaterial and methodsâ) including reads from both EU17 and EU18 libraries. Horizontal lines representmitochondrial chromosomes with genes as predicted by MFannot and lengths above them. Star (â ) depicts the nad1 gene that is split to twocontigs most likely because of poor quality of contig endsZáhonová et al. BMC Biology (2021) 19:103 Page 5 of 14our results suggest that EU17 and EU18 likely represent anovel basal lineage of euglenids or a basal euglenozoanlineage. The uncertain placement could be attributed tothe poor data representation, and/or the limited taxonsampling of basal euglenids and/or euglenozoans.Concatenated mitochondrial gene phylogeny placesEU17/18 as a deep-branching euglenozoanWe identified 21 and seven ancestrally conservedmitochondria-encoded proteins in EU17/18 and EU2, re-spectively (Fig. 3). Full-length rns and rnl genes wereidentified in EU17/18 and a very small part of rnl (corre-sponding to the 3â half [LSU-R] of the E. gracilis rnl[30]) was identified in EU2. Along with the standardmitochondrial genes, we identified a large number (24)of open reading frames (ORFs), that, when translated,have no similarity to any protein in the GenBank data-base. Furthermore, no protein domains were predictedby InterProScan, except transmembrane domains in 12of them (Additional file 2: Fig. S4). One of these ORFscould represent an extremely divergent nad6, which isexpected to be found as it is rarely lost or transferred tothe nucleus [40]. No additional ORFs were identified inEU2 (Fig. 3). In both SAGs, several genes encoded TGAcodons as tryptophan (W), a feature found in all knowneuglenozoans [28, 33, 41â43], further supporting theFig. 4 Concatenated mitochondrial phylogenetic analysis confirms EU17/18 as a member of an early-diverging euglenozoan lineage.Mitochondrial-encoded protein sequences were predicted from EU17/18 and EU2 mitochondrial contigs and subjected to phylogenetic analysis.The alignment, based on a previously published study including 15 mitochondrion-encoded proteins [33], contained 37 taxa and 4325 aminoacid positions. EU17/18 and EU2 had 8.35% and 38.81% missing data, respectively. The maximum likelihood tree, estimated under two models, LG+ C20 + F + Î (C20) and LG + F + I + G4 (LG; the best-fitting model as determined by IQ-TREE), with 1000 standard bootstraps (BS) and 1000ultrafast bootstraps (UFB). The tree topology shown is from the C20 analysis. Support values for < 50% BS and < 75% UFB are denoted by a dash(-), whereas an asterisk (*) marks a topology that does not exist in a particular analysis. Fully supported nodes are shown as black circlesZáhonová et al. BMC Biology (2021) 19:103 Page 6 of 14conclusion that the TGA-W genetic code change is anancestral feature of euglenozoans. In addition, trnK waspredicted to be encoded in EU17/18, and trnM in EU2(Fig. 3).Since the position of EU17/18 in nuclear phylogenieswas not fully resolved, we sought to reconstruct a mito-chondrial phylogeny using a set of 15 proteins used pre-viously [33]. In this concatenated alignment, EU17/18had only 8.35% missing data, whereas in EU2 38.81%was missing (in comparison, the model species E. gracilishad 32.42% missing data). The mitochondrial phylogenyshows euglenozoans, inclusive of EU17/18, as a fullysupported monophyletic clade with fully supportedeuglenid (EU2 branching deeply within), kinetoplastid,and diplonemid clades (Fig. 4). Glycomonada (kineto-plastids + diplonemids) were also retrieved as monophy-letic with nearly full support (99/99% BS/UFB using LG+ C20 + F + Î model, and 98/100% BS/UFB using LG +F + I + G4 model as determined by IQ-Tree as best-fitting model). Support for a monophyletic euglenozoanclade excluding EU17/18 was moderately supported (74/89% BS/UFB [LG + C20 + F + Î], 72/92% BS/UFB [LG+ F + I + G4]). These data further suggest that EU17and EU18 represent an early-branching euglenozoanlineage. We repeated the analysis including the highlydivergent atp6 from EU17/18 identified using Phyre2.This phylogeny placed EU17/18 as a basal euglenid(Additional file 2: Fig. S5). However, the support for thisposition was low (72/85% [LG + C20 + F + Î], 52/65%BS/UFB [LG + F + I + G4]), and many internal euglenidbranches remained unresolved. While this placement issuggestive of a euglenid identity of EU17/18, the extremedivergence of atp6 sequences in euglenozoans could alsocontribute to long-branch attraction artefacts. Moremitochondrial genomes from deep-branching euglenidslike petalomonads and ploeotids are required to improvethe resolution of the deep-branching relationships withineuglenozoans.EU17/18 and EU2 bear surprising mtDNA architecture andgene contentSince architectures of mitochondrial genomes differ sig-nificantly among euglenozoan lineages [26], we investi-gated the mtDNA contigs from EU2 and EU17/18assemblies. Unlike other discobans, mitochondrial gen-ome structures in euglenozoans exhibit diverse organiza-tions [22]. The ancestral state from which these uniqueorganizations evolved was likely a circular, gene-rich,bacteria-like mitochondrial genome. To date, no inter-mediates between the divergent extant euglenozoan ar-chitectures and their ancestral state have beendiscovered. Both EU2 and EU17/18 exhibited contigs en-coding several mitochondrial genes, unscrambled, andwithout need for RNA editing (Fig. 3). To confirm thesegene arrangements, we inspected read coverage by map-ping trimmed reads onto contigs (see âMaterials andmethodsâ). Like previously sequenced SAGs [4], themitochondrial to nuclear gene coverage ratios were veryhigh in our euglenozoan SAGs (Additional file 1: TableS2), which is probably due to a combination of highmtDNA copy numbers in euglenozoans [44] as well asbiassed amplification during SAG preparation. Neverthe-less, read mapping showed a similar level of coveragethroughout all contigs, supporting the predicted genomearrangements (Fig. 3).All euglenozoans encode several subunits of respira-tory complexes in their mitochondrial genome. EuglenidmtDNAs (e.g., E. gracilis and Eutreptiella gymnastica)encode only eight proteins: an extremely divergent ATPsynthase subunit 6 (atp6), cytochrome b (cob), threesubunits of cytochrome c oxidase (cox1, cox2, and cox3),and three subunits of NADH dehydrogenase (nad1,nad4, and nad5) [28, 45]. Diplonemids and kinetoplas-tids additionally encode seven subunits of NADH de-hydrogenase (nad2, nad3, nad4L, nad6, nad7, nad8, andnad9) and kinetoplastids also possess two mitoribosomalproteins (rps3 and rps12) as well as a protein of un-known function (murf2) [19, 46]. Diplonemids furtherencode two proteins with unknown function (y4 and y7),although the former might represent a highly divergentsubunit of the respiratory chain complexes or a mitori-bosomal protein [32, 33, 47]. So far, no euglenozoanmtDNA was found to encode a tRNA gene [26, 48].Small and large subunits of mitoribosomal RNAs (rnsand rnl) of kinetoplastids are extremely short [49â51].In euglenids, rns and rnl are each split into two halvesthat are separately transcribed, though they appear tofold into more conventional secondary structures [30].Those in diplonemids are composed of one or morefragments, called modules (up to three and four for rnsand rnl, respectively, depending on species) [32, 33]. Re-gardless of the number of modules, diplonemidsâ rns areabout 390 nt long, which is only one third of the lengthof those in kinetoplastids.In the EU2 mitochondrial contigs, we were able toidentify the identical protein-coding repertoire of genesas E. gracilis mtDNA with one exception (cox3) (Fig. 3).Several genes were identified on a single contig, namelyatp6, cob, cox1, cox2, nad4, nad5, and surprisingly, apredicted tRNAMet(CAU) gene (Fig. 3; Additional file 2:Fig. S6). nad1 and an rnl fragment were found on twoseparate contigs. Since EU2 is undoubtedly a euglenid(Figs. 1, 2, and 4; Additional file 2: Fig. S3), its gene ar-rangement suggests that the mitochondrial genomearchitecture in euglenids is not well reflected in the soleinvestigated species, E. gracilis [28, 30].In EU17/18, we identified atp6, cob, all three cox sub-units, 10 nad subunits (namely nad1, nad2, nad3, nad4,Záhonová et al. BMC Biology (2021) 19:103 Page 7 of 14nad4L, nad5, nad7, nad8, nad9, and nad11), six mitori-bosomal proteins (rpl2, rpl14, rps2, rps12, rps13, andrps14), full-length rnl and rns, and a single tRNALy-s(UUU) gene (trnK; Fig. 3; Additional file 2: Fig. S6). Wewere unable to identify nad6, which may have either di-verged beyond recognition (e.g., atp6 in E. gracilis [45])or have independently transferred to the nucleus [52â57]. Unlike any other sequenced euglenozoan, EU17/18exhibited full-length rnl and rns genes that could be eas-ily aligned with ribosomal sequences of E. coli (Add-itional file 2: Fig. S7).The gene repertoire and genome architecture ofEU17/18 implies that the ancestral euglenozoan mtDNAwas more complex than anticipatedâin line with nu-merous convergent simplifications of mitochondrial genecontent across the eukaryotes [6]. Our phylogenetic re-constructions suggest that EU17/18 represents either adeep-branching euglenozoan or a basal member of theeuglenids. Either way, the euglenozoan ancestor can beinferred to have had a circular-mapping single mito-chondrial chromosome encoding at least 16 electrontransport chain components, seven mitoribosomal pro-teins (i.e., rpl2, rpl14, rps2, rps3, rps12, rps13, andrps14), full-length rRNA genes, and at least one tRNA.Euglenozoans exhibit a complex history of endosymbiontgene transfer of mitoribosomal proteinsAlthough genes encoding components of the electrontransport chain and mitoribosomal proteins are retainedin the mtDNA of most eukaryotes, these genes can belost or transferred to the nucleus [58]. In sampled eugle-nozoans other than EU17/18, most protein componentsof mitoribosomes are encoded in the nuclear genome[19]. The only two exceptions are rps3 and rps12 genesin mtDNA of kinetoplastids, which could be retained indiplonemids but are too divergent to detect [19, 33]. Inthe well-characterized mitoribosome of the kinetoplastidT. brucei, the short length of mito-rRNAs is supple-mented by 127 mitoribosomal proteins, assembled by anumber of dedicated factors, making it the largestknown ribosome [19, 59, 60]. While the T. brucei mitori-bosome contains some conserved subunits widely dis-tributed in other eukaryotes, it also incorporatesnumerous euglenozoa- and kinetoplastid-specific sub-units [60â62]. The same applies for the E. gracilis mitor-ibosome, predicted to be composed of 108 proteins, alsowith euglenozoa- and euglenid-specific subunits present[63]. Indeed, there are eight eukaryotic mitoribosomalsubunits in E. gracilis, which are absent from T. brucei(L1, L6, L18, L38, L56, S7, S13, and S28), and 16eukaryotic subunits retained in T. brucei, but likely lack-ing in E. gracilis (L5, L29, L30, L33, L35, L41, L42, L48,S14, S21, S26, S30, S33, S37, S38, and Fyv4) [63, 64]. Wesought to identify mitoribosomal genes encoded in thenuclear genome of EU17/18, but identified only one can-didate rps protein (EG_transcript_13898 homologue)and two candidate rpl proteins (EG_transcript_7639 andEG_transcript_26116 homologues) (Additional file 1:Table S3). However, even if these proteins are ortholo-gous, there is no guarantee that they will perform thesame function as those in other euglenozoans. Fromwhere novel euglenozoan mitoribosomal proteinsemerged and diversified is still unknown, but EU17/18has provided a starting point for this investigation. Asmore sequences from the EU17/18 lineage become avail-able, we will begin to grasp the origins of the divergentcellular features exhibited by euglenozoans.ConclusionsOur data strongly suggest that SAG EU17/18 representsthe earliest-branching lineage of known euglenozoans,or perhaps a novel euglenid lineage with ancestral fea-tures. EU17/18 exhibits several unique mitochondrialcharacteristics, enabling us to infer the ancestral eugle-nozoan mtDNA coding content and reconstruct thetransformations that occurred within each major eugle-nozoan lineage (Fig. 5). From an ancestralheterolobosean-like mtDNA with a standard geneticcode, several major changes can be traced (Fig. 5 â ).Prior to the last common euglenozoan ancestor theTGA-W genetic code change in mtDNA occurred andseveral genes were lost or transferred to the nucleus(â¡). The ancestral euglenozoan mtDNA was most prob-ably a circular-mapping chromosome encoding atp6,cob, cox1-3, nad1, 2, 3, 4, 4 L, 5, 6, 7, 8, 9, and 11, rpl2,14, rps2, 3, 12, 13, 14, full-length rns and rnl, as well assome tRNA genes (â¡). When tRNA genes were fullytransferred to the nucleus remains unclear as the eugle-nid EU2 retains at least one predicted tRNA.After divergence of EU17/18 from all other euglenozo-ans, rps2, rpl2, and nad11 were transferred to the nu-cleus (â¢). From this point, major changes likelyoccurred in the rns and rnl genes, which coincided withthe emergence of a number of novel mitoribosomal pro-teins [19, 59, 63]. In euglenids this can be seen by splitrns and rnl genes â£, whereas in glycomonads, this isseen in their accelerated divergence and drastic shorten-ing â¤. In euglenids, nad2, 3, 4 L, 6, 7, 8, and 9 were ei-ther lost or transferred to the nucleus, as only atp6, cob,cox1, cox2, cox3, nad1, nad4, and nad5 were retained inthe mtDNA â£ (more euglenid mtDNA sequences areneeded to confirm this inference) [63]. In kinetoplastidsand diplonemids, the mitochondrial coding complementis nearly overlapping in electron transport chain compo-nents, both retaining atp6, cob, cox1-3, nad1, 2, 3, 4, 4 L,5, 6, 7, 8, and 9 â¤. Kinetoplastids additionally retainrps3 and rps12 [19, 46]. rps13 and rpl14 are encoded inthe mtDNA of EU17/18, appear to be lost outright inZáhonová et al. BMC Biology (2021) 19:103 Page 8 of 14glycomonads â¤ but transferred to the nucleus in eugle-nids â£. Similarly, rps14 was lost in euglenids â£ butlikely transferred to the nucleus in the lineage leading toglycomonads â¤.In conclusion, SAG EU17/18 has provided signifi-cant insight into the origin and diversification of theeuglenozoan mtDNA gene complement (Fig. 6). Theevolutionary reconstruction presented herein showsthat the extreme complexity of mtDNA organizationand transcript processing exhibited by most eugle-nozoans had rather simple origins, an unexpectedfinding for this group known for its highly divergentmitochondrial characteristics. Our data further dem-onstrate that single-cell approaches are necessary foridentifying basal protist lineages whose incorporationinto the tree of life is essential for understandingcellular evolution in particular and eukaryote biologyin general.Material and methodsSample origin, genome amplification, and sequencingWe chose to sequence a set of 20 heterotrophic fla-gellate SAGs that were previously isolated and ampli-fied, and their V9 18S sequences were Sangersequenced [4]. V9 18S placement revealed theirphylogenetic affinity to euglenozoans [4]. These SAGsoriginated from cells isolated from the deep chloro-phyl maximum in the North Pacific Ocean as re-ported previously [4]. In the current study, weprepared sequencing libraries with Nextflex RapidDNA library preparation kit (BIOO Scientific) and 12cycles of PCR amplification. Libraries (250 bp paired-Fig. 5 The evolutionary history of mtDNA in euglenozoans. Mitochondrial traits were mapped onto a schematic phylogenetic tree based on thisand previous studies [2, 28, 30, 32, 43]. â The ancestral discobid-like mtDNA. â¡ For the last common ancestor of euglenozoans, mtDNA wasrepresented by a circular-mapping chromosome, meaning of TGA codon was changed to code for W, and genes for atp1, 3, 8, and 9, sdh3, and 4,and majority of mitoribosomal proteins were transferred to the nucleus. â¢ After separation of EU17/18, rps2, rpl2, and nad11 underwentendosymbiotic gene transfer (EGT). â£ For the common euglenid ancestor, mtDNA was fragmented to chromosomes with single genes andmito-rRNAs were split to halves. Majority of genes encoding NADH dehydrogenase, rps12, and 13, and rpl14 were transferred to the nucleus, andrps14 was lost. â¤ For the glycomonads, genes were split to modules and rRNAs were significantly shortened. While rps14 underwent EGT, rps13and rpl14 were lost completely. â¥ Mitochondrial genome structure of early-branching euglenids is currently unknown. â¦ For the SAG EU2,chromosomes encoding single genes were joined to a possibly circular molecule, resembling the ancestral state. â§ Diplonemids acquired diverseRNA editing mechanism to produce mature transcripts. â¨ After separation of SAG D1, mtDNA of the rest of kinetoplastids was catenated intointerlocked mini- and maxicircles. Primary transcripts are processed by U-insertion/deletion RNA editing to restore the coding sequenceZáhonová et al. BMC Biology (2021) 19:103 Page 9 of 14end) were sequenced on a HiSeq 2500 using Rapidrun SBS v2 reagents (Illumina).Single-cell genome assemblyEach sequence library corresponding to a SAG was assem-bled using the SAGAWE pipeline available at https://github.com/guyleonard/sagawe. Briefly, libraries werequality controlled and adapter trimmed using Trim Gal-ore! v0.6.4 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore), digitally normalised using BBNorm(part of the BBTools suite v37.93; https://jgi.doe.gov/data-and-tools/bbtools/) to compensate for uneven coverage ofmultiple displacement amplification, reads merged usingBBMerge (part of the BBTools suite) to create sets of lon-ger reads, and then assembled with SPAdes v3.13.1 [65] insingle-cell mode under default parameters. Post assemblystatistics were computed to provide general assembly statsusing QUAST v5.0.2 [66], genome completeness estimatesusing BUSCO v3.0.1 [67] software, contamination checkedusing BlobTools v1.0 [68] and BLAST v2.2.31+ [69]against NCBI nucleotide database, and coverage estimatedvia read mapping using BWA v0.7.17-r1188 [70] and Qua-limap v2.2.2-dev [71]. For the reassembly of EU17/18, thesame pipeline was employed using reads from both EU17and EU18 libraries.Phylogenetic analysis of 18S rDNA and concatenatednuclear genesSmall subunit ribosomal DNA (18S rDNA) was ex-tracted from each assembly by BLASTn searchesusing a set of euglenozoan 18S sequences as queries.Extracted 18S rDNAs of SAGs were aligned usingMAFFT v7.458 [72] under FFT-NS-i strategy (asdetermined with --auto) with 18S rDNAs of organ-isms from all major eukaryotic groups [7] and fromother discobans. To remove poorly aligned positionstrimAl v1.4 [73] (-gt 0.8) was used. Maximum likeli-hood analysis was performed with IQ-TREE v1.6.8[74] under the GTR + Î model. Branch supports wereobtained by non-parametric (BS) and ultrafast (UFB)bootstrap approximation methods [75] with 100 and1000 replicates, respectively.Assemblies EU17/18 and SAG EU2 were queried for20 nucleus-encoded proteins [39]. Found proteins werealigned in the respective single-gene alignment usingMAFFT v7.458 [72] under L-INS-I strategy (as deter-mined with --auto), and trimmed with trimAl v1.4 [73](-gt 0.5). Single-gene trees were inferred from trimmedalignments using the best-fitting substitution model asdetermined by IQ-TREE v1.6.8 [74], and supportassessed with 1000 ultrafast bootstrap replicates [75].Single-gene trees were visually inspected to identify con-taminant and paralogous sequences. The final trimmedand concatenated dataset consisted of 20 genes from 62discobans and had 10,366 amino acid positions. A max-imum likelihood phylogeny was estimated in IQ-TREEv1.6.8 [74] under two models: LG + C60 + F + Î andLG4X. For the LG + C60 + F + Î model, 200 BS and1000 UFB replicates were used, and for the LG4X model,1000 replicates were estimated with both bootstrappingmethods.Mitochondrial genome analysis and concatenatedphylogenetic analysisReassembly EU17/18 and SAG EU2 were searched formitochondrial protein- and rRNA-coding genes usingFig. 6 The mitochondrial genome of EU17/18 encodes the most complete mitochondrial repertoire of any known euglenozoan. Mitochondrion-and nucleus-encoded genes are shown as black and grey rectangles, as explained in graphical legend. For each gene, number of precursortranscript is indicated by number in the upper half of the rectangle. In case of diplonemids, the range indicates representatives with the smallestand highest number of modules as identified previously [32, 33]. Presence of any type of RNA editing is shown in blue in the lower half of therectangle. The number in parentheses after the lineage name represents number of sequenced speciesZáhonová et al. BMC Biology (2021) 19:103 Page 10 of 14Diplonema papillatum [32] and Euglena gracilis [28] se-quences as tBLASTn and BLASTn queries, respectively.The identity of found sequences was confirmed byBLAST searches against the NCBI non-redundant data-base. Larger mitochondrial contigs of EU2 and EU17/18were analysed by MFannot (https://megasun.bch.umontreal.ca/cgi-bin/dev_mfa/mfannotInterface.pl). Pro-tein domains were predicted by InterProScan search[76]. The protein translations of identified ORFs weresubjected to search using Phyre2 [77] to identify moredivergent homologues.For presentation purposes, tRNA and rRNA sequenceswere aligned using the built-in aligner of the GeneiousPrime v2020.2.3 software [78] and adjusted in a graph-ical editor.Identified mitochondrial protein-coding genes wereadded to a dataset from a previous investigation [33].Datasets were aligned with MAFFT v7.458 [72] under L-INS-I strategy (as determined with --auto), and trimmedwith trimAl v1.4 [73] (-gt 0.5). Single-gene trees were in-ferred from the alignments using the best-fitting substitu-tion model as determined by IQ-TREE v1.6.8 [74] and1000 UFB replicates. The final trimmed concatenateddataset consisted of 15 genes from 37 discobans and had4324 and 4348 amino acid positions with and withoutatp6 from EU17/18, respectively. A maximum likelihoodtree was inferred with IQ-TREE v.1.6.8 [74] under the LG+ F + I + G4 model (determined as the best-fitting modelby IQ-TREE) and 1000 BS and 1000 UFB replicates.Homology searchingAncestral and euglenozoan-specific subunits of themitoribosome were searched using the previously pub-lished dataset of E. gracilis [63]. Candidate homologueswere subjected to a reciprocal BLAST search to validateorthology. A list of proteins that were searched is avail-able in Additional file 1: Table S3.Supplementary InformationThe online version contains supplementary material available at https://doi.org/10.1186/s12915-021-01035-y.Additional file 1: Table S1. General statistics of assembled SAGs. TableS2. Proportion of mtDNA in SAGs. Table S3. List of proteins searched inEU17/18 reassembly. +, presence; +?, uncertain presence; -, absence.Additional file 2: Fig. S1. BlobTools plots showing contamination ofseveral sequenced SAGs. The bacterial contamination is shown as bluecircles, while sequences with eukaryotic signal are in magenta. Forcomparison, BlobTools plots for SAGs EU17 and EU18 are also shown.Fig. S2. 18S rDNA phylogeny of eukaryotes. The Maximum Likelihoodphylogenetic tree was estimated from an alignment containing 131 taxaand 1551 nucleotide positions under the GTR + Î model with standardbootstrapping (BS) and ultrafast bootstrapping (UFB). Support values areshown if â¥ 50% and â¥ 75% for BS and UFB, respectively. Fully supportednodes are shown as black circles. Fig. S3. 18S rDNA phylogeny ofeuglenozoans. The Maximum Likelihood phylogenetic tree was estimatedfrom an alignment containing 368 taxa and 1269 nucleotide positionsunder the GTR + Î model with standard bootstrapping. Support valuesare shown if â¥ 50%. Fig. S4. Predicted domains in EU17/18 mtDNA-encoded ORFs. ORFs annotated by MFannot (https://megasun.bch.umontreal.ca/cgi-bin/dev_mfa/mfannotInterface.pl) were submitted to anInterProScan [76] search. Predicted domains are highlighted as explainedin the graphical legend. Fig. S5. Concatenated mitochondrial phylogen-etic analysis including atp6 from EU17/18. The alignment contained 37taxa and 4348 amino acid positions, with EU17/18 missing 3.65% of data.The Maximum Likelihood tree was estimated under two models, LG +C20 + F + Î (C20) and LG + F + I + G4 (LG; the best-fitting model as de-termined by IQ-TREE), with 1000 standard bootstraps (BS) and 1000 ultra-fast bootstraps (UFB). The tree topology shown is from the C20 analysis.Support values for < 50% BS and < 75% UFB are denoted by a dash (-),whereas an asterisk (*) marks a topology that does not exist in a particu-lar analysis. Fully supported nodes are shown as black circles. Fig. S6.Characterization of tRNAs encoded in mtDNA of EU17/18 and EU2. a-b)Sequences of trnK (a) and trnM (b) were aligned with mitochondriallyencoded tRNAs of other species of Discoba. Residue shading indicates se-quence conservation. c-d) Secondary structures of trnK (c) and trnM (d) aspredicted by tRNAScan-SE. Double and triple bonds are depicted as dark-and light-blue circles, respectively. Anticodons are highlighted with agreen background. Since all other known euglenozoans import all tRNAsinto mitochondria from the nucleus [48], we built tRNA alignments withhomologues from the mtDNAs of other discobans to take into accountdifferent evolutionary pressures and mutational rates in nuclei and mito-chondria [52]. The identity across nine trnK and 20 trnM sequences was38.7% and 15.8%, respectively (a-b). Predicted secondary structures re-sembled other tRNAs supporting their functionality (c-d). While most eu-karyotes have at least some tRNA mitochondrial-encoded, the long-standing paradigm was that euglenozoans and unrelated apicomplexans(which share with euglenozoans a range of unique features [79]) importall tRNAs from the cytosol [80]. This has significant consequences, sincethe bacterial-type translation system has to cope solely with theeukaryotic-type tRNAs [81]. Fig. S7. Mitoribosomal RNAs of EU17/18. Se-quences of rns (a) and rnl (b) genes, as predicted by MFannot, werealigned with sequences of Escherichia coli. E. coli sequences were ob-tained from http://rna.ucsc.edu/rnacenter/ribosome_images.html, andtheir predicted domains [82, 83] are shown as magenta and blue boxesbelow the sequences. Nucleotide identities are shown by black back-ground with white nucleotides.AcknowledgementsWe thank ZdenÄk Paris (Institute of Parasitology, Äeské BudÄjovice) andMatúš Valach (Université de Montréal) for their help with tRNA and rRNAinvestigation, respectively.Authorsâ contributionsKZ and JGW conceived and designed the project. KZ, GLa, SDS, GLe, andJGW analysed the data and performed bioinformatic and phylogeneticanalyses. JGW, TAR, and JL supervised the project. KZ and JGW wrote themanuscript. All authors contributed to the editing of the final manuscript.The authors read and approved the final manuscript.FundingThis work was supported by the Czech Grant Agency (20-07186S), ERC CZ(LL1601), and the ERD funds of the Czech Ministry of Education (16_019/0000759) to JL. TAR is supported by a Royal Society University ResearchFellowship (URFR191005). Computational resources were supplied by theproject âe-Infrastruktura CZâ (LM2018140).Availability of data and materialsAll data needed to evaluate the conclusions in the paper are present in thepaper and/or the Supplementary Information files. Raw sequencing readshave been previously deposited at NCBI Sequence Read Archive (SRP102236)under NCBI BioProject PRJNA379597 [4]. Final assemblies are available fromFigshare at https://figshare.com/s/de8998f2e6cc71de6702. BLAST server forassemblies is accessible at http://evocellbio.com/SAGdb/zahonova_et_al/.Additional data related to this paper may be requested from the authors.Záhonová et al. BMC Biology (2021) 19:103 Page 11 of 14DeclarationsEthics approval and consent to participateNot applicable.Consent for publicationNot applicable.Competing interestsThe authors declare no competing interests.Author details1Institute of Parasitology, Biology Centre, Czech Academy of Sciences, ÄeskéBudÄjovice (Budweis), Czech Republic. 2Faculty of Science, Charles University,BIOCEV, Vestec, Czech Republic. 3Department of Botany, University of BritishColumbia, Vancouver, Canada. 4Center for Mechanisms of Evolution,Biodesign Institute, School of Life Sciences, Arizona State University, Tempe,USA. 5Department of Zoology, University of Oxford, Oxford, UK. 6Faculty ofSciences, University of South Bohemia, Äeské BudÄjovice (Budweis), CzechRepublic.Received: 1 March 2021 Accepted: 22 April 2021References1. Sibbald SJ, Archibald JM. More protist genomes needed. Nat Ecol Evol.2017;1(5):145. https://doi.org/10.1038/s41559-017-0145.2. 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Single-cell amplified genome
Evolution
Mitochondrial ribosome
Phylogeny
Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans
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