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Edited by W. Ford Doolittle, Dalhousie University, Halifax, Canada, and approved January 10, 2020 (received for review June 25, 2019)
Although aerobic respiration is a hallmark of eukaryotes, a few uni- have highly divergent genome structures, with large multipartite
cellular lineages, growing in hypoxic environments, have second- circular mt chromosomes and unusually high evolutionary rates (8,
arily lost this ability. In the absence of oxygen, the mitochondria of 9). To gain further insight into the evolution of the myxozoan
these organisms have lost all or parts of their genomes and evolved mt genome, we studied two closely related freshwater species,
into mitochondria-related organelles (MROs). There has been
debate regarding the presence of MROs in animals. Using deep
sequencing approaches, we discovered that a member of the Significance
Cnidaria, the myxozoan Henneguya salminicola, has no mitochon-
drial genome, and thus has lost the ability to perform aerobic Mitochondrial respiration is an ancient characteristic of eukary-
cellular respiration. This indicates that these core eukaryotic fea- otes. However, it was lost independently in multiple eukaryotic
tures are not ubiquitous among animals. Our analyses suggest lineages as part of adaptations to an anaerobic lifestyle. We
that H. salminicola lost not only its mitochondrial genome but also show that a similar adaptation occurred in a member of the
nearly all nuclear genes involved in transcription and replication of Myxozoa, a large group of microscopic parasitic animals that are
the mitochondrial genome. In contrast, we identified many genes closely related to jellyfish and hydroids. Using deep sequencing
that encode proteins involved in other mitochondrial pathways approaches supported by microscopic observations, we present
and determined that genes involved in aerobic respiration or mi- evidence that an animal has lost its mitochondrial genome. The
tochondrial DNA replication were either absent or present only as myxozoan cells retain structures deemed mitochondrion-related
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pseudogenes. As a control, we used the same sequencing and organelles, but have lost genes related to aerobic respiration
annotation methods to show that a closely related myxozoan, and mitochondrial genome replication. Our discovery shows
Myxobolus squamalis, has a mitochondrial genome. The molecular that aerobic respiration, one of the most important metabolic
results are supported by fluorescence micrographs, which show pathways, is not ubiquitous among animals.
the presence of mitochondrial DNA in M. squamalis, but not in H.
salminicola. Our discovery confirms that adaptation to an anaerobic Author contributions: P.C., J.L.B., and D.H. designed research; D.Y., S.D.A., and D.H. per-
environment is not unique to single-celled eukaryotes, but has also formed research; D.Y., J.L.B., and D.H. contributed new reagents/analytic tools; D.Y., M.N.,
E.S.C., H.P., and D.H. analyzed data; and D.Y., S.D.A., and D.H. wrote the paper.
evolved in a multicellular, parasitic animal. Hence, H. salminicola
The authors declare no competing interest.
provides an opportunity for understanding the evolutionary transi-
tion from an aerobic to an exclusive anaerobic metabolism. This article is a PNAS Direct Submission.
Published under the PNAS license.
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Cnidaria mitochondrial evolution | mitochondria-related organelle | Data deposition: Voucher paratype material was deposited at the US National Parasite
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MRO cristae Collection, Smithsonian Institution, Washington, DC (https://collections.nmnh.si.edu/
search/iz/) under the following accession numbers: Henneguya salminicola myxospores:
USNM1611578 (from genome sample) and USNM1611579 (from transcriptome sample);
Henneguya salminicola and Myxobolus squamalis (SI Appendix, (15). This view is supported by calculations using only the most
Fig. S1), both of which are parasites of salmonid fish (10–12). conserved CEGMA genes, which have higher recovery in both
H. salminicola and M. squamalis (76.9% and 56.9%, respectively).
Results Assembly of the mt genomes revealed striking differences
We assembled transcriptomes and genomes from both species between the two parasites. For M. squamalis, we successfully
using identical protocols and computational pipelines. Our phylo- recovered a circular mt genome composed of a single chromo-
genetic analyses based on 78 nuclear ribosomal protein-encoding some, which phylogenetic analyses confirmed was myxozoan (SI
genes from taxa representative of eukaryotic diversity confirmed Appendix, Supplementary Results and Figs. S5 and S6). Similar to
that the organisms we sequenced are closely related myxozoans, other myxozoans (8), the M. squamalis mt genome lacked tRNAs,
and not contaminants (Fig. 1 and SI Appendix, Fig. S2). The and has a fast evolutionary rate (SI Appendix, Supplementary Re-
genome assembly statistics revealed that H. salminicola has a sults and Figs. S5 and S6). In stark contrast, we could not identify
more complete assembly with higher coverage and more pre- any mt sequence among the contigs of H. salminicola, despite the
dicted protein sequences than M. squamalis (Table 1 and SI higher quality of that assembly compared with that of M. squa-
Appendix, Figs. S3 and S4). Targeted searches in the genomes malis. To identify whether DNA was present in the myxozoan
identified 75/78 nuclear ribosomal protein genes, which sug- mitochondria, we stained living multicellular developing stages of
gested that the completeness is >90% for both species. However, M. squamalis and H. salminicola with DAPI (Fig. 2). Cells of M.
estimates of genome completeness using the Core Eukaryotic squamalis showed the characteristic eukaryotic staining of both
Genes Mapping Approach (CEGMA) (13) recovered only 53.6% of nuclei and mitochondria (as much smaller blue dots; Fig. 2A),
core eukaryotic genes for H. salminicola and 37.5% for M. squamalis. whereas H. salminicola showed only nuclear staining (Fig. 2B).
We hypothesize that the fast evolutionary rates of myxozoans (14) The microscopy results, together with the lack of mt contigs in
reduced our ability to detect many common eukaryotic genes, a the genome and transcriptome assemblies, supported our central
challenge also known with other fast-evolving eukaryotic lineages hypothesis that this animal has lost its mt genome. Electron
Yahalomi et al. PNAS | March 10, 2020 | vol. 117 | no. 10 | 5359
Table 1. Assembly statistics, presence of mt genome, and number of nuclear-encoded mt genes identified for myxozoan genomes
(gen.) and transcriptomes (trans.)
K. iwatai T. kitauei M. cerebralis
H. salminicola M. squamalis (14) (18) (14)
*There are two additional proteins, involved in pyruvate metabolism and present in all Myxozoa, that appear also in other metabolic pathways.
microscopy images, however, showed mt-like double membrane H. salminicola, and was absent from the H. salminicola tran-
organelles with cristae in H. salminicola (Fig. 2C and SI Appendix, scriptome assembly, whereas we identified homologous contigs in
Fig. S7) and M. squamalis (SI Appendix, Fig. S8). Accordingly, all other myxozoan transcriptomes (Dataset S3). The presence of
genes involved in cristae organization were also detected in the a pseudogene copy of this polymerase has several implications.
genome of both species, in particular DNAJC11 and MTX1, First, it supports our central conclusion that H. salminicola has lost
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which have been linked to the presence of cristae (16, 17) (Dataset its mtDNA, as it has no mtDNA replication machinery. Second, it
S1). Together, these results confirm that an MRO without an mt shows that the absence of protein homologs in this species is the
genome, but with cristae, is present in this species. result of pseudogenization, and not an assembly artifact.
In animals, most of the mt proteome is encoded in the nucleus. The loss of the mt genome should impact aerobic respiration,
Accordingly, we identified 51 and 57 genes involved in key mt since animal mt genomes code for essential proteins of the
metabolic pathways (e.g., amino acid, carbohydrate, or nucleo- electron-transport chain (20). To verify whether the loss of the
tide metabolism) in H. salminicola and M. squamalis, respectively mt genome meant loss of aerobic respiration in H. salminicola, we
(Fig. 3, Table 1, and Dataset S2). This suggests that the MROs of searched for homologs of known Drosophila nuclear genes that
H. salminicola still perform diverse metabolic functions, similar typically encode ∼100 proteins from the mt electron-transport
to the mitochondria of M. squamalis. In contrast, almost all chain complexes (Fig. 3 and SI Appendix, Supplementary Meth-
nuclear-encoded proteins involved in mt genome replication and ods). Our searches of all myxozoan genomes available revealed
translation were absent from the H. salminicola genome. Using a that nuclear genes for only seven of these mt proteins remain in H.
database of 118 such nuclear-encoded genes in Drosophila, we salminicola, whereas 18 to 25 are present in other myxozoans (Fig.
identified 41 to 58 homologous mt genes in M. squamalis and 3, Table 1, and Dataset S2). Specifically, all complex I, III, and IV
among published myxozoan data (14, 18), but only six of these genes that we identified in other myxozoans are absent in H.
genes in H. salminicola (Table 1 and Dataset S3). In addition, we salminicola (Fig. 3B, Dataset S2, and SI Appendix, Supplementary
calculated that H. salminicola does not have a faster evolutionary Results and Fig. S10) or present as pseudogenes (SI Appendix,
rate than other myxozoans, which might otherwise have pre- Fig. S9). Since complex IV interacts with O2 molecules, we
cluded gene discovery (Fig. 1 and SI Appendix, Fig. S2). conclude that H. salminicola might not be capable of standard
Interestingly, in H. salminicola, we found that the mt DNA cellular aerobic respiration. In concurrence with the absence of
polymerase subunit gamma-1 (19) gene is a pseudogene, as it the complexes that pump protons into the mitochondrial in-
contains three point mutations that create premature stop codons termembrane space (i.e., complexes I, III, and IV), most genes
(SI Appendix, Fig. S9). Furthermore, this gene is not expressed in that encode the Fo subunit of the adenosine triphosphate (ATP)
EVOLUTION
Oxidative phosphorylation
Oxidative phosphorylation Present Absent
H2 synthesis
C Mitochondrial / MRO pathways
Propionate
TCA cycle
Glycine
present in selected species
Cristae
ASCT
PNO
PDH
AOX
DNA
SCS
ACS
PFO
PFL
CIV
CIII
Legend
CV
CII
CI
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Fig. 3. Comparison between the pathways present in (A) a typical aerobic mitochondrion and (B) the H. salminicola MRO. (C) Mitochondrial/MRO pathways present
in selected species (see refs. 1 and 2). The presence and absence of organellar genomes are indicated. ACS, acetyl-CoA synthetase; acetate AOX, alternative oxidase;
ASCT, acetate succinyl-CoA transferase; DNA pol, mtDNA polymerase; RNA pol, mtDNA-dependent RNA polymerase; CI-CV, respiratory complexes I-V; C, cytochrome
c; PDH, pyruvate dehydrogenase; PFL, pyruvate formate lyase; PFO, pyruvate ferredoxin oxidoreductase; PNO, pyruvate NADPH oxidoreductase; SCS, succinyl-CoA
synthetase; TCA cycle, tricarboxylic acid cycle; UQ, ubiquinone; e−, electrons; H+, protons; ψ indicates the presence of a pseudogene in the nuclear genome.
synthase complex (i.e., the proton channel of complex V) are also times independently, some of them present striking similarities
missing in H. salminicola (Dataset S4), while being present in (1, 2). Not only have MROs often lost the same mt pathways
Myxobolus (Dataset S4). This suggests that a proton gradient is (e.g., pyruvate dehydrogenase or electron transport chain en-
absent across the inner organelle membrane in H. salminicola. In zymes) but also, in several cases, homologous enzymes, such as
contrast, for complex II, which is part of the Krebs cycle, and for the hydrogenases or pyruvate formate lyases, have been acquired
F1 subunit of the ATP synthase, H. salminicola encodes a similar independently by horizontal gene transfer. These enzymes allow
number of protein coding genes as other myxozoans (Dataset S4). ATP production by anaerobic pyruvate metabolism and H2 syn-
thesis. MROs with such abilities are called hydrogen-producing
Discussion mitochondria or hydrogenosomes, the latter having lost their
Structurally, H. salminicola has lost its mt genome, but has retained ability to utilize oxygen (Fig. 3C) (1, 2).
an organelle that resembles a mitochondrion. However, as mito- As our H. salminicola assemblies did not contain any hydrogenase
chondria are defined based on the use of oxygen as electron ac- or other genes of prokaryotic origin (Fig. 3C and Dataset S5), we
ceptor (21), and usually the presence of an mt genome (but see conclude that the MROs in H. salminicola are not hydrogenosomes.
ref. 22), we conclude that H. salminicola possesses MROs rather The presence of cristae in H. salminicola’s MRO is surprising since
than true mitochondria. Although MROs have evolved several these membrane invaginations are usually absent in anaerobic
Yahalomi et al. PNAS | March 10, 2020 | vol. 117 | no. 10 | 5361
MROs (1, 16, 17). However, we note that the MROs of H. visualized under a Leica DMR compound fluorescence microscope at 630×
salminicola share these characteristics with the MRO of the and 1,000× magnification.
apicomplexan Cryptosporidium muris, which has also lost com-
Electron Microscopy. Fresh parasite pseudocysts were dissected from the tissue
plexes I, III, and IV, but possesses an alternative oxidase and
of a single host and then fixed in a solution comprising 1% glutaraldehyde
retains cristae (Fig. 3C) (23). The presence of cristae together with and 2% paraformaldehyde in 0.1 M phosphate buffer. Larger pseudocysts
the identification of pseudogenes suggest that the loss of mtDNA were sliced to permit penetration of the fixative. Fixed tissue was then
and aerobic respiration may be a recent evolutionary event in the stained with osmium tetroxide and then uranyl acetate, before being
Henneguya lineage. Future experiments are needed to better dehydrated in a graded alcohol series and embedded in Epon resin. Ultrathin
characterize the metabolic energy pathways of H. salminicola. sections were mounted on copper grids and examined using a Helios 650 FEG
However, such experiments are challenging because it is currently dual-beam SEM (Thermo Scientific) in transmission mode, at the Oregon State
not possible to culture H. salminicola in the laboratory. University Electron Microscope Facility.
Similar to most Myxozoa, H. salminicola likely alternates be-
tween two hosts (6). In its fish host, it undergoes proliferation Filtering and Assembling the Genomic and Transcriptomic Data. Stringent
filtering, involving multiple steps, was performed to eliminate host
and sporogenesis in pseudocysts within the white muscle (11), a
and bacterial contamination from M. squamalis and H. salminicola data. This
tissue known to have anaerobic metabolism (24). While the obli- involved mapping reads to the corresponding host genomes and several
gate invertebrate host of H. salminicola is unknown, it is probably rounds of BLAST searches against the National Center for Biotechnology In-
an annelid from the family Naididae, based on known life cycles of formation nucleotide database (SI Appendix, Supplementary Methods). Reads
related myxozoans (25). Members of the Naididae can grow and from contaminant sequences were eliminated and the remaining DNA and
reproduce in anoxic environments (26). As all protists that have RNA reads assembled using IDBA (version 1.1.1) (34) and Trinity (35), re-
lost their mt genomes live in anaerobic environments, we specu- spectively (SI Appendix, Supplementary Methods).
late that the loss of the mt genome in H. salminicola was driven by
low-oxygen environments in both of its hosts. Assembly of the M. squamalis mt Genome and Absence of mt Sequences in
H. salminicola. Local blastn and tblastn searches using cnidarian (including
Loss of superfluous genes likely conveys an evolutionary ad-
published myxozoan) mt genome and protein sequences, respectively, were
vantage, as it has been shown that the bioenergetic cost of a gene performed against our myxozoan assemblies of M. squamalis and H. salmi-
is higher in small genomes (27). Myxozoans have smaller ge- nicola. After manual inspection of all sequences with E-values <1e−1, no
nomes [22 to 180 Mb (14, 18)] than free-living Cnidaria [>250 mitochondrial sequence could be identified for H. salminicola. In contrast, a
Mb (28, 29)]. Therefore, the loss of the mt genome and associ- putative mitochondrial contig was identified for M. squamalis. The coverage
ated nuclear genes involved in its replication and electron of the mt genome was 185, about twice the nuclear coverage. To further
pathways may be advantageous for a myxozoan living in anaer- search the H. salminicola data, Hidden Markov Model (HMM) profiles were
obic environments. However, the loss of useless genes by random built based on alignments of myxozoan mt proteins, using HMMer3.0 (36).
drift cannot be excluded. Interestingly, our results also open the These profiles were used to search protein predictions of H. salminicola by
Maker2 v2.31.10 (37) (see SI Appendix, Supplementary Methods, for details
way to new treatment options against this pathogen, since an-
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1. A. J. Roger, S. A. Muñoz-Gómez, R. Kamikawa, The origin and diversification of mi- 26. P. Famme, J. Knudsen, Anoxic survival, growth and reproduction by the freshwater
tochondria. Curr. Biol. 27, R1177–R1192 (2017). annelid, Tubifex sp., demonstrated using a new simple anoxic chemostat. Comp. Bio-
2. C. W. Stairs, M. M. Leger, A. J. Roger, Diversity and origins of anaerobic metabolism in chem. Physiol. A Comp. Physiol. 81, 251–253 (1985).
mitochondria and related organelles. Philos. Trans. R. Soc. Lond. B Biol. Sci. 370, 27. M. Lynch, G. K. Marinov, The bioenergetic costs of a gene. Proc. Natl. Acad. Sci. U.S.A.
20140326 (2015). 112, 15690–15695 (2015).
EVOLUTION
3. J. M. Bernhard et al., Metazoans of redoxcline sediments in Mediterranean deep-sea 28. J. A. Chapman et al., The dynamic genome of Hydra. Nature 464, 592–596 (2010).
hypersaline anoxic basins. BMC Biol. 13, 105 (2015). 29. N. H. Putnam et al., Sea anemone genome reveals ancestral eumetazoan gene rep-
4. R. Danovaro et al., The first metazoa living in permanently anoxic conditions. BMC ertoire and genomic organization. Science 317, 86–94 (2007).
Biol. 8, 30 (2010). 30. S. Löfmark, C. Edlund, C. E. Nord, Metronidazole is still the drug of choice for treat-
5. R. Danovaro et al., The challenge of proving the existence of metazoan life in per- ment of anaerobic infections. Clin. Infect. Dis. 50 (suppl. 1), S16–S23 (2010).
manently anoxic deep-sea sediments. BMC Biol. 14, 43 (2016). 31. E. Kayal et al., Phylogenomics provides a robust topology of the major cnidarian lineages
6. B. Okamura, A. Gruhl, J. L. Bartholomew, “An introduction to myxozoan evolution, ecology and insights on the origins of key organismal traits. BMC Evol. Biol. 18, 68 (2018).
and development” in Myxozoan Evolution Ecology and Development, B. Okamura, A. 32. S. D. Atkinson, J. L. Bartholomew, T. Lotan, Myxozoans: Ancient metazoan parasites
Downloaded from https://www.pnas.org by 187.255.193.154 on May 30, 2023 from IP address 187.255.193.154.
Gruhl, J. L. Bartholomew, Eds. (Springer International Publishing, 2015), pp. 1–20. find a home in phylum Cnidaria. Zoology (Jena) 129, 66–68 (2018).
7. I. Fontes, S. L. Hallett, T. A. Mo, “Comparative epidemiology of myxozoan diseases” 33. M. V. Olson, When less is more: Gene loss as an engine of evolutionary change. Am. J.
in Myxozoan Evolution Ecology and Development, B. Okamura, A. Gruhl, Hum. Genet. 64, 18–23 (1999).
J. L. Bartholomew, Eds. (Springer International Publishing, 2015), pp. 317–341. 34. Y. Peng, H. C. M. Leung, S. M. Yiu, F. Y. L. Chin, “IDBA–A practical iterative de Bruijn
8. D. Yahalomi et al., The multipartite mitochondrial genome of Enteromyxum leei graph de novo assembler” in Research in Computational Molecular Biology. RECOMB
(Myxozoa): Eight fast-evolving megacircles. Mol. Biol. Evol. 34, 1551–1556 (2017). 2010, B. Berger, Ed. (Lecture Notes Computer Science, 2010), vol. 6044, pp. 426–440.
9. F. Takeuchi et al., The mitochondrial genomes of a myxozoan genus Kudoa are ex- 35. B. J. Haas et al., De novo transcript sequence reconstruction from RNA-seq using the
tremely divergent in Metazoa. PLoS One 10, e0132030 (2015). Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–1512 (2013).
10. I. Fiala, P. Bartošová-Sojková, C. M. Whipps, “Classification and phylogenetics of 36. J. Mistry, R. D. Finn, S. R. Eddy, A. Bateman, M. Punta, Challenges in homology search:
Myxozoa” in Myxozoan Evolution Ecology and Development, B. Okamura, A. Gruhl, J. HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res. 41, e121 (2013).
L. Bartholomew, Eds. (Springer International Publishing, 2015), pp. 85–110. 37. C. Holt, M. Yandell, MAKER2: An annotation pipeline and genome-database manage-
11. F. F. Fish, Observations on Henneguya salminicola Ward, a myxosporidian parasitic in ment tool for second-generation genome projects. BMC Bioinformatics 12, 491 (2011).
Pacific salmon. J. Parasitol. 25, 169–172 (1939). 38. E. P. Nawrocki, S. R. Eddy, Infernal 1.1: 100-fold faster RNA homology searches. Bio-
12. T. M. Polley, S. D. Atkinson, G. R. Jones, J. L. Bartholomew, Supplemental description informatics 29, 2933–2935 (2013).
of Myxobolus squamalis (Myxozoa). J. Parasitol. 99, 725–728 (2013). 39. N. Dierckxsens, P. Mardulyn, G. Smits, NOVOPlasty: De novo assembly of organelle
13. G. Parra, K. Bradnam, I. Korf, CEGMA: A pipeline to accurately annotate core genes in genomes from whole genome data. Nucleic Acids Res. 45, e18 (2017).
eukaryotic genomes. Bioinformatics 23, 1061–1067 (2007). 40. R. M. Waterhouse et al., BUSCO applications from quality assessments to gene pre-
14. E. S. Chang et al., Genomic insights into the evolutionary origin of Myxozoa within diction and phylogenomics. Mol. Biol. Evol. 35, 543–548 (2018).
Cnidaria. Proc. Natl. Acad. Sci. U.S.A. 112, 14912–14917 (2015). 41. G. W. Vurture et al., GenomeScope: Fast reference-free genome profiling from short
15. A. Karnkowska et al., A eukaryote without a mitochondrial organelle. Curr. Biol. 26, reads. Bioinformatics 33, 2202–2204 (2017).
1274–1284 (2016). 42. G. Marçais, C. Kingsford, A fast, lock-free approach for efficient parallel counting of
16. M. A. Huynen, M. Mühlmeister, K. Gotthardt, S. Guerrero-Castillo, U. Brandt, Evolu- occurrences of k-mers. Bioinformatics 27, 764–770 (2011).
tion and structural organization of the mitochondrial contact site (MICOS) complex 43. L. S. Gramates et al.; The FlyBase Consortium, FlyBase at 25: Looking to the future.
and the mitochondrial intermembrane space bridging (MIB) complex. Biochim. Bio- Nucleic Acids Res. 45, D663–D671 (2017).
phys. Acta 1863, 91–101 (2016). 44. D. D’Elia et al., The MitoDrome database annotates and compares the OXPHOS nu-
17. S. A. Muñoz-Gómez et al., Ancient homology of the mitochondrial contact site and clear genes of Drosophila melanogaster, Drosophila pseudoobscura and Anopheles
cristae organizing system points to an endosymbiotic origin of mitochondrial cristae. gambiae. Mitochondrion 6, 252–257 (2006).
Curr. Biol. 25, 1489–1495 (2015). 45. O. Rackham, T. R. Mercer, A. Filipovska, The human mitochondrial transcriptome and
18. Y. Yang et al., The genome of the myxosporean Thelohanellus kitauei shows adapta- the RNA-binding proteins that regulate its expression. Wiley Interdiscip. Rev. RNA 3,
tions to nutrient acquisition within its fish host. Genome Biol. Evol. 6, 3182–3198 (2014). 675–695 (2012).
19. M. A. Graziewicz, M. J. Longley, W. C. Copeland, DNA polymerase γ in mitochondrial 46. V. Muthye, D. V. Lavrov, Characterization of mitochondrial proteomes of non-
DNA replication and repair. Chem. Rev. 106, 383–405 (2006). bilaterian animals. IUBMB Life 70, 1289–1301 (2018).
20. D. V. Lavrov, W. Pett, Animal mitochondrial DNA as we do not know it: Mt-genome or- 47. S. M. Adl et al., Revisions to the classification, nomenclature, and diversity of eu-
ganization and evolution in nonbilaterian lineages. Genome Biol. Evol. 8, 2896–2913 (2016). karyotes. J. Eukaryot. Microbiol. 26, 12691 (2018).
21. M. Müller et al., Biochemistry and evolution of anaerobic energy metabolism in eu- 48. B. Roure, N. Rodriguez-Ezpeleta, H. Philippe, SCaFoS: A tool for selection, concatena-
karyotes. Microbiol. Mol. Biol. Rev. 76, 444–495 (2012). tion and fusion of sequences for phylogenomics. BMC Evol. Biol. 7 (suppl. 1), S2 (2007).
22. U. John et al., An aerobic eukaryotic parasite with functional mitochondria that likely 49. J. Castresana, Selection of conserved blocks from multiple alignments for their use in
lacks a mitochondrial genome. Sci. Adv. 5, eaav1110 (2019). phylogenetic analysis. Mol. Biol. Evol. 17, 540–552 (2000).
23. T. Makiuchi, T. Nozaki, Highly divergent mitochondrion-related organelles in anaer- 50. N. Lartillot, H. Philippe, A Bayesian mixture model for across-site heterogeneities in
obic parasitic protozoa. Biochimie 100, 3–17 (2014). the amino-acid replacement process. Mol. Biol. Evol. 21, 1095–1109 (2004).
24. I. A. Johnston, Studies on the swimming musculature of the rainbow trout. J. Fish Biol. 51. N. Lartillot, H. Brinkmann, H. Philippe, Suppression of long-branch attraction artefacts in the
7, 459–467 (1975). animal phylogeny using a site-heterogeneous model. BMC Evol. Biol. 7 (suppl. 1), S4 (2007).
25. J. D. Alexander, B. L. Kerans, M. El-Matbouli, S. L. Hallett, L. Stevens, “Annelid-myxosporean 52. N. Lartillot, N. Rodrigue, D. Stubbs, J. Richer, PhyloBayes MPI: Phylogenetic re-
interactions” in Myxozoan Evolution Ecology and Development, B. Okamura, A. Gruhl, J. construction with infinite mixtures of profiles in a parallel environment. Syst. Biol. 62,
L. Bartholomew, Eds. (Springer International Publishing, 2015), pp. 217–234. 611–615 (2013).
Yahalomi et al. PNAS | March 10, 2020 | vol. 117 | no. 10 | 5363