Morales, J., Hashimoto, M., Williams, T., Hirawake-Mogi, H.,Makiuchi, T., Tsubouchi, A., Kaga, N., Taka, H., Fujimura, T., Koike,M., Mita, T., Bringaud, F., Concepción, J. L., Hashimoto, T., Embley,T. M., & Nara, T. (2016). Differential remodelling of peroxisomefunction underpins the environmental and metabolic adaptability ofdiplonemids and kinetoplastids. Proceedings of the Royal Society B:Biological Sciences, 283(1830).https://doi.org/10.1098/rspb.2016.0520

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Differential remodelling of peroxisome function underpins the environmental

and metabolic adaptability of diplonemids and kinetoplastids

Short title: Gluconeogenic compartment in diplonemids

Jorge Morales1,9, Muneaki Hashimoto1,11, Tom A. Williams2,3,11, Hiroko Hirawake-Mogi1,11,

Takashi Makiuchi1,10, Akiko Tsubouchi1, Naoko Kaga4, Hikari Taka4, Tsutomu Fujimura4, Masato

Koike5, Toshihiro Mita1, Frédéric Bringaud6, Juan L. Concepción7, Tetsuo Hashimoto8, T. Martin

Embley2 and Takeshi Nara1*

1Department of Molecular and Cellular Parasitology, 4Division of Proteomics and Biomolecular

Science, and 5Department of Cellular and Molecular Neuropathology, Juntendo University

Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

2Institute for Cell and Molecular Biosciences, Catherine Cookson Building, Framlington Place,

Newcastle University, Newcastle upon Tyne NE2 4HH, UK

3School of Earth Sciences, University of Bristol, Bristol BS8 1TG, UK.

6Centre de Résonance Magnétique des Systèmes Biologiques (RMSB) UMR 5536 CNRS

Université de Bordeaux, France

7Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes,

Mérida 5101, Venezuela

8Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai,

Tsukuba, Ibaraki 305-8572, Japan

1

*Correspondence: tnara@juntendo.ac.jp

Footnote

9Present address: Emmy Noether-Gruppe “Microbial Symbiosis and Organelle Evolution”,

Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany

10Present address: Department of Infectious Diseases, Tokai University School of Medicine,

Isehara, Kanagawa 259-1193, Japan

11These authors contributed equally to this work.

2

Abstract

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic

biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved

specialised peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway

as well as other enzymes, which underpin the physiological flexibility of these major human

pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most

abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalisation of

gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine

diplonemid, Diplonema papillatum. Our results suggest that peroxisome modification was

already underway in the common ancestor of kinetoplastids and diplonemids, and raise the

possibility that the central importance of gluconeogenesis to carbon metabolism in the

heterotrophic free-living ancestor may have been an important selective driver. Our data indicate

that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a

factor in the success of their free-living euglenozoan relatives.

Keywords

organelle evolution; metabolic compartmentalisation; peroxisomes; glycolysis; diplonemids;

kinetoplastids

3

1. Introduction

Glycolysis (the Embden–Meyerhof-Parnas pathway) and gluconeogenesis are common to all

domains of life and play fundamental roles in essential cellular processes. Glycolysis consists of

10 enzymatic steps and generates ATP, NADH, and intermediate metabolites for a variety of

metabolic processes, while gluconeogenesis produces glucose 6-phosphate (G6P) from other

carbon sources including lipids and amino acids [1]. Glycolysis takes place in the cytosol but

glycolytic enzymes have also been associated with the plasma membrane of erythrocytes [2] and

with mitochondria [3, 4]. In addition, enzyme isoforms with non-glycolytic roles are found to

locate to other compartments [5].

Relocation of the first 7 of 10 glycolytic enzymes to microbody-like organelles, called

glycosomes, was first demonstrated in the kinetoplastid parasite, Trypanosoma brucei [6], and

subsequently in other kinetoplastids [7]. So far the kinetoplastids, which comprise bodonids and

trypanosomatids, are the only organism known to sequester a successive glycolytic cascade into

organelles. These enzymes are targeted to glycosomes by a peroxisomal targeting signal (PTS),

implying that glycosomes originated as modified peroxisomes. Glycosomes also contain enzymes

of other pathways including gluconeogenesis, the pentose phosphate pathway, energy/redox

metabolism, and nucleotide synthesis [7]. Glycosomes are thus a classic example of how complex

and physiologically important metabolic pathways can be relocated during evolution.

The relocation of entire metabolic pathways in eukaryotes is rarely observed, because

isolation of the individual pathway enzymes by transfer from one compartment to another can

abolish otherwise tightly coordinated enzymatic cascades. To overcome these difficulties, a

“minor-mistargeting” mechanism has been proposed to allow for the dual location of pathways

4

[8]. According to this hypothesis, the imperfect targeting of PTS-tagged enzymes to peroxisomes

would allow for the retention of functional glycolysis in the cytosol until the newly routed

peroxisomal enzymes could reconstitute the pathway. This hypothesis provides a plausible

mechanism for how functional retargeting could occur, but does not identify the physiological

conditions or selective pressures that might drive the initial evolution of compartmentalisation.

The phylum Euglenozoa currently contains the euglenoids, diplonemids and kinetoplastids,

with diplonemids the sister group of kinetoplastids and euglenoids the outgroup to both [9]. In

euglenoids, glycolysis occurs in the cytosol but some enzymes are also located to its secondary

plastid to participate in the Calvin cycle, as in plants and green algae [10, 11]. In the free-living

marine diplonemid Diplonema papillatum, we have previously reported that fructose-1,6-

biphosphate aldolase (FBPA), the 4th enzyme of glycolysis, has a type-2 PTS (PTS2) and is

compartmentalized [12], suggesting that the transition to glycosomes may have already occurred

in the common ancestor of diplonemids and kinetoplastids.

2. Materials and methods

(a) Organisms

Diplonema papillatum (strain ATCC® 50162) and the kinetoplastid Trypanosoma cruzi

(Tulahuen stock), were grown axenically in ATCC® 1532 and 1029 media, respectively. ATCC

1532® medium contains 0.1% (w/v) tryptone and 1% horse serum, corresponding to 10 mM

amino acid ingredients and 40-60 µM glucose, respectively. ATCC 1029® LIT medium is a rich

medium that contains 6 mM glucose and 0.9% (w/v) liver infusion broth/0.5% tryptone,

5

equivalent to > 100 mM amino acid ingredients. All experiments were carried out using the

standard medium unless otherwise stated.

(b) Draft genome sequencing of D. papillatum

D. papillatum genomic DNA was extracted using standard protocols, purified on agarose gel to

remove mitochondrial DNA, and used for genome sequencing. The D. papillatum genomic DNA

was sequenced using a HiSeq 2000 apparatus (Illumina K.K., Tokyo, Japan) by Takara Bio Inc.,

Shiga, Japan and Eurofins Genomics K.K., Tokyo, Japan. Total reads were 21.5 Gb and the

estimated genome size was 176.5 Mb. The D. papillatum open reading frames for genes of

interest were identified using TBLASTN. We predicted PTS1 by the Prosite pattern PS00342 for

the C-terminal tripeptides, [-(STAGCN)-(RKH)-(LIVMAFY)], and the PTS 1 Predictor

(http://mendel.imp.ac.at/pts1/) [13]; we required a positive identification from both methods to

diagnose the presence of a bona fide PTS. The presence of a PTS2 was manually inspected based

on the N-terminal nonameric consensus sequence, (RK)-(LIV)-X5-(QH)-(LA) [14]. The T.

brucei glycosomal proteins obtained by detailed proteomic analysis were used as references [15].

(c) Phylogenetics

We used the D. papillatum sequences as queries in BLASTP searches against the other

euglenozoan genomes and a broad selection of outgroups. The Euglena gracilis PFK sequence

was obtained from http://euglenadb.org/. Sequences were aligned with Meta-Coffee [16]. Poorly-

aligning regions were masked using the “automated1” option in trimAl [17]. Phylogenies were

built using the LG [18], C20, and C60 [19] in PhyloBayes. The trees inferred under the three

methods were very similar, with no differences impacting on our inferences of the relationships

6

among the euglenozoans, and the consensus trees in figures 1, S1 to S5 and S8 to S10 were

inferred using the C60 model.

(d) Indirect immunofluorescence analysis

Antisera to glycolytic enzymes were obtained as described in supplementary materials and

methods. Indirect immunofluorescence analysis was performed as described [12]. Briefly, cells

were fixed with 2% paraformaldehyde, probed with antisera to each glycolytic enzyme, and then

treated with anti-rabbit Alexa-fluor 488-conjugate (Life Technologies), followed by

counterstaining of the nucleus with Hoechst 33342 dye. Fluorescence was observed using

fluorescence microscopy (Axio Imager M2, Carl Zeiss Co. Ltd., Tokyo, Japan).

(e) Glucose and amino acid consumption

D. papillatum cells of the mid-log phase were cultured in ATCC® 1532 medium supplemented

with 6 mM D-glucose, while T. cruzi cells were cultured in LIT medium. The glucose

concentration of the culture supernatant was measured using a Glucose-Oxidase assay kit

(Sigma-Aldrich). Amino acid consumption by cells was evaluated by the increase of NH4+ in the

culture supernatant using the Ammonia assay kit (Sigma-Aldrich).

(f) Metabolic labeling

Cells in the mid-log phase of growth were starved in artificial seawater (ATCC® 1405 HESNW

medium) for 2 hrs and then incubated with 6 mM 13C6 D-glucose or 6 mM 13C5 L-glutamine

(Sigma-Aldrich) in artificial seawater for 0, 4, and 8 hrs or with 30 mM 13C6 D-glucose in

ATCC® 1532 medium for 72 hrs without starvation. The detailed experimental procedures for

7

extraction of metabolites and analysis by CE-Q-TOFMS and LC-MS were described in the

supplementary materials and methods.

3. Results

(a) Identification of peroxisome targeting signals in the glycolytic enzymes of D. papillatum

To investigate glycolytic compartmentalisation in diplonemids, we sequenced the genome of D.

papillatum and identified all of the genes for glycolysis (table 1). We identified putative PTS for

2 types of hexose kinase, namely a high-affinity type hexokinase (HK) and a low-affinity type

glucokinase (GLK), and for phosphoglucose isomerase (PGI) and phosphoglycerate kinase

(PGK), similar to kinetoplastid homologues. We identified two homologues of

phosphofructokinase (PFK), PFK1 and PFK2, in the genome assembly. PFK1 possesses a

predicted PTS1 but is likely a pseudogene or a bacterial contaminant (see below). We also found

a PTS for triose-phosphate isomerase (TIM), despite of the absence of an apparent PTS in the

kinetoplastid TIM. By contrast, putative PTS were absent from the predicted coding sequences of

PFK2, phosphoglycerate mutase (PGAM, 2,3-bisphosphoglycerate-independent type), enolase

(ENO), pyruvate kinase (PK) and two isoforms of glyceraldehyde-3-phosphate dehydrogenase

(GAPDH).

Phylogenetic analysis demonstrated that most of these enzymes (HK, GLK, PGI, PGK,

ENO and PK) were present in the common ancestor of D. papillatum and kinetoplastids (figures

S1, S2, and S3). The phylogeny for TIM was weakly supported and hence cannot reject the

hypothesis of a single common origin for TIM (figure S1d). In contrast, three glycolytic enzymes

8

– PFK1 and PFK2, the two isoforms of GAPDH, and PGAM – appear to have separate

evolutionary origins for D. papillatum and the kinetoplastids (figures 1, S1e, and S4).

The protein phylogeny for PFK revealed that E. gracilis and kinetoplastids are

monophyletic with high posterior support (PP = 0.99), suggesting their shared evolutionary origin

(clade A, figure 1). By contrast, D. papillatum PFK2 clusters with a PFK homologue from the

haptophyte Emiliania huxleyi (PP = 1) and is nested within a different eukaryotic clade (Clade B,

PP = 0.99), suggesting replacement of the ancestral euglenozoan PFK by lateral gene transfer. D.

papillatum PFK1 detected in our genome assembly is likely a pseudogene or a bacterial

contaminant: it does not group with other eukaryotic PFKs in the tree; the putative pfk1 gene

lacks a splice acceptor site at its 5’-untranslated region, which is essential for trans-splicing in

euglenozoans; and we were unable to detect any PFK activity in the D. papillatum lysate (table

1). In summary, our data suggest that PTS-driven relocation of most, but not all, glycolytic

enzymes had already occurred in the common ancestor of diplonemids and kinetoplastids.

We also investigated the presence of a PTS in the non-glycolytic enzymes of D. papillatum.

Among 18 PTS-harboring enzymes specific to kinetoplastid glycosomes, only three of these

enzymes were also predicted to have PTS in D. papillatum, which participate in gluconeogenesis

and energy metabolism (tables 1 and S1). These include the gluconeogenic fructose-1,6-

biphosphatase (FBPase) that catalyzes the reverse reaction to PFK, ATP-producing glycerol

kinase (GK) and pyruvate phosphate dikinase (PPDK). None of the enzymes identified for the

Diplonema pentose phosphate pathway (PPP), pyrimidine biosynthesis, or purine salvage were

predicted to locate to peroxisomes. Notably, despite of the presence of PTS1, the protein

phylogeny of FBPase revealed different evolutionary origins between D. papillatum and

kinetoplastids, implying independent LGT events and convergent PTS-driven relocation of

9

FBPase in D. papillatum and kinetoplastids (table 1 and figure S5). Our data suggest that

metabolic compartmentalisation originated in association with glycolysis, gluconeogenesis and

energy metabolism in the common ancestor of D. papillatum and kinetoplastids.

(b) Localisation of PTS-harboring glycolytic enzymes in the peroxisomes of D. papillatum

To investigate the expression and confirm the cellular location of the D. papillatum glycolytic

enzymes, we raised antisera to 11 glycolytic enzymes including GLK. Western blot analysis of

the D. papillatum extracts showed the expected band for all enzymes tested, with the exception of

the two homologues of PFK and HK (figure S6a). The absence of the signals for both PFK1 and

PFK2 suggests that neither of the two PFK homologues is expressed or functional in the culture

condition of D. papillatum, consistent with the lack of PFK activity. To confirm the absence of

HK, we measured hexose kinase activity in D. papillatum cell extracts and found that the Km

value for glucose of the native enzyme harboring hexose kinase activity was 0.33 mM, whereas

the Km values of the recombinant HK and GLK were 0.064 and 0.66 mM, respectively. These

results suggest that the native hexose kinase activity is likely attributable to GLK in D.

papillatum under the experimental conditions.

Indirect immunofluorescence analysis (IFA) was performed to investigate the cellular

location of the glycolytic enzymes expressed in D. papillatum. Our previous study showed that

our mouse antiserum to D. papillatum FBPA detected a single band on western blots of the

membrane-rich fraction (10,500 x g sediment) from the D. papillatum extract and also

demonstrated the punctate pattern in the cell by IFA [12]. Immuno-electron microscopy using the

same antiserum also detected labeling of FBPA inside single-membrane compartments with the

characteristic morphology of peroxisomes (figure S6b). Therefore, we used FBPA as a

10

peroxisomal marker. The signals for GLK, PGI, TIM, and PGK co-localised with that of FBPA,

indicating their shared peroxisomal location (figure 2). The signal for the anti-GAPDH1 antibody

gave a patchy distribution in the cytosol but did not co-localise with FBPA, consistent with the

absence of a PTS. PTS-lacking PGAM, ENO, and PK were also exclusively detected in the

cytosol of D. papillatum, similar to their kinetoplastid PTS-lacking homologues.

(c) Preference of amino acids as carbon source in D. papillatum

The failure to detect PFK activity in D. papillatum suggests that the glycolytic enzymes together

with FBPase play a replenishing role for gluconeogenesis in this organism. Notably, D.

papillatum can grow with a trace level of glucose. The modified ATCC 1532 medium

supplemented with 1% dialyzed fetal bovine serum (Thermo Fisher Scientific Inc.), which

corresponds to < 2.7 µM glucose, can also support the normal growth of D. papillatum (data not

shown). However, the growth of D. papillatum even in the absence of glucose does not

necessarily indicate the loss of glucose uptake and glycolytic activity. Therefore, we investigated

firstly the relative preference of carbon sources in D. papillatum. Consistent with our hypothesis,

D. papillatum does not significantly consume glucose (6 mM), but preferentially uses amino

acids (figure 3a, left). This is demonstrated by the significant production of ammonium in the

presence of glucose during early exponential growth. By contrast, epimastigotes (an insect form)

of the parasitic kinetoplastid Trypanosoma cruzi prefer glucose to amino acids, as previously

reported [20]. In experiments with T. cruzi (figure 3a, right), the levels of glucose decreased

significantly at day 3 to 6 along with exponential growth and became undetectable (< 10 µM) at

day 7, while the levels of ammonium remained constantly by day 5 and then increased

11

significantly. Because amino acids are the major nutritional components in the diet of phagocytic

heterotrophs like D. papillatum, this preference likely reflects nutritional adaptation.

(d) Gluconeogenesis is the central carbon metabolism in D. papillatum

To investigate the metabolic fate of amino acids, we performed tracer experiments using 6 mM

13C-uniformly labeled (13C5, +5 carbons) L-glutamine, and D. papillatum cells which had been

starved in artificial seawater for 2 hours prior to labeling. We detected specific enrichment with

+5 carbon in the α-ketoglutarate fraction, indicating that glutamine is readily catabolized and

incorporated into metabolites of the TCA cycle of D. papillatum (figures 3b and S7a). The

presence of +5 and +6 carbons in the citrate fraction also indicated an active TCA cycle and the

recycling of labeled oxaloacetate. The time-dependent increase of 13C-labeling with +1 to +6

carbons for hexose phosphates provided evidence of active gluconeogenesis. In addition, the

extensive metabolic dilution of 13C-isotopologues in the hexose 6-phosphate fractions indicated

the presence of an internal carbon source. Quantitative metabolomics demonstrated that D.

papillatum contains higher amounts of free amino acids when compared to T. cruzi, suggesting

that they may serve as a carbon reservoir (table S2). We conclude that amino acids are the

preferred carbon/energy source for D. papillatum and that they are catabolized via the TCA cycle

and can subsequently be anabolized via gluconeogenesis.

(e) The pentose phosphate pathway complements PFK-deficiency in the glycolytic process

Metabolic labeling using 6 mM 13C6-D-glucose as a sole external carbon source showed

enrichment with +1 to +5 carbons in the hexose phosphate fractions, indicating the occurrence of

glucose catabolism in D. papillatum at a very low rate (figures 3c and S7b). Importantly,

12

sedoheptulose 7-phosphate (S7P), an intermediate metabolite of PPP, was significantly labeled

with +1 to +6 carbons (figure 3c). Glucose uptake by D. papillatum was further investigated by

cells incubated for 72 hrs in normal medium containing 30 mM 13C6-glucose. Under these

conditions, the levels of +6 carbon were significantly lower than that of +3 carbon in the hexose

phosphate fractions (figure S7c). These data suggest that G6P is catabolized via the PPP to

produce triose phosphates and their subsequent catabolites, which can then be recycled to form

hexose phosphate species. Quantitative determination of the glycolytic metabolites also showed a

relative accumulation of hexose phosphates in D. papillatum compared to T. cruzi, regardless of

the nutritional conditions (table S2), suggesting the predominance of anabolism towards the

production of hexose phosphates. Taken together, these data demonstrate that glycolysis does not

significantly contribute to central carbon metabolism in D. papillatum and that gluconeogenesis

from amino acids is the dominant process.

(f) Retargeting of non-glycolytic pathway enzymes to peroxisomes occurred in the common

ancestor of kinetoplastids

To investigate the evolution of kinetoplastid glycosomes after the split from the diplonemid

lineage, we inferred additional phylogenies for PTS-harboring enzymes specific to kinetoplastids.

Both the phylogeny of adenylate kinase – which catalyzes the reversible interconversion of 2

ADP into ATP plus AMP – and of inosine 5-monophosphate dehydrogenase – a purine salvage

enzyme – revealed that the glycosomal forms of these enzymes have been acquired laterally from

bacteria in kinetoplastids (figures S8 and S9). Thus, kinetoplastids encode two copies of these

genes: an ancestral copy shared with D. papillatum, and a glycosomal copy obtained by LGT.

The phylogeny of a PPP enzyme, transketolase, showed monophyly of D. papillatum and

13

kinetoplastids within the eukaryotic clade, indicating that the ancestral forms of this enzyme were

retargeted to the glycosomes in the lineage leading to kinetoplastids (figure S10). The phylogeny

of other PTS-targeted enzymes was ambiguous, largely due to a lack of resolution commonly

encountered in single-gene trees. We conclude that the transfer to the glycosomes of non-

glycolytic enzymes, including those for pyrimidine biosynthesis [21], occurred on the branch

leading to the kinetoplastids after their split from the diplonemid lineage (figure 4).

4. Discussion

Recent advances in our sampling of microbial eukaryotic diversity have begun to shed light on

the unique biological features of diplonemids, not only as a relative of important human

pathogens but also as a major protistan group in ocean plankton [22, 23]. The ecological success

of diplonemids as marine plankton [23] suggests that independence from glucose accompanied

by peroxisome remodelling is a viable strategy for heterotrophic life. Our draft genome

sequencing of Diplonema provides an important source of genomic data for this group.

The phylogeny of PFK revealed that kinetoplastid and E. gracilis PFKs share the same

evolutionary origin. Therefore, it is likely that, as well as euglenoids, a common ancestor of

diplonemids and kinetoplastids utilized PFK to perform glycolysis. Whether this ancestral PFK

had already relocated to peroxisomes in their common ancestor is, as yet, unclear. By contrast, D.

papillatum PFK2 shares the same origin with a PFK homologue of the haptophyte, E. huxleyi. E.

huxleyi is the most prominent marine cosmopolitan phytoplankton and often causes

coccolithophore blooms [24]. It is possible that marine heterotrophs like Diplonema feed on

14

haptophytes, which may have been an evolutionary source of LGT; alternatively, both E. huxleyi

and D. papillatum may have obtained these PFK genes by LGT from other members of clade B.

Our data demonstrate that the remodelling of peroxisome function already occurred in the

common ancestor of diplonemids and kinetoplastids, and involved the retargeting of at least a

subset of glycolytic enzymes and GK and PPDK. It is unclear whether FBPase also relocated to

peroxisomes in their common ancestor, because both FBPases have different origins. Retargeting

to peroxisomes of ATP-producing GK and PPDK may be related to the requirement of ATP

hydrolysis in the first seven enzymatic steps of glycolysis, in terms of the maintenance of ATP

homeostasis inside the peroxisomes. That is, the maintenance of the ATP levels by the action of

GK, acting in the reverse direction, and PPDK in the peroxisomes might have played an

important role in maintaining the efficiency of glycolysis and gluconeogenesis in these

organisms.

As with enzymatic composition of the ancestral glycosomes, the primary metabolic status

in the common ancestor of diplonemids and kinetoplastids is still unclear. Our metabolome data

indicate that D. papillatum lacks a fully functional glycolytic pathway. By contrast, our

phylogenetic analyses suggest that the ancestor of diplonemids and kinetoplastids likely retained

glycolysis. A possible explanation is that the gluconeogenic state of Diplonema may not be an

ancestral, but is instead a derived feature, which was accompanied by nutritional adaptations to

heterotrophic life followed by the loss of expression and activity of PFK.

From a metabolic viewpoint, it seems rational that ancestral glycosomes harbored the first

seven enzymes of glycolysis and the corresponding enzymes of gluconeogenesis for completion

of a contiguous enzymatic cascade (figure 4). D. papillatum may represent an interesting case

whereby nutritional adaptations leading to glucose-independent metabolism have evolved,

15

resulting in peroxisomes housing gluconeogenesis (“gluconeosomes”). At the same time,

preservation of glycolytic activity in kinetoplastids might have potentiated adaptation to a

different mode of life, parasitism. Parasitism has developed repeatedly among kinetoplastids, and

this adaptability is made possible by the compartmentalisation into glycosomes of metabolic

processes that have to undergo drastic and rapid changes during some transitions in the complex

life cycles [25]. Hence, the sequestering of core metabolic pathways into peroxisomes, begun in

the common ancestor of diplonemids and kinetoplastids, may have laid the foundations for the

success of a major clade of medically and economically important parasites. Indeed, high levels

of glucose (in the millimolar range) are present in the blood and body fluids of vertebrates, which

can be readily utilized by hemoflagellates as carbon source.

Data accessibility. The D. papillatum genome was deposited in DDBJ/EMBL/GenBank under

the accession LMZG00000000. D. papillatum sequences for relevant enzymes reported here have

been deposited individually at DDBJ (accession numbers AB970479 - AB970481, AB970483 -

AB970501, LC127115, LC127507). The datasets supporting this article have been uploaded as

part of the supplementary material.

Competing interest. We have no competing interests.

Author contributions. T.N. designed research; J.M., M.H., T.A.W., H.H.-M., A.T., N.K., H.T.,

T.F., M.K., and T.N. performed research; J.M., T.A.W. and T.N. analyzed data; F.B. and J.L.C.

provided materials; T.A.W., T.Ma., T.Mi., F.B., J.L.C, and T.H. helped to draft manuscript;

T.A.W. and T.M.E edited the paper; and J.M. and T.N. wrote the paper.

16

Acknowledgements. We thank Paul Michels for critical reading and the Euglena gracilis

nucleotide sequencing consortium (http://euglenadb.org/) for E. gracilis PFK sequence.

Funding. T.N. acknowledges support from grants-in-aid for scientific research (24390102 and

25670205) and from the Foundation of Strategic Research Projects in Private Universities

(S1201013) from the Ministry of Education, Culture, Sport, Science, and Technology, Japan

(MEXT). T.M.E. acknowledges support from the Wellcome Trust and the European Advanced

Investigator Programme. T.A.W. is supported by a Royal Society University Research

Fellowship.

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20

Table 1. Characteristics of glycolytic enzymes of Diplonema papillatum (Dp).

Figure Legends

Figure 1. Diplonema papillatum lacks an orthologue of the glycosome-targeted

phosphofructokinase (PFK) gene found in other euglenozoans. A Bayesian consensus phylogeny

of PFK was inferred under the C60 model in PhyloBayes. E. gracilis and kinetoplastid PFKs

form a monophyletic group (clade A) with high posterior support (PP = 0.99). By contrast, D.

papillatum PFK2 clusters with a PFK homologue from the haptophyte Emiliania huxleyi (PP = 1)

and is nested within the clade “B” (PP = 0.99). Branch lengths are proportional to the expected

number of substitutions.

Figure 2. Cellular localisation of glycolytic enzymes in Diplonema papillatum using

immunofluorescence microscopy. Cells were labeled with polyclonal antisera to GLK, PGI, TIM,

GAPDH, PGK, PGAM, ENO, or PK (Target, red) and with antisera to D. papillatum FBPA as a

peroxisomal marker (FBPA, green). Images of the same cells were merged together with the

labels of Hoechst 33342 (blue) to visualize the nucleus (Merge). DIC, differential interference

contrast image; Merge, merged image. Scale bar = 10 µm.

Figure 3. Carbon metabolism in Diplonema papillatum. (a) Consumption of glucose and amino

acids in the culture media of D. papillatum (left) and Trypanosoma cruzi epimastigotes (right).

Production of ammonium, a catabolite of amino acids, denotes the consumption of amino acids.

(b and c) Metabolome analysis of D. papillatum. D. papillatum cells were incubated in

21

conditioned marine water for 2 hrs and then supplemented with 13C5-L-glutamine (b) or 13C6-D-

glucose (c) for 0, 4, and 8 hrs. The Y-axis indicates the relative abundance (%) of each

isotopomer of the relevant metabolite. The X-axis represents the number of 13C in the

isotopomers. Data for all metabolites in these experiments are shown in figures S7a and S7b,

respectively. Data points and error bars represent the mean ± s.d. of three independent

experiments.

Figure 4. A proposed metabolic model and peroxisome remodelling in Euglenozoa. (a)

Peroxisomes (Px) and mitochondria (Mt) have been present in the last eukaryotic common

ancestor (LECA), with cytosolic location of glycolysis (blue arrows), gluconeogenesis (red

arrows), pentose phosphate pathway (PPP), pyrimidine biosynthesis (Pyr), and purine salvage

(Pur). PFK and FBPase (FBP) are critical allosteric enzymes in glycolysis and gluconeogenesis,

respectively. (b) After the divergence of the lineage leading to Euglenoids, the peroxisomes of the

common ancestor of diplonemids and kinetoplastids were remodelled through the sequestration

of a subset of glycolytic enzymes, possibly FBPase, and others (boxed in yellow). In these

remodelled compartments, both glycolysis and gluconeogenesis were functional. (c) The

ancestral diplonemids or Diplonema lost glycolysis accompanied by the loss of PFK activity as

suggested in our analysis. (d) The ancestral kinetoplastid further sequestered into peroxisomes a

part of enzymes for PPP, Pyr, Pur, and others such as PEPCK and NADH-fumarate reductase

(boxed in blue).

22

Table 1. Characteristics of glycolytic enzymes of Diplonema papillatum.

EnzymePTS

Monophyly* Activity†D. papillatum T. cruzi

1. HK PTS2 PTS2 Yes (0.85)0.92‡

1. GLK PTS1 PTS1 Yes (0.96)2. PGI PTS1 PTS1 Yes (0.93) 39.13. PFK PTS1/ND PTS1 No (0.99) ND3. FBPase (gluconeogenic) PTS1 PTS1 No (1.00) 4.74. FBPA PTS2 PTS2 Yes (1.00)§ 14.25. TIM PTS1 ND Unresolved 123.26. GAPDH ND PTS1/ND No (0.90) 48.57. PGK PTS1 PTS1/ND Yes (0.81) 103.78. PGAM ND ND No (1.00) 2,1009. ENO ND ND Yes (0.99) 3.710. PK ND ND Yes (0.98) 15.1

*Monophyly of D. papillatum and kinetoplastids with posterior probability in parenthesis based on the relevant protein phylogeny (figures 1 and supplementary figures).

†nmol/min/mg protein of the D. papillatum cell extract (see also supplemental material). ‡The activities of HK (high-affinity type) and GLK (low-affinity type) are indistinguishable. §Ref. 12. ND; not detected.

Euglenozoa

Euglenozoa

Euglenozoa(Excavata)

Bacteria

Bacteria

Bacteria

Bacteria

Bacteria

Bacteria

Bacteria

Bacteria

Euglenozoa (Excavata)

Archaea

Archaea

Methanocella paludicola SANAERheinheimera nanhaiensis E407-8

Alteromonas macleodii str. Deep ecotypeAlteromonas macleodii

Methanospirillum hungatei JF-1Methanoculleus marisnigri JR1

Magnetococcus marinus MC-1Trichomonas vaginalis G3 (Excavata)

Saccharomyces cerevisiae S288cSaccharomyces cerevisiae AWRI1631Neurospora crassa

Haemonchus contortusLactococcus lactis

Succinivibrionaceae bacterium WG-1Oceanimonas sp. GK1

Haemophilus sputorum CCUG 13788Haemophilus ducreyi 35000HP

Actinobacillus ureaeBuchnera aphidicola (Cinara tujafilina)

Escherichia coliThalassiosira pseudonana CCMP1335

Phaeodactylum tricornutumAureococcus anophagefferens

Guillardia theta CCMP2712Emiliania huxleyiCoraliomargarita akajimensis

Agrobacterium tumefaciens F2Sinorhizobium fredii

Bodo saltans PFK1Trichomonas vaginalis (Excavata)

Trichomonas vaginalis G3Trichomonas vaginalis G3

Trichomonas vaginalisPelosinus fermentans DSM 17108

Naegleria gruberi strain NEG-MNaegleria fowleri (Excavata)

Syntrophobacter fumaroxidansAccumulibacter phosphatis

Desulfobacca acetoxidansDesulfobacter postgatei 2ac9

Desulfobacterium autotrophicumUncultured Desulfobacterium sp.Desulfococcus oleovorans

Diplonema papillatum PFK1Waddlia chondrophila 2032/99

Paramecium tetraurelia strain d4-2Toxoplasma gondii ME49

Plasmodium falciparum 3D7Cryptosporidium hominisToxoplasma gondii GT1

Plasmodium falciparum 3D7Cryptosporidium hominis

Ectocarpus siliculosusOryza sativa Japonica Group

Arabidopsis thalianaArabidopsis thaliana

Toxoplasma gondii ME49Toxoplasma gondii

Oryza sativa Japonica GroupOryza sativa

Oryza sativa Japonica GroupOryza sativaArabidopsis thalianaArabidopsis thaliana

Giardia intestinalis (Excavata)Treponema phagedenis F0421

Treponema pallidumTreponema caldaria

Solobacterium moorei F0204Holdemanella biformis

Spirochaeta thermophila DSM 6578

Entamoeba histolyticaBorellia burgdoferi

Entamoeba histolytica (Amoebozoa)Monocercomonoides sp. (Oxymonadida, Excavata)

Euglena gracilisTrypanosoma cruziTrypanosoma brucei

Leishmania major strain FriedlinCrithidia fasciculata

Bodo saltans PFK2Nakamurella multipartita

Emiliania huxleyi CCMP1516 (Haptophyta)Diplonema papillatum PFK2

Phaeodactylum tricornutum CCAP 1055/1 (Stramenopiles)Phaeodactylum tricornutum (Stramenopiles)

Ectocarpus siliculosus (Stramenopiles)Ectocarpus siliculosus (Stramenopiles)Chlamydomonas reinhardtii (Plantae)

Tetrahymena thermophila SB210 (Alveolata)Paramecium tetraurelia (Alveolata)

Arabidopsis thaliana (Plantae)Arabidopsis thaliana (Plantae)

Nicotiana tomentosiformis (Plantae)

1

0.78

0.991

0.99

0.52

0.99

0.991

1

0.55

0.57 1

0.930.99

1

0.53

0.99

0.93

10.92

0.971 1 1

0.99

0.99

0.731

0.990.98

1

1

0.730.98

11

1

0.99

0.78

0.5

0.94

0.92

0.99

0.920.91

0.87

1

0.71

0.9511

0.98

0.93

10.81

10.99

1

0.96

0.991

0.7

1

0.970.92

0.99

0.91

0.970.99

11

1

1

0.64

0.99

1

0.59

1

0.99

1

0.98

1

0.99 10.2

Clade A

Clade B

Bacteria

DIC FBPA Target Merge

PGI

TIM

PGK

GAPDH

GLK

PGAM

ENO

PK

Time (days) Time (days)

0

15

10

5

2 84 106

Con

cent

ratio

n (m

M)

104

105

106

107

Cel

l cou

nt (/

ml,

----

)D. papillatum T. cruzi

10

10

10

Cel

l cou

nt (/

ml,

----

)

Glucose 6-phosphate

0 hr 4 hr 8 hr

20

10

15

0

5

20

10

30

0

40

20

50

30

0

10

(c)R

elat

ive

abun

danc

e (%

)R

elat

ive

abun

danc

e (%

)20

10

15

0

5

(b)

+1

20

10

15

0

5

0 hr 4 hr 8 hr

20

10

15

0

5

Sedoheptulose7-phosphate

Glucose 6-phosphate

Fructose 6-phosphate

Citrateα-Ketoglutarate

(a)

Rel

ativ

e ab

unda

nce

(%)

Rel

ativ

e ab

unda

nce

(%)

– – Glucose – – Ammonium 0

5

10

15

Con

cent

ratio

n (m

M)

+1 +2 +3 +4 +5 +6 +2 +3 +4 +5

+1 +2 +3 +4 +5 +6 +1 +2 +3 +4 +5 +640

+1 +2 +3 +4 +5 +6

+1 +2 +3 +4 +5 +6

2 84 106

+7

6

7

8

– – Glucose – – Ammonium

Diplonemids(D. papillatum) Kinetoplastids

Euglenoids

LECA

Mt

Glucose

Glucose-P

PEP

Amino acids

PPP

Pyr

Pur

PFKFBP

Px

(a)

Px

Glucose

PFK

PPP

Pyr

Mt

Glucose-P

PEP

PPDKGK

FBP

Amino acids

Pur

(c)

Px

PPP

Pyr

Pur

Mt

Glucose-P

PEP

Amino acids

PFKFBP

PPDKADK

GK

Glucose

PEPCK

(d)

Px

PPP

Pyr

Mt

Glucose-P

PEP

PPDKGK

Amino acids

Pur

(b)

PFKFBP

Glucose

Differential remodelling of peroxisome function underpins the environmental

and metabolic adaptability of diplonemids and kinetoplastids

Jorge Morales, Muneaki Hashimoto, Tom A. Williams, Hiroko Hirawake-Mogi, Takashi

Makiuchi, Akiko Tsubouchi, Naoko Kaga, Hikari Taka, Tsutomu Fujimura, Masato Koike,

Toshihiro Mita, Frédéric Bringaud, Juan L. Concepción, Tetsuo Hashimoto, T. Martin Embley

and Takeshi Nara

Supplementary material

Supplementary materials and methods

(a) Enzyme activity

Cells of the mid-log phase were collected, washed twice with 10% (v/v) mannitol and

resuspended in 50 mM Tris-HCl/2 mM EDTA pH 7 with protease inhibitor cocktail (Complete,

Mini, Roche Diagnostics K.K., Tokyo, Japan). Cells were disrupted by sonication, centrifuged at

26,000 x g for 20 minutes, and the supernatant was used to measure the enzymatic activity

described as follows; hexokinase and glucokinase [1], phosphoglucose isomerase [2],

phosphofructokinase [3], fructose-1,6-biphosphate aldolase [4], GAPDH [5], triose-phosphate

isomerase [6], phosphoglycerate kinase [7], phosphoglycerate mutase [8], enolase [9], pyruvate

kinase [10], and fructose-1,6-biphosphatase [11].

(b) Antibodies

The mouse antiserum to D. papillatum FBPA was produced as described [12]. To obtain antisera

to glycolytic enzymes, the cDNA clones of interest were obtained by PCR, cloned in pET151/D-

TOPO vector, and expressed in BL21(DE3)Star cells (Life Technologies Japan Inc., Tokyo,

Japan). The recombinant proteins were purified using His•Bind® Resin (Merck KGaA,

Darmstadt, Germany) and used for raising antisera in rabbits or mice. Immunization of animals

and collection of antisera were in part carried out by Medical & Biological Laboratories Co., Ltd.,

Nagoya, Japan. All animal experiments were carried out in accordance with the fundamental

guidelines for animal experiments under the jurisdiction of the Ministry of Education, Culture,

Sports, Science and Technology (Notice No. 71, 2006), and approved by the Committee for

Animal Experimentation of Juntendo University with the Approval No. 270081.

(c) Western blots

20 µg of protein from the cell supernatant were separated onto a SuperSep® Ace 10-20 % gel

(Wako Pure Chemical Industries, Ltd., Osaka, Japan) and transferred to a polyvinylidene fluoride

membrane under semi-dry conditions at 20 V for 30 minutes. After blocking with 5% fetal

bovine serum (FBS) in phosphate-buffered saline containing 0.25 % tween20 (PBST), the

membranes were incubated overnight at 4°C with the antisera to each glycolytic enzyme in 1%

FBS/PBST, washed with PBST, and incubated with HRP-conjugated secondary antibody

(Jackson ImmunoResearch Laboratories Inc., PA), followed by chemiluminescent development.

The reaction was visualized using ImageQuant LAS-4000 min (GE Healthcare Japan Inc., Tokyo,

Japan).

(d) Electron microscopy

Cells of the late-log phase were harvested, washed twice in artificial marine water, and fixed as

described [13]. Ultrathin sections were stained with lead citrate and uranyl acetate and examined

with a transmission electron microscope at 75 kV (Hitachi H-7100, Hitachi High-Technologies

Co. Ltd., Tokyo, Japan). For immunoelectron microscopy, cells were fixed using 4%

paraformaldehyde and 1% glutaraldehyde in PBS at 4ºC for 2 hrs, dehydrated with ethanol, and

embedded in LRWhite at -20ºC for 4 days under UV-light. Ultrathin sections were incubated

with 50 mM NH4Cl in PBS for 2 hours, blocked with 3% BSA in PBS/0.025 % tween20 for 1 hr,

and incubated with antisera to D. papillatum FBPA [12] for 1 hr. Cells were then incubated with

gold-labeled anti-mouse antibody (10 nm particle, Sigma-Aldrich Co. LLC., MO), counterstained

with uranyl acetate, and observed using Hitachi H-7100.

(e) Metabolic labeling

The mid-log phase of cells were starved in artificial seawater for 2 hrs and then incubated with 6

mM 13C6 D-glucose or 6 mM 13C5 L-glutamine (Sigma-Aldrich) in artificial seawater for 0, 4,

and 8 hrs or with 30 mM 13C6 D-glucose in normal culture medium for 72 hrs without starvation.

Cells in the mid-log phase of growth were starved in artificial seawater for 2 hrs and then

incubated with 6 mM 13C6 D-glucose or 6 mM 13C5 L-glutamine (Sigma-Aldrich) in artificial

seawater for 0, 4, and 8 hrs or with 30 mM 13C6 D-glucose in normal culture medium for 72 hrs

without starvation. Cells were then washed twice with 10% w/v mannitol and treated with

methanol by vortexing for 1 min. After removing the debris by centrifugation, the supernatant

was extracted with 1:0.4 of chloroform and milli-Q water by vortexing for 1 min. The aqueous

phase was filtered through an Ultrafree MC PHLCC HMT filter (Merck KGaA, Darmstadt,

Germany) having a cut-off of 5 kDa at 9,400 x g for 2 hrs to remove proteins. The eluate

containing the metabolites was evaporated and dissolved in 0.05 ml of Milli-Q water. CE-Q-

TOFMS analysis was performed using an Agilent CE system combined with a Q-TOFMS

(Agilent Technologies, Palo Alto, CA, USA) as described [14-16]. The data obtained by CE-Q-

TOFMS analysis were preprocessed using MassHunter workstation software Qualitative Analysis

Version B.06.00. Each metabolite was identified and quantified based on the peak information

including m/z, migration time, and peak area. The quantified data were then evaluated for

statistical significance by a Wilcoxon signed-rank test. Alternatively, liquid chromatography-

coupled mass spectrometry (LC-MS) was performed on a TSQ Quantum Ultra AM with the

analysis software Xcalibur 2.0.6 software (Thermo Fisher Scientific, San Jose, CA) coupled to a

Gilson-HPLC system and a Gilson 234 autosampler (Gilson Inc., WI) as described [17].

(f) Metabolome analysis

Metabolome measurements were carried by Human Metabolome Technology Inc., Tsuruoka,

Japan. Briefly, cells were collected in mid-log phase, washed twice with 10% mannitol, and

resuspended in methanol containing internal standards (H3304-1002, Human Metabolome

Technologies, Inc., Tsuruoka, Japan) followed by ultrasonication, to inactivate enzymes. The

metabolites were further extracted and finally dissolved in 50 µL of Milli-Q water for CE-MS

analysis.

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Table S1. Predicted PTS in non-glycolytic enzymes of Diplonema papillatum.

Enzyme PTS*

D. papillatum T. brucei

Gluconeogenesis

Malate dehydrogenase (MDH), glycosomal ND PTS1 Phosphoenolpyruvate carboxykinase (PEPCK) ND PTS1

Pentose phosphate pathway 6-phosphogluconolactonase ND PTS1 Ribokinase ND PTS1 Transketolase ND PTS1

Energy/redox metabolism

Adenylate kinase ND PTS1 Glycerol 3-phosphate dehydrogenase ND PTS1 Glycerol kinase (GK) PTS1 PTS1 Isocitrate dehydrogenase ND PTS1 NADH-dependent fumarate reductase ND PTS1 Pyruvate phosphate dikinase (PPDK) PTS1 PTS1

Pyrimidine biosynthesis/purine salvage

Adenine phosphoribosyltransferase ND PTS1 Guanylate kinase ND PTS1 Hypoxanthine-guanine phosphoribosyltransferase ND PTS1 Inosine-5'-monophosphate dehydrogenase ND PTS1 Orotate phosphoribosyltransferase (OPRT) ND PTS1† Orotidine monophosphate decarboxylase (OMPDC) ND PTS1†

*PTS1 represents being double positive for both the C-terminal consensus tripeptides, (STAGCN)-(RKH)-(LIVMAFY), and a category of “targeted” or “twilight zone” using the PTS1 predictor (see materials and methods). †Fused in OMPDC-OPRT. ND; No PTS detected.

Table S2. Metabolites of carbon metabolism in Diplonema papillatum. Metabolites (pmole/108 cells) T. cruzi D. papillatuma D. papillatumb D. papillatumc Glycolysis Hexose phosphate group

Glucose 6-phosphate 161 21,166 37,182 6,168 Glucose 1-phosphate 77 4,393 5,773 1,435 Fructose 6-phosphate 348 7,895 13,324 2,739 Fructose 1,6-bisphosphate 158 3,490 2,203 769

Triose phosphate group

Dihydroxyacetone phosphate 129 11,779 5,944 2,600 Glyceraldehyde 3-phosphate ND 186 ND ND 3-Phosphoglyceric acid 1,707 4,080 4,442 1,003 2-Phosphoglyceric acid 267 638 777 189 Phosphoenolpyruvic acid 1,036 564 903 ND

Ratio of total hexose-P:triose-P 744:3149

(1:4.2) 36944:17247

(2.1:1) 58482:12066

(4.8:1) 11111:3792

(2.9:1) TCA cycle

Citric acid 330 1,705 894 401 cis-aconitic acid ND 924 532 237 Isocitric acid ND 70,382 53,107 21,083 α-ketoglutaric acid ND 4,397 4,412 3,180 Succinic acid 35,203 108,395 113,315 22,092 Fumaric acid 126 6,454 2,204 1,492 Malic acid 535 22,612 15,545 9,739

Pentose phosphate pathway

6-Phosphogluconic acid 21 73 ND ND Ribulose 5-phosphate 144 1,859 1,906 511 Ribose 5-phosphate 38 585 638 128 Sedoheptulose 7-phosphate 282 1,729 5,199 443 Erythrose 4-phosphate ND 572 353 ND

Nucleotides

ATP 8,578 6,944 9,481 1,247 ADP 8,010 19,800 19,395 6,637 AMP 8,028 50,665 23,739 14,455 cAMP 15 ND ND ND GTP 2,264 1,228 2,116 607 GDP 2,196 4,572 4,143 2,223 GMP 1,208 6,643 3,884 3,248 UTP 4,974 1,984 2,259 290 UDP 5,083 4,847 4,626 1,502 UMP 5,571 11,936 5,445 2,657 CTP 583 139 400 ND CDP 360 302 528 280 CMP 457 2,560 2,407 1,424

a ATCC® 1405 artificial seawater supplemented with 10 % v/v horse serum. b ATCC® 1532 medium. c ATCC® 1532 medium supplemented with 6 mM D-glucose. ND, not detected.

Table S2. Continued. Metabolites (pmole/108 cells) T. cruzi D. papillatuma D. papillatumb D. papillatumc Others

Pyruvic acid 126 1,372 2,351 1,820 Lactic acid 11,800 82,962 84,245 20,330

Amino acids β-Ala 147 1,191,232 Glu 14,748 842,686 Lys 20,288 453,660 Pro 17,256 423,095 Ala 212,561 367,248 Gln 86 317,801 Arg 11,335 217,878 Gly 99,373 137,363 Val 36,898 134,090 His 200 109,362 Ser 507 98,449 Thr 338 85,611 Leu 3,968 78,799 Tyr 929 73,437 Asn 1,700 53,480 Asp 1,578 51,883 Ile 15,529 35,984 Phe 808 35,481 Trp 290 15,796 Met 244 12,534 Cys ND 569

a ATCC® 1405 artificial seawater supplemented with 10 % v/v horse serum. b ATCC® 1532 medium. c ATCC® 1532 medium supplemented with 6 mM D-glucose. ND, not detected.

Euglenozoa

Bacteria

Ruegeria sp.Micromonas pusilla

Geobacter sp.Synechococcus sp.Microcoleus chthonoplastes

Sideroxydans lithotrophicusXanthomonas albilineans

Metaseiulus occidentalisAcanthamoeba castellanii

Trichomonas vaginalis ATrichomonas vaginalis B

Naegleria gruberiTrypanosoma cruzi

Leishmania majorDiplonema papillatum

Dictyostelium discoideum

0.96

0.66

0.97

0.82 0.91

1

0.62

0.99

0.96 1

0.2

(a) Hexokinase (b) Glucokinase

Euglenozoa

Euglenozoa

BacteriaArchaea

Bacteria

Mus musculusHomo sapiens

Drosophila melanogasterDictyostelium discoideumSaccharomyces cerevisiae

Neurospora crassaDiplonema papillatum

Phaeodactylum tricornutumTrichomonas vaginalis

Naegleria gruberiTrimastix pyriformis

Giardia lambliaEctocarpus siliculosus

Cyanidioschyzon merolaeTrypanosoma cruzi

Trypanosoma bruceiLeishmania major

Tetrahymena thermophilaParamecium tetraurelia

Toxoplasma gondiiPlasmodium falciparum

Cryptosporidium parvumOryza sativa

Chlamydomonas reinhardtiiArabidopsis thaliana

Marinomonas sp. MED121Lautropia mirabilis

group III euryarchaeote KM3-28-E8Magnetococcus marinusGeobacter daltonii

Lactobacillus helveticusTrichodesmium erythraeum

Thermodesulfatator indicusPatulibacter sp. I11

0.58

0.81

0.5

0.96

0.54

0.71

0.521 0.98

0.78

1

0.92 0.640.94

10.7

0.97

0.99 0.5 0.91

0.2

(d) Triosephosphate isomerase

Euglenozoa

Bacteria

Bacteria

BacteriaBacteria

Bacteria

Streptomyces sp.Gloeobacter violaceus

Trypanosoma cruziTrypanosoma brucei

Leishmania majorDiplonema papillatum

Saccharomyces cerevisiaeNeurospora crassa

Aspergillus nidulansSyntrophobacter fumaroxidans

Dictyostelium discoideumDesulfovibrio africanusDrosophila melanogaster

Mus musculusHomo sapiens

Methylotenera mobilisTrimastix pyriformis

Magnetococcus marinusPhaeodactylum tricornutumEntamoeba histolytica

Fibrisoma limiEscherichia coli

Photobacterium damselaeVibrio metschnikoviiVibrio mimicusVibrio cholerae

Chloroherpeton thalassiumChloroherpeton thalassium

Ectocarpus siliculosusChlamydomonas reinhardtii

Oryza sativaArabidopsis thaliana

Toxoplasma gondiiToxoplasma gondii

Tetrahymena thermophilaParamecium tetraureliaParamecium tetraurelia

Plasmodium falciparumCryptosporidium hominisCryptosporidium parvum

Thalassiosira pseudonanaCyanidioschyzon merolae

1

0.63

0.58

0.93 1

0.98

0.7

11

0.991

0.7

0.81

0.96

1 11

1

1

0.591

0.99

1

1

0.67

11

0.67 1

0.980.2

(c) Phosphoglucose isomerase

Euglenozoa

Bacteria

Bacteria

Trimastix pyriformisToxoplasma gondii

Plasmodium falciparumCryptosporidium hominis

Trypanosoma bruceiTrypanosoma cruzi

Leishmania majorDiplonema papillatum

Chlamydomonas reinhardtiiMus musculus

Drosophila melanogasterSaccharomyces cerevisiae

Neurospora crassaTreponema primitiaDysgonomonas gadeiBacteroides sp.

Arabidopsis thalianaOryza sativa

Pelosinus fermentansDesulfotomaculum gibsoniae

Clostridium cellulolyticum

0.54

0.88

10.74

0.76

0.850.99

0.94

0.99

0.7

0.64

0.98

0.930.99

11

0.99

0.950.2

Bacteria

Euglenozoa

Euglenozoa

BacteriaArchaea

Bacteria

Sorangium cellulosum So ce 56Methylococcus capsulatuss tr.Bath

Dechloromonas aromatica RCBHoldemania filiformis DSM12042

Giardia lamblia ATCC50803Entameoba histolytica

Trimastix pyriformisDiplonema papillatum

Chlamydomonas reinhardtiiTrypanosoma cruzi Trypanosoma brucei

Leishmania majorArabidopsis thaliana

Oryza sativa Japonica GroupSlackia piriformis YIT12062

Silicibacterlacus caerulensis ITI-1157Methanosaeta harundinacea 6Ac

Trichomonas vaginalis G3Encephalitozoon cuniculi

Aspergillus nidulansNeurospora crassa OR74A

Fischerella sp. JSC-11

0.67 0.97

0.98

0.99

1

0.67

0.98

11

0.99

0.98

0.540.63

0.580.9

0.2

(e) Phosphoglycerate mutase

Euglenozoa

Bacteria

BacteriaArchaea

Paramecium tetraureliaEntamoeba histolytica

Encephalitozoon cuniculiDictyostelium discoideumCyanidioschyzon merolae

Arabidopsis thalianaToxoplasma gondii

Toxoplasma gondii ME49Plasmodium falciparum

Ustilago maydisSaccharomyces cerevisiae S288c

Aspergillus nidulansNeurospora crassa OR74A

Mus musculusMus musculusHomo sapiens

Drosophila melanogasterGiardia lamblia

Trimastix pyriformisTrypanosoma cruzi Trypanosoma brucei bruceiLeishmania major

Diplonema papillatumDictyostelium discoideum AX4

Cryptosporidium parvumCryptosporidium hominis TU502Chlamydomonas reinhardtii

Thalassiosira pseduonanaPhaeodactylum tricornutum

Ectocarpus siliculosusArabidopsis thaliana

Oryza sativaNaegleria gruberi

Trichomonas vaginalisTrichomonas vaginalis

Methanococcus voltaeA3Thermofilum pendens Hrk5

Korarchaeum cryptofilumScardovia wiggsiaePelobacter carbinolicus

Lactobacillus plantarumMagnetococcus marinus MC-1

Euglena gracilisEuglena gracilis

Glaciecola sp. 4H-3-7+YE-5Dechloromonas aromatica

Microcystis aeruginosaCandidatus Caldiarchaeum subterraneum

0.91

0.690.99

0.5

0.971

1

0.73

0.75

0.92

0.80.75

0.9910.99

1

0.99

0.910.97 0.99

0.99

11

0.570.97

0.88

0.75

0.90.53

0.860.76

1

0.610.2

Archaea

Euglenozoa

(f) Enolase

Figure S1. Consensus phylogenetic trees for glycolytic enzymes. (a) Hexokinase; (b) glucokinase; (c) phophoglucose isomerase; (d) triose phosphate isomerase; (e) phophoglycerate mutase; and (f) enolase. Euglenozoa, bacteria and archaea are labeled in green, brown, and gray, respectively. The detailed methods to reconstruct the phylogenetic trees are described in the materials and methods.

Archaea

Archaea

Bacteria

Bacteria

Bacteria

Euglenozoa

Euglenozoa

Bacteria

Euglenozoa

Bacteria

Bacteria

Bacteria

Methanosarcina barkeri str.FusaroMethanosaeta harundinacea 6Ac

Methanocella conradii HZ254Methanocella conradii HZ254

uncultured marine crenarchaeote HF4000 ANIW93H17Cenarchaeum symbiosum A

Candidatus Nitrosoarchaeum limnia SFB1Nitrosopumilus maritimus SCM1Candidatus Nitrosopumilus salaria BD31

Candidatus Nitrosoarchaeum koreensis MY1Candidatus Caldiarchaeum subterraneum

Vulcanisaeta distributa DSM14429Candidatus Korarchaeum cryptofilumOPF8

Metallosphaera cuprina Ar4Sulfolobus solfataricusSulfolobus islandicus REY15A

Caldivirga maquilingensis IC167Ignicoccus hospitalis KIN4I

Aeropyrum pernix K1Acidilobuss accharovorans 34515

Thermofilum pendensHrk5Encephalitozoon cuniculi

Phaeodactylum tricornutumCCAP10551Pelobacter carbinolicus DSM2380

Methanoculleus marisnigri JR1Methanoculleus bourgensis MS2

Methanosphaerula palustris E19cMethanoregula boonei 6A8

Methanocella paludicola SANAEMethanocella arvoryzae MRE50

Magnetococcus marinus MC1Trypanosoma cruzi strain CL BrenerEctocarpus siliculosus

Gloeobacter violaceus PCC7421Euglena gracilis

Trypanosoma cruzi strain CL BrenerLeishmania major strain Friedlin

Trypanosoma cruzi strain CL BrenerTrypanosoma cruzi strain CL Brener

Trypanosoma bruceiTrypanosoma brucei brucei

Trypanosoma bruceiLeishmania major strain FriedlinDiplonema papillatum

Clostridium novyi NTClostridium botulinum C str.StockholmClostridium perfringens SM101

Clostridium botulinum B str.Eklund17BClostridium sp.7243FAAClostridium beijerinckii NCIMB8052

Chlamydomonas reinhardtiiTrimastix pyriformis

Cryptosporidium parvumPlasmodium falciparum

Naegleria gruberiToxoplasma gondii VEG

Tetrahymena thermophilaParamecium tetraurelia strain d42

Paramecium tetraureliaTrichomonas vaginalis

Saccharomyces cerevisiae S288cUstilago maydis

Aspergillus nidulansNeurospora crassa OR74A

Homo sapiensMus musculus

Mus musculusHomo sapiens

Drosophila melanogasterDrosophila melanogaster

Dictyostelium discoideumAX4Thalassiosira pseduonana

Entamoeba histolyticaEuglena gracilis

Giardia lamblia ATCC50803Candidatus Accumulibacter phosphatis clad .e IIA str.UW1

Coriobacter iaceaebacterium JC110Collinsella stercoris DSM13279

Collinsella tanakaei YIT12063Collinsella intestinalis DSM13280

Coriobacterium glomerans PW2Olsenella uli DSM7084

Atopobium vaginae PB189T14Atopobium vaginae DSM15829

Atopobium rimae ATCC49626Atopobium parvulum DSM20469

Ectocarpus siliculosusEctocarpus siliculosus

Phaeodactylum tricornutumEctocarpus siliculosus

Chlamydomonas reinhardtiiCyanidioschyzon merolae

Toxoplas magondii ME49Ectocarpus siliculosus

Phaeodactylum tricornutum CCAP10551Ectocarpus siliculosus

Prochlorococcus marinus str. MIT9515Prochlorococcus marinus str. MIT9202

Fischerella sp. JSC11Raphidiopsis brookii D9Cylindrospermopsis raciborskii CS505

Cyanothece sp. PCC7822Oryza sativa Japonica GroupOryza sativa Indica GroupArabidopsis thalianaOryza sativa Japonica GroupOryza sativa Indica Group

Arabidopsis thalianaArabidopsis thalianaStigmatella aurantiaca DW431

Corallococcus coralloides DSM2259Chondromyces apiculatusDSM436

Anaeromyxobacter sp. KAnaeromyxobacter sp. Fw1095

gammaproteobacteriaFrancisella philomiragia subsp. philomiragia ATCC25015

Methylococcus capsulatus str.BathLautropia mirabilis ATCC51599

Oxalobacter formigenes OXCC13Herminiimonas arsenicoxydans

Cupriavidus basilensis OR16Burkholderia ambifaria AMMD

Rhodomicrobium vannielii ATCC17100Hyphomicrobium sp. MC1Methylocystis sp. SC2

Methylocystis sp. ATCC49242Methylobacterium populi BJ001

Rhodospirillum centenum SWCommensalibacter intestini A911

Acetobacter aceti NBRC14818Acetobactert ropicalis NBRC101654

0.99

0.990.84

1

1

10.52

0.98

0.59

0.99

0.51

0.73

0.860.92

0.991

1

0.99

0.93

0.980.99

0.890.99

0.99

0.55

0.78

0.81

0.99

0.990.83

0.990.99

0.86

0.990.89

0.94

0.950.98

0.56

0.78

0.7

0.50.53 0.99

0.99

0.71

0.59

0.850.99

0.99

0.75

0.990.99

0.89

0.99

0.99

0.810.9

0.96

0.93

0.89

0.99

0.99

0.99

0.71

0.750.99 0.99

0.99

0.52

0.980.99

0.96

0.53

0.510.83

0.7

0.87

0.98

0.95

0.95

0.99

0.820.85

0.94

0.87

0.99

0.94

0.990.99

0.96

0.99

0.99

0.991

0.611

0.99

0.76 0.99

0.97

0.92

0.990.89

0.99

0.990.99

0.2

Figure S2. Consensus phylogenetic tree for phosphoglycerate kinase. The methods and labeling are as in figure S1.

Archaea

Bacteria

Bacteria

Euglenozoa

Archaea

Bacteria

Archaea

Bacteria

Bacteria

Entamoeba histolyticaTrichomonas vaginalis

Trichomonas vaginalisG3Synechococcus sp. RS9916

Prochlorococcus marinus str.MIT9515Naegleria gruberi

Methanocella paludicola SANAEMethanocella conradii HZ254

Methanocella arvoryzae MRE50Giardia lamblia ATCC50803

Giardia lamblia ATCC50803Trimastix pyriformis

Cyanothece sp.PCC 7822Haliangium ochraceum DSM14365

Stigmatella aurantiaca DW4/3-1Corallococcus coralloides DSM2259

Candidatus KorarchaeumcryptofilumOPF8ThermofilumpendensHrk5

Candidatus Caldiarchaeum subterraneumToxoplasma gondii

Plasmodium falciparum 3D7Ectocarpus siliculosus

Pseudogulbenkiania sp. NH8BHerbaspirillum sp. GW103

Herbaspirillum sp. CF444Granulibacter bethesdensis CGDNIH1

Azospirillum sp.B510Azospirillum lipoferum 4B

Acidianus hospitalis W1Metallosphaera yellowstonensis MK1

Drosophila melanogasterCyanidioschyzon merolae

Arabidopsis thalianaParamecium tetraurelia

Tetrahymena thermophilaNaegleria gruberi

Vibrio metschnikovii CIP69.14Moritella sp. PE36Moritellamarina ATCC15381

Ectocarpus siliculosusUstilago maydis

Saccharomyce scerevisiae S288cSaccharomyces cerevisiae S288c

Aspergillus nidulansNeurospora crassa OR74A

Encephalitozoon cuniculiMus musculusHomo sapiensMus musculusDrosophila melanogaster

Trypanosoma cruzi strain CL BrenerTrypanosoma brucei brucei strain 927/4 GUTat10.1

Leishmania major strain FriedlinDiplonema papillatum

Dictyostelium discoideumDictyostelium discoideum AX4

Olsenellasp.oraltaxon 809 str.F0356Coriobacterium glomerans PW2

Collinsella tanakaei YIT12063Paenibacillus sp. oraltaxon 786 str.D14Paenibacillus alvei DSM29

Brevibacillus laterosporus LMG15441Cryptosporidium parvum

Toxoplasma gondii ME49Plasmodium falciparum 3D7

Tetrahymena thermophilaTetrahymena thermophila

Paramecium tetraurelia strain d4-2Thalassiosira pseudonana

Phaeodactylum tricornutum CCAP1055/1Phaeodactylum tricornutum CCAP1055/1

Ectocarpus siliculosusChlamydomonas reinhardtii

Chlamydomonas reinhardtiiChlaymdomonas reinhardtii

Oryza sativa Japonica GroupArabidopsis thaliana

Oryza sativa Indica GroupArabidopsis thaliana

0.74

0.99

0.98

0.950.97

0.51

0.78

0.51 0.99

0.79

0.6

0.970.92

0.96

0.990.99

0.64 0.99

0.55

0.77

0.87

0.99

0.99

0.94

0.91

0.98

0.99

0.99

0.99

0.87

0.99

0.8

0.98 0.99

0.91

0.99

0.990.99

0.980.99

0.95

0.94

0.97

0.99

0.70.99

0.93

0.99

0.99

0.96

0.98

0.850.86

0.94

0.890.2

Figure S3. Consensus phylogenetic tree for pyruvate kinase. The methods and labeling are as in figure S1.

Euglenozoa

Euglenozoa

Euglenozoa

Euglenozoa

Euglenozoa

Bacteria

Bacteria

Bacteria

Bacteria

Bacteria

Archaea

Archaea

Archaea

Bacteria

Acetivibrio cellulolyticus CD2Clostridium thermocellum ATCC 27405

Clostridium clariflavum DSM 19732Paludibacter propionicigenes WB4

Clostridium papyrosolvens DSM 2782Clostridium sp. MSTE9

Clostridium leptum DSM 753Clostridium methylpentosum DSM 5476

Anaerotruncus colihominis DSM 17241Giardia lamblia

Entamoeba histolyticaPhaeodactylum tricornutum

Tetrahymena thermophilaTetrahymena thermophila

Paramecium tetraureliaParamecium tetraurelia strain d4 2

Trypanosoma bruceiTrypanosoma cruzi strain CL Brener

Leishmania major strain FriedlinEncephalitozoon cuniculi

Giardia lamblia ATCC 50803Toxoplasma gondii VEGToxoplasma gondiiToxoplasma gondii

Phaeodactylum tricornutum CCAP 1055 1Ectocarpus siliculosusThalassiosira pseudonana

Phaeodactylum tricornutum CCAP 1055 1Phaeodactylum tricornutum CCAP 1055 1

Ectocarpus siliculosusEctocarpus siliculosus

Naegleria gruberiDiplonema papillatum GAPDH2

Diplonema papillatum GAPDH1Diplonema sp. ATCC 50225

Syntrophus aciditrophicus SBTrypanosoma cruzi strain CL Brener

Trypanosoma cruzi strain CL BrenerTrypanosoma cruzi strain CL Brener

Trypanosoma cruzi strain CL BrenerTrypanosoma cruzi strain CL Brener

Trypanosoma bruceiXenorhabdus bovienii SS 2004

Morganella morganii subsp. morganii KTYersinia enterocolitica subsp. enterocolitica 8081Hafnia alvei ATCC 51873Erwinia tasmaniensis Et1 99

Synechococcus sp. WH 5701Synechococcus sp. CB0101Cyanobium sp. PCC 7001

Polaribacter irgensii 23 PNitrosospira sp. TCH716

Magnetospirillum magnetotacticum MS 1Magnetospirillum gryphiswaldense MSR 1

Hydra magnipapillatablood disease bacterium R229

Hylemonella gracilis ATCC 19624Cupriavidus metallidurans CH34

Toxoplasma gondii VEGPlasmodium falciparum

Ectocarpus siliculosusCryptosporidium parvum Iowa II

Saccharomyces cerevisiae S288cSaccharomyces cerevisiae S288c

Chlamydomonas reinhardtiiChlamydomonas reinhardtiiChlamydomonas reinhardtii

Arabidopsis thalianaOryza sativa Indica Group

Arabidopsis thalianaCyanidioschyzon merolae

Ustilago maydisNeurospora crassa OR74A

Mus musculusHomo sapiensHomo sapiens

Homo sapiensHomo sapiens

Mus musculusMus musculus

Drosophila melanogasterDrosophila melanogaster

Dictyostelium discoideum AX4Oryza sativa Japonica Group

Oryza sativa Japonica GroupOryza sativa Japonica Group

Arabidopsis thalianaArabidopsis thaliana

Microcystis aeruginosa NIES 843Cyanothece sp. PCC 7425

SAR324 cluster bacterium SCGC AAA001 C10SAR324 cluster bacterium JCVI SC AAA005

Leishmania majorTrypanosoma cruzi strain CL BrenerTrypanosoma brucei brucei strain 927 4 GUTat10.1Trypanosoma brucei brucei

Euglena gracilisPelobacter carbinolicus DSM 2380

Geobacter uraniireducens Rf4Aspergillus nidulans

Methanoculleus bourgensis MS2Halopiger xanaduensis SH 6

Natronobacterium gregoryi SP2Halobiforma lacisalsi AJ5

Natronobacterium gregoryi SP2Halobiforma lacisalsi AJ5

Halogranum salarium B 1Halogranum salarium B 1

Halalkalicoccus jeotgali B3Phaeodactylum tricornutum CCAP 1055 1

Ectocarpus siliculosusDiplonema sp. ATCC 50225

Rhynchopus sp. ATCC 50231Diplonema sp. ATCC 50225

Chlamydomonas reinhardtiiuncultured archaeon

Thermofilum pendens Hrk 5Candidatus Korarchaeum cryptofilum OPF8

delta proteobacterium NaphS2Geobacter uraniireducens Rf4

Geobacter sp. M21Geobacter sp. M18

Geobacter daltonii FRC 32alpha proteobacterium IMCC14465

Tistrella mobilis KA081020 065Parvularcula bermudensis HTCC2503

Azospirillum lipoferum 4BAzospirillum brasilense Sp245

Chlamydomonas reinhardtiiActinomyces urogenitalis DSM 15434

Candidatus Aquiluna sp. IMCC13023Renibacterium salmoninarum ATCC 33209

Arthrobacter sp. Rue61aArthrobacter globiformis NBRC 12137

Arthrobacter chlorophenolicus A6Actinomyces sp. oral taxon 448 str. F0400

Trimastix pyriformisTrichomonas vaginalis G3

Trichomonas vaginalis G3Trichomonas vaginalis G3

uncultured archaeon GZfos1D1Aciduliprofundum boonei T469

0.97

0.65

0.98

0.9

0.960.99

0.86

0.82

0.67

0.94

0.980.99

0.99

1

0.78

0.951

0.97

0.950.79 0.97

0.84

0.840.96

0.870.9

0.94

0.95

0.990.99

0.97

0.69

0.95

0.62

0.99

0.99

0.64 0.99 0.99

0.990.99

0.99

0.89

0.9

0.530.99

0.99

10.88

0.54

0.96

0.56

0.991

0.85

0.950.99

0.75

0.77

0.94

0.980.99

0.99

0.87

0.95

0.61

0.99

0.8

0.99

0.990.98

0.99

0.890.96

0.91

0.81

0.91

1

0.99

0.97

1

0.990.97

1

0.66

0.99

0.99

0.69 10.93

0.72

0.62

0.550.99 1

0.990.51

0.51 1

0.510.99

0.78

0.990.96

0.990.69

0.64

10.99

0.99

0.75

0.2

Figure S4. Consensus phylogenetic tree for glyceraldehyde-3-phosphate dehydrogenase. The methods and labeling are as in figure S1.

Euglenozoa (FBPase)

Bacteria

Euglenozoa (FBPase)

Euglenozoa (SBPase)

Bacteria

Archaea

Diplonema papillatumDesulfobulbus propionicus

delta proteobacterium MLMS-1Desulfurivibrio alkaliphilus

delta proteobacterium NaphS2Desulfovibrio aespoeensis

Desulfovibrio salexigensDesulfovibrio vulgaris

Toxoplasma gondiiToxoplasma gondii

Synechococcus sp. PCC7335Synechococcus sp. JA-3-3Ab

Synechococcus elongatusCylindrospermopsis raciborskii

Raphidiopsis brookiiFischerella sp. JSC-11Nostoc punctiformeAnabaena variabilisNostoc sp. PCC7120

Synechocystis sp. PCC6803Hippea maritima

Calditerrivibrio nitroreducensDeferribacter desulfuricans

Sorangium cellulosumDesulfurobacterium thermolithotrophum

Thermovibrio ammonificansThermodesulfobium narugense

Thermodesulfobium narugenseSulfurihydrogenibium yellowstonense

Sulfurihydrogenibium azorenseHydrogenivirga sp.

Tetrahymena thermophilaThalassiosira pseudonana

Phaeodactylum tricornutumEctocarpus siliculosus

Mus musculusMus musculus

Mus musculusHomo sapiens

Mus musculusHomo sapiensHomo sapiens

Drosophila melanogasterDictyostelium discoideum

Tetrahymena thermophilaTetrahymena thermophila

Cyanidioschyzon merolaePhaeodactylum tricornutum

Ectocarpus siliculosusPhaeodactylum tricornutum

Phaeodactylum tricornutumEctocarpus siliculosus

Phaeodactylum tricornutumEctocarpus siliculosus

Ectocarpus siliculosusOryza sativaArabidopsis thaliana

Chlamydomonas reinhardtiiOryza sativaOryza sativaArabidopsis thaliana

Oryza sativaOryza sativa

Oryza sativaOryza sativa

Arabidopsis thalianaUstilago maydis

Trypanosoma cruziTrypanosoma brucei

Leishmania majorSaccharomyces cerevisiaeSaccharomyces cerevisiae

Neurospora crassaAspergillus nidulans

Methylotenera versatilisMagnetospirillum magneticum

Sulfitobacter sp.Roseobacter sp. GAI101

Octadecabacter arcticusAgrobacterium rhizogenes

Agrobacterium rhizogenesPseudogulbenkiania sp. NH8BMethyloversatilis universalis

Oxalobacter formigenesSutterella parvirubra

Neisseria mucosaNeisseria weaveri

Neisseria meningitidisNeisseria meningitidis 93004Neisseria meningitidis MC58

Saccharophagus degradansPseudoalteromonas haloplanktis

Idiomarina sp. A28LShewanella baltica OS223Shewanella baltica OS195

Marinomonas mediterraneaMarinomonas sp. MWYL1

Oceanobacter sp. RED65Rhodovulum sp. PH10

Caulobacter sp. AP07Caulobacter sp. K31

Streptomyces coelicoflavusMycobacterium tusciae

Nocardia cyriacigeorgicaMicrolunatus phosphovorus

Pseudonocardia dioxanivoransMethanoplanus petrolearius

Halopiger xanaduensisNatrinema pellirubrum

Natronomonas pharaonisHaloarcula marismortuiHaloarcula hispanica

Haloferax mediterraneiHaloferax volcanii

Natrialba magadiiHaloterrigena turkmenica

Chlamydomonas reinhardtiiTrypanosoma cruzi

Trypanosoma bruceiTetrahymena thermophila

Paramecium tetraureliaParamecium tetraurelia

Paramecium tetraureliaParamecium tetraurelia

Toxoplasma gondiiPhaeodactylum tricornutum

Neurospora crassaEctocarpus siliculosus

Ectocarpus siliculosusChlamydomonas reinhardtii

Chlamydomonasr einhardtiiChlamydomonas reinhardtii

Arabidopsis thaliana

0.62

0.52

0.77

0.79

0.75

0.99

11

0.67 10.98

0.790.99

0.870.99

1

0.83

0.991

0.830.85 1

0.990.99

0.96

1

0.990.99

1

0.84

0.990.99

0.99

11

10.9

0.940.99

0.781

0.98

0.87

0.70.68

0.61

1

0.66

0.99

11

11

1

0.970.98

0.99

0.99

0.830.96

10.99

11

1

0.95

0.990.51

0.99

1

0.82

0.93

0.95

0.68

0.99

0.86 1

0.74

0.82

0.66

0.56

1

1

0.760.99

0.97

0.93

0.87

0.99

0.71

1

0.550.66 0.93

0.83

1

1

1

0.92

0.821

0.991

0.99

0.98

0.60.98

0.991

1

0.88

0.2

Figure S5. Consensus phylogenetic tree for fructose-1,6-bisphosphatase (FBPase). Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme for pentose phosphate pathway. The methods and labeling are as in figure S1.

(a)

C

E

F

D

D

N

M

A

M

B

B

Peroxisome

HK PGI GAPDH1 TIM PGK PGM PK ENO GLK

kDa

75-

50-

37-

25- 21-

15-

100-

150-

E F

Figure S6. Expression of glycolytic enzymes in D. papillatum. (a) Western blot analysis of the D. papillatum extracts using antisera to the relevant enzymes. The expected size of each protein is shown by a triangle in each panel. (b) Ultrastructure of D. papillatum. Peroxisomes (arrow) are present as dense bodies surrounded by a single-membrane (B, a magnified image of the inset of A). C-F, Immunoelectron microscopic observations of FBPA within peroxisomes. Note that gold particles are specifically detected inside peroxisomes (arrow). (D, E, and F are magnified images of the insets of C). N; nucleus. M; mitochondrion, P; peroxisome. Scale bar = 0.5 μm.

(b)

PFK2 PFK1

Rel

ativ

e ab

unda

nce

(%)

(a) 6 mM 13C5-L-glutamine

Rel

ativ

e ab

unda

nce

(%)

Rel

ativ

e ab

unda

nce

(%)

(b) 6 mM 13C6-D-glucose

(c) 30 mM 13C6-D-glucose

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Glucose 6-phosphate

0 hr 4 hr 8 hr

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Fructose 6-phosphate

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Fructose 1,6-isphosphate

0

5

10

15

20

+1 +2 +3

3-phospho-glycerate

0

5

10

15

20

+1 +2 +3

Phosphoenol-pyruvate

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Glucose 6-phosphate

0 hr 4 hr 8 hr

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Fructose 6-phosphate

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Fructose 1,6-bisphosphate

0

5

10

15

20

+1 +2 +3

3-phospho-glycerate

0

10

20

30

40

+1 +2 +3

Phosphoenol-pyruvate

0

5

10

15

20

+1 +2 +3

Lactate

0 hr 4 hr 8 hr

0

10

20

30

40

50

+1 +2 +3 +4 +5

α-ketoglutarate

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Citrate

0

10

20

30

+1 +2 +3 +4

Malate

0

10

20

30

40

+1 +2 +3 +4 +5 +6 +7

Sedoheptulose 7-phosphate

0

5

10

15

20

+1 +2 +3

Lactate

0 hr 4 hr 8 hr

0

5

10

15

20

+1 +2 +3 +4

Succinate

0

5

10

15

20

+1 +2 +3 +4

Fumarate

0

5

10

15

20

+1 +2 +3 +4

Malate

0

10

20

30

40

+1 +2 +3 +4 +5 +6 +7

Sedoheptulose 7-phosphate

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Citrate

0

10

20

30

+1 +2 +3 +4 +5 +6

Glucose 6-phosphate

12C 13C

0

10

20

30

+1 +2 +3 +4 +5 +6

Fructose 6-phosphate

0

10

20

30

+1 +2 +3 +4 +5 +6

Fructose 1,6-bisphosphate

0

10

20

30

40

+1 +2 +3

DHAP

0

10

20

30

+1 +2 +3

3-phospho-glycerate

0

5

10

15

20

+1 +2 +3

Lactate

0

5

10

15

20

+1 +2 +3 +4 +5 +6

Citrate

12C 13C

0

5

10

15

20

+1 +2 +3 +4

Succinate

0

5

10

15

20

+1 +2 +3 +4

Malate

0

10

20

30

40

+1 +2 +3 +4 +5

Ribose 5-phosphate

0

10

20

30

+1 +2 +3 +4 +5

Ribulose 5-phosphate

0

10

20

30

40

+1 +2 +3 +4 +5 +6 +7

Sedoheptulose 7-phosphate

Figure S7. Metabolomic analysis of 13C-labeled metabolites in D. papillatum. D. papillatum cells were incubated with 6 mM 13C5-glutamine for 0, 4, and 8 hrs (a), 6 mM 13C6-D-glucose for 0, 4, and 8 hrs (b), or 30 mM 13C6-D-glucose for 72 hrs (c). The Y-axis indicates the relative abundance (%) of each isotopomer of the relevant metabolite. The X-axis represents the number of 13C in the isotopomers. Data points and error bars represent the mean ± s.d. of three independent experiments.

Euglenozoa (glycosomal)

Euglenozoa

Euglenozoa

Euglenozoa

Bacteria

Bacteria

Bacteria

Leptotrichia hofstadiiStreptomyces sp. NRRL S 87

Desulfotalea psychrophilaEuglena gracilis

Desulfonatronum thiodismutansClostridiales bacterium VE202 09

Francisella philomiragiaTrypanosoma brucei brucei TREU927

Trypanosoma grayiTrypanosoma cruzi CL Brener

Trypanosoma brucei brucei TREU927Phytomonas sp. isolate Hart1

Leishmania major FriedlinStrigomonas culicis

Angomonas deaneiNecropsobacter rosorum

Pasteurella sp. FF6Haemophilus sp. FF7

Pseudomonas aeruginosaMethylococcaceae bacterium Sn10 6

Burkholderiales bacterium 1 1 47Acinetobacter soli

Toxoplasma gondiiNeospora caninum Liverpool

Diplonema papillatumBlastocystis hominis

Phytophthora infestans T30 4Saprolegnia parasitica

Aphanomyces invadansTrypanosoma grayiTrypanosoma cruzi CL Brener

Trypanosoma brucei brucei TREU927Strigomonas culicis

Phytomonas sp. isolate Hart1Leptomonas pyrrhocoris

Leishmania major FriedlinAngomonas deanei

Volvox carteri f. nagariensisHelicosporidium sp. ATCC 50920

Ectocarpus siliculosusOstreococcus tauri

Micromonas sp. RCC299Bathycoccus prasinos

Arabidopsis thalianaRicinus communisOryza sativa Japonica Group

Glycine sojaAegilops tauschii

Oxytricha trifallaxGaldieria sulphuraria

Rhizopus delemar RA 99 880Coprinopsis cinerea cytosolic

Wallemia sebi CBS 633.66Cryptococcus neoformans var. grubii H99

Trichophyton soudanense CBS 452.61Podospora anserina S mat+

Saccharomyces cerevisiae S288cCandida albicans 12C

Salpingoeca rosettaCapsaspora owczarzaki ATCC 30864

Solenopsis invictaDrosophila melanogaster

Danaus plexippusPlutella xylostella

Bombyx moriXenopus laevis mitochondrial 2

Homo sapiens mitochondrialAscaris suum

Acanthamoeba castellanii str. NeffDictyostelium discoideum

0.99

0.99

0.991

0.94

0.771

1

0.98

0.9

0.630.94

0.56

1

1

0.98

0.560.99

1

0.99

0.99

1

0.76

0.68

0.67

0.910.99

0.53

0.95

0.96

1

0.990.950.99

0.5

0.95

0.59

0.76

0.991

0.74

0.92

0.990.9 1

0.59

0.931

10.64

0.950.64

0.76

1

0.530.2

Figure S8. Consensus phylogenetic tree for adenylate kinase. Note that E. gracilis enzyme and the glycosomal enzymes of kinetoplastids are nested independently within the bacterial clade (PP = 0.99), while D. papillatum and cytosolic kinetoplastid homologues cluster in the eukaryotic subtree (PP = 0.99). The methods and labeling are as in figure S1.

Euglenozoa(glycosomal)

Euglenozoa

Bacteria

Bacteria

Archaea

Clostridium botulinum A ATCC 3502Thermoanaerobacter tengcongensis

Lactococcus lactisBacillus subtilis

Helicobacter pylori 26695Thermus thermophilus HB27

Spirochaeta thermophila DSM 6192Pyrococcus furiosus DSM 3638

Tetrahymena thermophilaParamecium tetraurelia

Trypanosoma bruceiTrypanosoma cruziLeishmania major

Fibrobacter succinogenesChlorobium tepidum

Fusobacterium nucleatum ATCC 25586Bacteroides thetaiotaomicron

Thermotoga maritima MSB8Aquifex aeolicus

Neisseria gonorrhoeaeEscherichia coli

Acidobacterium capsulatumGeobacter sulfurreducens

Phytophthora infestansToxoplasma gondii

Plasmodium falciparum 3D7Thalassiosira pseudonana

Phaeodactylum tricornutumGuillardia theta

Dictyostelium discoideumEmiliania huxleyi

Cyanidioschyzon merolaeEuglena gracilis

Trypanosoma cruzi BrenerTrypanosoma brucei

Leishmania major

Diplonema papillatum

Saccharomyces cerevisiaeMus musculusHomo sapiens

Drosophila melanogasterMonosiga brevicollis

Caenorhabditis elegansChlamydomonas reinhardtii

Zea maysArabidopsis thaliana

0.56

0.91

1

0.94

0.5

0.99

0.75 11

1

0.59

0.7

0.93

0.99

1

0.85

1

0.54

0.68

0.99

1

0.7

0.63

0.99

0.97

0.81 1

0.93

0.710.59

1

0.660.99

10.2

Figure S9. Consensus phylogenetic tree for inosine-5-monophosphate dehydrogenase. Note that glycosomal isoforms of kinetoplastids are nested within the bacterial clade, while E. gracilis, D. papillatum and cytosolic kinetoplastid homologues are monophyletic and cluster in the eukaryotic clade, consistent with the organismal tree (PP = 1). The methods and labeling are as in figure S1.

Figure S10. Consensus phylogenetic tree for transketolase. Note that the euglenozoan enzymes are monophyletic (PP = 1). The methods and labeling are as in figure S1.

Euglenozoa

Bacteria

Bacteria

Mycobacterium tuberculosisTrichomonas vaginalis

Giardia lambliaGeobacter metallireducens

Deinococcus radioduransChlorobium tepidum

Synechocystis sp. PCC 6803Chloroflexus aurantiacus

Chlamydia trachomatisBorrelia burgdorferi B31

Bacillus subtilisThermotoga maritima MSB8

Bacteroides thetaiotaomicronAquifex aeolicus

Agrobacterium sp. H13 3Neisseria gonorrhoeae FA 1090

Naegleria gruberiZea mays

Cyanidioschyzon merolaeToxoplasma gondii

Phytophthora sojaeSaccharomyces cerevisiae

Capsaspora owczarzaki ATCC 30864Caenorhabditis elegans

Solenopsis invictaHomo sapiensBos taurus

Arabidopsis thalianaDiplonema papillatum

Trypanosoma cruzi strain CL BrenerTrypanosoma brucei brucei TREU927Phytomonas sp. isolate EM1

Leishmania major strain FriedlinStrigomonas culicisAngomonas deanei

0.87

0.780.69

0.67

0.97

1

0.99

0.61

0.62

0.78

1

0.99 1

1

1

0.651

0.79

0.770.2