1.
2.
3.
4.
Burki F. 2014 The eukaryotic tree of life from a
global phylogenomic perspective. Cold Spring Harb.
Perspect. Biol. 6, a016147. (doi:10.1101/cshperspect.
a016147)
Burki F, Roger AJ, Brown MW, Simpson AGB. 2019
The new tree of eukaryotes. Trends Ecol. Evol. 35,
43–55. (doi:10.1016/j.tree.2019.08.008)
Keeling PJ, Burki F. 2019 Progress towards the tree
of eukaryotes. Curr. Biol. 29, R808–R817. (doi:10.
1016/j.cub.2019.07.031)
Bleidorn C. 2016 Third generation sequencing:
technology and its potential impact on evolutionary
biodiversity research. Syst. Biodivers. 14, 1–8.
(doi:10.1080/14772000.2015.1099575)
5.
6.
7.
Vincent AT, Derome N, Boyle B, Culley AI, Charette
SJ. 2017 Next-generation sequencing (NGS) in the
microbiological world: how to make the most of
your money. J. Microbiol. Methods. 138, 60–71.
(doi:10.1016/j.mimet.2016.02.016)
Lax G, Eglit Y, Eme L, Bertrand EM, Roger AJ,
Simpson AGB. 2018 Hemimastigophora is a
novel supra-kingdom-level lineage of eukaryotes.
Nature 564, 410–414. (doi:10.1038/s41586-0180708-8)
Kolisko M, Boscaro V, Burki F, Lynn DH, Keeling PJ.
2014 Single-cell transcriptomics for microbial
eukaryotes. Curr. Biol. 24, R1081–R1082. (doi:10.
1016/j.cub.2014.10.026)
8.
Strassert JFH, Jamy M, Mylnikov AP, Tikhonenkov
DV, Burki F. 2019 New phylogenomic analysis of the
enigmatic phylum Telonemia further resolves the
eukaryote tree of life. Mol. Biol. Evol. 36, 757–765.
(doi:10.1093/molbev/msz012)
9. Zhao S, Burki F, Brte J, Keeling PJ, Klaveness D,
Shalchian-Tabrizi K. 2012 Collodictyon—an ancient
lineage in the tree of eukaryotes. Mol. Biol. Evol.
29, 1557–1568. (doi:10.1093/molbev/mss001)
10. Yabuki A, Kamikawa R, Ishikawa SA, Kolisko M, Kim
E, Tanabe AS, Kume K, Ishida K-I, Inagaki Y. 2014
Palpitomonas bilix represents a basal cryptist
lineage: insight into the character evolution in
Cryptista. Sci. Rep. 4, 4641. (doi:10.1038/srep04641)
Proc. R. Soc. B 287: 20201538
barthelonids
royalsocietypublishing.org/journal/rspb
gain of
but after assessing the data from stain PAP020, this particular
event needs to be pushed back at least to the common ancestor
of fornicates and barthelonids, as strain PAP020 and multiple
early branching CLOs (e.g. C. membranifera) share ACS2. It is
noteworthy that acquisition of ACS2 may extend back to the
last common metamonad ancestor, since a possibly directly
related ACS2 is also present in Paratrimastix (electronic supplementary material, figure S5). Secondly, as barthelonids are
distantly related to D. brevis and diplomonads, loss of substrate-level phosphorylation in barthelonid MROs, if this is
the case, can be assumed to have occurred independently
from the loss in the common ancestor of D. brevis and diplomonads (highlighted by blue diamonds in figure 5). Further,
barthelonids and the common ancestor of D. brevis and
diplomonads seem to have accommodated the loss of
MRO-localized substrate-level phosphorylation via possessing
evolutionarily distinct ACS homologues (ACS2 and ACS1,
represented by yellow and red lines, respectively, in figure 5).
Finally, pyruvate metabolism might have been relocated from
the MRO to the cytosol in strain PAP020 as seen in G. intestinalis
[54–56].
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
modification of Sato’s method. J. Electron Microsc.
35, 304–306. (doi:10.1093/oxfordjournals.jmicro.
a050582)
Sato T. 1968 A modified method for lead staining of
thin sections. J. Electron Microsc. (Tokyo) 17,
158–159. (doi:10.1093/oxfordjournals.jmicro.
a049610)
Engel SR et al. 2014 The reference genome
sequence of Saccharomyces cerevisiae: then and
now. G3 Genes Genomes Genet. 4, 389–398.
(doi:10.1534/g3.113.008995)
Fukasawa Y, Tsuji J, Fu SC, Tomii K, Horton P, Imai
K. 2015 MitoFates: improved prediction of
mitochondrial targeting sequences and their
cleavage sites. Mol. Cell. Proteomics 14, 1113–1126.
(doi:10.1074/mcp.M114.043083)
Kume K, Amagasa T, Hashimoto T, Kitagawa H.
2018 NommPred: prediction of mitochondrial and
mitochondrion-related organelle proteins of
nonmodel organisms. Evol. Bioinform. 14, 1–12.
(doi:10.1177/1176934318819835)
Li B, Dewey CN. 2011 RSEM: accurate transcript
quantification from RNA-Seq data with or without a
reference genome. BMC Bioinf. 12, 323. (doi:10.
1186/1471-2105-12-323)
Kulda J, Nohýnková E, Cepicka I. 2017
Retortamonadida (with notes on Carpediemonaslike organisms and Caviomonadidae). In Handbook
of the protists, 2nd ed., pp. 1247–1278. Berlin,
Germany: Springer International Publishing.
Yubuki N, Simpson AGB, Leander BS. 2013
Comprehensive ultrastructure of Kipferlia bialata
provides evidence for character evolution within the
Fornicata (Excavata). Protist 164, 423–439. (doi:10.
1016/j.protis.2013.02.002)
Yubuki N, Inagaki Y, Nakayama T, Inouye I. 2007
Ultrastructure and ribosomal RNA phylogeny of the
free-living heterotrophic flagellate Dysnectes brevis
n. gen., n. sp., a new member of the Fornicata.
J. Eukaryot. Microbiol. 54, 191–200. (doi:10.1111/j.
1550-7408.2007.00252.x)
Simpson AGB, Patterson DJ. 1999 The ultrastructure
of Carpediemonas membranifera (Eukaryota) with
reference to the ‘excavate hypothesis’.
Eur. J. Protistol. 35, 353–370. (doi:10.1016/S09324739(99)80044-3)
Tovar J, León-Avila G, Sánchez LB, Sutak R, Tachezy J,
Van Der Giezen M, Hernández M, Müller M, Lucocq
JM. 2003 Mitochondrial remnant organelles of Giardia
function in iron-sulphur protein maturation. Nature
426, 172–176. (doi:10.1038/nature01945)
Cenci U et al. 2018 Nuclear genome sequence of the
plastid-lacking cryptomonad Goniomonas avonlea
provides insights into the evolution of secondary
plastids. BMC Biol. 16, 137. (doi:10.1186/s12915018-0593-5)
Burki F et al. 2009 Large-scale phylogenomic
analyses reveal that two enigmatic protist lineages,
Telonemia and Centroheliozoa, are related to
photosynthetic chromalveolates. Genome Biol. Evol.
1, 231–238. (doi:10.1093/gbe/evp022)
Inagaki Y, Nakajima Y, Sato M, Sakaguchi M,
Hashimoto T. 2009 Gene sampling can bias multi-
Proc. R. Soc. B 287: 20201538
24. Ronquist F et al. 2012 Mrbayes 3.2: efficient
Bayesian phylogenetic inference and model choice
across a large model space. Syst. Biol. 61, 539–542.
(doi:10.1093/sysbio/sys029)
25. Grabherr MG et al. 2011 Full-length transcriptome
assembly from RNA-Seq data without a reference
genome. Nat. Biotechnol. 29, 644–652. (doi:10.
1038/nbt.1883)
26. Haas BJ et al. 2013 De novo transcript sequence
reconstruction from RNA-seq using the Trinity
platform for reference generation and analysis.
Nat. Protoc. 8, 1494–1512. (doi:10.1038/nprot.
2013.084)
27. Tanifuji G, Takabayashi S, Kume K, Takagi M,
Nakayama T, Kamikawa R, Inagaki Y, Hashimoto T.
2018 The draft genome of Kipferlia bialata reveals
reductive genome evolution in fornicate parasites.
PLoS ONE 13, e0194487. (doi:10.1371/journal.pone.
0194487)
28. Yabuki A, Gyaltshen Y, Heiss AA, Fujikura K, Kim E.
2018 Ophirina amphinema n. gen., n. sp., a new
deeply branching discobid with phylogenetic affinity
to jakobids. Sci. Rep. 8, 16219. (doi:10.1038/
s41598-018-34504-6)
29. Leger MM et al. 2017 Organelles that illuminate the
origins of Trichomonas hydrogenosomes and Giardia
mitosomes. Nat. Ecol. Evol. 1, 92. (doi:10.1038/
s41559-017-0092)
30. Stamatakis A. 2014 RAxML version 8: a tool for
phylogenetic analysis and post-analysis of large
phylogenies. Bioinformatics 30, 1312–1313. (doi:10.
1093/bioinformatics/btu033)
31. Wang HC, Minh BQ, Susko E, Roger AJ. 2018
Modeling site heterogeneity with posterior mean
site frequency profiles accelerates accurate
phylogenomic estimation. Syst. Biol. 67, 216–235.
(doi:10.1093/sysbio/syx068)
32. Lartillot N, Philippe H. 2004 A Bayesian mixture
model for across-site heterogeneities in the aminoacid replacement process. Mol. Biol. Evol. 21,
1095–1109. (doi:10.1093/molbev/msh112)
33. Lartillot N, Philippe H. 2006 Computing
Bayes factors using thermodynamic integration.
Syst. Biol. 55, 195–207. (doi:10.1080/106351
50500433722)
34. Lartillot N et al. 2007 Suppression of long-branch
attraction artefacts in the animal phylogeny using a
site-heterogeneous model. BMC Evol. Biol. 7, S4.
(doi:10.1186/1471-2148-7-S1-S4)
35. Shimodaira H. 2002 An approximately
unbiased test of phylogenetic tree selection. Syst.
Biol. 51, 492–508. (doi:10.1080/106351
50290069913)
36. Shimodaira H, Hasegawa M. 2001 CONSEL: for
assessing the confidence of phylogenetic tree
selection. Bioinformatics 17, 1246–1247. (doi:10.
1093/bioinformatics/17.12.1246)
37. Susko E, Field C, Blouin C, Roger AJ. 2003
Estimation of rates-across-sites distributions in
phylogenetic substitution models. Syst. Biol. 52,
594–603. (doi:10.1080/10635150390235395)
38. Hanaichi T, Sato T, Iwamoto T, Malavasi-Yamashiro
J, Hoshino M, Mizuno N. 1986 A stable lead by
royalsocietypublishing.org/journal/rspb
11. Burki F, Kaplan M, Tikhonenkov DV, Zlatogursky V,
Minh BQ, Radaykina LV, Smirnov A, Mylnikov AP,
Keeling PJ. 2016 Untangling the early diversification
of eukaryotes: a phylogenomic study of the
evolutionary origins of Centrohelida, Haptophyta
and Cryptista. Proc. R. Soc. B 283, 20152802.
(doi:10.1098/rspb.2015.2802)
12. Janouškovec J, Tikhonenkov DV, Burki F, Howe AT,
Rohwer FL, Mylnikov AP, Keeling PJ. 2017 A new
lineage of eukaryotes illuminates early
mitochondrial genome reduction. Curr. Biol. 27,
3717–3724. (doi:10.1016/j.cub.2017.10.051)
13. Brown MW et al. 2018 Phylogenomics places
orphan protistan lineages in a novel eukaryotic
super-group. Genome Biol. Evol. 10, 427–433.
(doi:10.1093/gbe/evy014)
14. Gawryluk RMR, Tikhonenkov DV, Hehenberger E,
Husnik F, Mylnikov AP, Keeling PJ. 2019 Nonphotosynthetic predators are sister to red algae.
Nature 572, 240–243. (doi:10.1038/s41586-0191398-6)
15. Kamikawa R et al. 2014 Gene content evolution in
discobid mitochondria deduced from the
phylogenetic position and complete mitochondrial
genome of Tsukubamonas globosa. Genome Biol.
Evol. 6, 306–315. (doi:10.1093/gbe/evu015)
16. Bernard C, Simpson AGB, Patterson DJ. 2000 Some
free-living flagellates (Protista) from anoxic habitats.
Ophelia 52, 113–142. (doi:10.1080/00785236.1999.
10409422)
17. Lee WJ. 2002 Some free-living heterotrophic
flagellates from marine sediments of Inchon and
Ganghwa Island, Korea. Korean J. Biol. Sci. 6,
125–143. (doi:10.1080/12265071.2001.9647643)
18. Lee WJ. 2006 Some free-living heterotrophic
flagellates from marine sediments of tropical
Australia. Ocean Sci. J. 41, 75–95. (doi:10.1007/
BF03022413)
19. Nakayama T, Marin B, Kranz HD, Surek B,
Huss VAR, Inouye I, Melkonian M. 1998
The basal position of scaly green flagellates among
the green algae (Chlorophyta) is revealed by
analyses of nuclear-encoded SSU rRNA sequences.
Protist 149, 367–380. (doi:10.1016/S14344610(98)70043-4)
20. Yabuki A, Inagaki Y, Ishida K. 2010 Palpitomonas
bilix gen. et sp. nov.: a novel deep-branching
heterotroph possibly related to Archaeplastida or
Hacrobia. Protist 161, 523–538. (doi:10.1016/j.
protis.2010.03.001)
21. Katoh K. 2002 MAFFT: a novel method for rapid
multiple sequence alignment based on fast Fourier
transform. Nucleic Acids Res. 30, 3059–3066.
(doi:10.1093/nar/gkf436)
22. Katoh K, Standley DM. 2014 MAFFT: iterative
refinement and additional methods. Methods Mol.
Biol. 1079, 131–146. (doi:10.1007/978-1-62703646-7_8)
23. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ.
2015 IQ-TREE: a fast and effective stochastic
algorithm for estimating maximum-likelihood
phylogenies. Mol. Biol. Evol. 32, 268–274. (doi:10.
1093/molbev/msu300)
Genet. Dev. 8, 616–623. (doi:10.1016/S0959437X(98)80028-2)
54. Lindmark DG. 1980 Energy metabolism of the
anaerobic protozoon Giardia lamblia. Mol. Biochem.
Parasitol. 1, 1–12. (doi:10.1016/01666851(80)90037-7)
55. Muller M et al. 2012 Biochemistry and evolution of
anaerobic energy metabolism in eukaryotes.
Microbiol. Mol. Biol. Rev. 76, 444–495. (doi:10.
1128/MMBR.05024-11)
56. Townson SM, Upcroft JA, Upcroft P. 1996
Characterisation and purification of pyruvate:
ferredoxin oxidoreductase from Giardia duodenalis.
Mol. Biochem. Parasitol. 79, 183–193. (doi:10.1016/
0166-6851(96)02661-8)
57. Yazaki E et al. 2020 Data from: Barthelonids
represent a deep-branching metamonad clade with
mitochondrion-related organelles predicted to
generate no ATP. Dryad Digital Repository. (doi:10.
5061/dryad.3tx95x6bn)
10
royalsocietypublishing.org/journal/rspb
gene phylogenetic inferences: the relationship
between red algae and green plants as a case
study. Mol. Biol. Evol. 26, 1171–1178. (doi:10.1093/
molbev/msp036)
52. Felsenstein J. 1978 Cases in which parsimony or
compatibility methods will be positively misleading.
Syst. Biol. 27, 401–410. (doi:10.1093/sysbio/
27.4.401)
53. Philippe H, Laurent J. 1998 How good
are deep phylogenetic trees? Curr. Opin.
Proc. R. Soc. B 287: 20201538
...