Unexpected mitochondrial genome diversity revealed by targeted single-cell genomics of heterotrophic flagellated protists

Abstract

Most eukaryotic microbial diversity is uncultivated, under-studied and lacks nuclear genome data. Mitochondrial genome sampling is more comprehensive, but many phylogenetically important groups remain unsampled. Here, using a single-cell sorting approach combining tubulin-specific labelling with photopigment exclusion, we sorted flagellated heterotrophic unicellular eukaryotes from Pacific Ocean samples. We recovered 206 single amplified genomes, predominantly from underrepresented branches on the tree of life. Seventy single amplified genomes contained unique mitochondrial contigs, including 21 complete or near-complete mitochondrial genomes from formerly under-sampled phylogenetic branches, including telonemids, katablepharids, cercozoans and marine stramenopiles, effectively doubling the number of available samples of heterotrophic flagellate mitochondrial genomes. Collectively, these data identify a dynamic history of mitochondrial genome evolution including intron gain and loss, extensive patterns of genetic code variation and complex patterns of gene loss. Surprisingly, we found that stramenopile mitochondrial content is highly plastic, resembling patterns of variation previously observed only in plants.

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Fig. 1: V9-nSSU phylogenetic mapping of Monterey Bay SAGs.
Fig. 2: Clade-specific maximum-likelihood subtrees showing subsections of the eukaryotic diversity sampled.
Fig. 3: Distribution and groupings of mitochondrial sequence coverage relative to estimated nuclear genome completeness.
Fig. 4: Uncharacterized mtDNAs from underrepresented eukaryotic groups.
Fig. 5: Comparison between mtDNA gene repertoires.
Fig. 6: Phylogenetic reconstruction of representative stramenopiles using concatenated conserved mitochondria-encoded electron transport chain proteins.

Data availability

Complete mtDNA sequences assembled from this study are available at GenBank under the accession numbers MK188935 to MK188947, MN082144 and MN082145. Sequencing data are available under NCBI BioProject PRJNA379597. Reads have been deposited at NCBI Sequence Read Archive with accession number SRP102236. Partial mtDNA contigs and other important contigs mentioned in the text are available from Figshare at https://doi.org/10.6084/m9.figshare.7314728. Nuclear SAG assemblies are available from Figshare at https://doi.org/10.6084/m9.figshare.7352966. A protocol is available from protocols.io at: https://doi.org/10.17504/protocols.io.ywpfxdn.

Code availability

The bioinformatic workflow is available at https://doi.org/10.5281/zenodo.192677; additional statistical analysis code is available at https://doi.org/10.6084/m9.figshare.9884309.

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Acknowledgements

We thank F. Lang and N. Beck for annotation assistance and access to an unreleased version of mfannot, D. Price for assistance with picozoan SAG data, and C. Dunn for discussions and encouragement. This project was supported by a Gordon and Betty Moore foundation grant (GBMF3307) to T.A.R., A.E.S., A.Z.W. and P.J.K. and a Philip Leverhulme Award (PLP-2014–147) to T.A.R.. Field sampling was supported by the David and Lucile Packard Foundation and GBMF3788 to A.Z.W., T.A.R. and A.M. are supported by Royal Society University Research Fellowships. J.G.W. was supported by the European Molecular Biology Organization Long-term Fellowship (ALTF 761–2014) co-funded by the European Commission (EMBOCOFUND2012, GA-2012–600394) support from Marie Curie Actions and a College for Life Sciences Fellowship at the Wissenschaftskolleg zu Berlin. R.R.-M. is supported by CONICYT FONDECYT 11170748. F.M. is supported by Genome Canada.

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J.G.W. performed bioinformatic and phylogenetic analyses and wrote the manuscript. R.R.-M. performed molecular biological analyses. A.M. performed bioinformatic and phylogenetic analyses and G.L. performed bioinformatic analyses. E.C. and C.P. collected the samples and performed flow cytometry. F.M. performed statistical and bioinformatic analyses. D.M. performed molecular biological experiments and generated biochemical reagents. K.M. performed genome sequencing. N.A.T.I. analysed genomic data. T.A.R. devised the project. J.G.W., A.E.S., P.J.K., A.Z.W. and T.A.R. supervised the project and wrote the manuscript. All authors contributed to the editing of the final manuscript.

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Correspondence to Jeremy G. Wideman or Thomas A. Richards.

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Extended data

Extended Data Fig. 1 Rank abundance curve of amplicon sequence variants (ASVs) from the Monterey Bay nSSU-V9 environmental census.

Relative abundances correspond to the mean relative abundance of each ASV in samples from two depths (20 m and 30 m) of eastern North Pacific station M2 (SAGs were recovered from 30 m depth). ASV sequences identical to V9 sequences from SAGs with recovered mitochondrial genomic information are represented by red circles; ASVs with no identical sequence match to V9 SAGs with mitochondrial data are represented by grey circles. For each ASV identical to a SAG V9, the corresponding SAG codenames are provided (in some cases there are multiple of each type). Samples are coloured according to taxonomic affiliation in V9 sorting. Blue, stramenopile; teal, hacrobian; purple, rhizarian; brown, opisthokont. See Supplemenatry Table 7 for details on ASV relative abundance.

Extended Data Fig. 2 Cox1 protein phylogeny.

Cox1 proteins were collected from representative eukaryote groups using BLAST26, aligned using MUSCLE81, and manually trimmed to a resulting 402 sites. We reconstructed the phylogeny of Cox1 using RAxML v8.2.1084 (100 bootstrap pseudoreplicates) under the LG model85. Maximum likelihood support values are indicated above each branch. The Cox1 from As1 grouped within the myzozoan alveolates within a fully supported clade comprising dinoflagellates, apicomplexans, and ‘chromerid’ algae. Picozoan M5584–11 Cox1 does not branch strongly with any eukaryotic group. Numbers in brackets indicate number of sequences collapsed.

Extended Data Fig. 3 Telonemid mtDNAs encode a putative rpl18 and retain partial synteny with the bacterial-like genomes of jakobids.

In all telonemid mitochondrial DNAs examined rps8, rpl6, and rpl18 were found in synteny as in mtDNAs of jakobids. Malawimonas jakobiformis is somewhat similar as rpl6 and rpl18 are found adjacent to one another. Genbank: Andalucia godoyi NC_021124.1, Histiona aroides NC_021125.1, Jakoba bahamiensis NC_021126.1, Jakoba libera NC_021127.1, Reclinomonas americana NC_001823.1, Seculamonas ecuadoriensis NC_021128.1, Malawimonas jakobiformis NC_002553.1. Small subunit ribosomal genes are coloured in pink, large subunit ribosomal genes in red, SecY in purple, and electron transport chain components in grey.

Extended Data Fig. 4 Thraustochytrid mtDNAs harbour a unique genetic code.

Alignment of mitochondria-encoded Cob proteins from Thraustochytrium aureum, Schizochytrium sp., and four putative thraustochytrid SAGs. Cob genes with internal stop codons were identified in mitochondrial contigs from each SAG and translated using the standard genetic code. These proteins were aligned using MUSCLE81 with proteins from publicly available thraustochytrid mtDNAs (KU183024.1 and AF288091.2). Positions occupied by TAG or TAA codons are marked with yellow asterisks and aligned most often with tyrosine or other hydrophobic residues (marked in orange). Relatively few TAA and TAG codons were conserved between genome sequences suggesting that these changes occurred during the recent radiation of this lineage.

Extended Data Fig. 5 Distribution of mitochondria-encoded tRNAs.

Comparison of mtDNA tRNA coding capacities from: new assemblies from this study (bold font), previously sequenced mtDNAs (regular font), and ancestral reconstructions (L-Dia- CA, Last Diaphoretickes Common Ancestor; L-Amo-CA, Last Amorphean Common Ancestor - including malawimonads and collodictyonids)); L-Jak-CA, Last Jakobid Common Ancestor; LECA, Last Eukaryote Common Ancestor. # symbols indicate incomplete mtDNA. Asterisks indicate genomes assembled from publicly available datasets. Black filled square, present; empty square, absent. Red filled squares indicate an independent codon reassignment. In some lineages extra tRNAs are also present other than the common tRNAs presented: a, I (uau), one cercozoan lineage (R32) contained a possible suppressor tRNA (gcaa); b, I (uau); c, L (caa); d, I (aau); e, L (gag), N (auu).

Extended Data Fig. 6 Gating strategy for cell sort 35 from which most SAGs originated.

A combination of gates (black polygons) was applied to select. a. cells larger than Synechococcus displaying low red fluorescence to exclude photosynthetic eukaryotes and b. cells stained with Oregon Green as compared to c. an unstained sample. The green rectangles show the position of 0.75 μm yellow-green beads.

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Wideman, J.G., Monier, A., Rodríguez-Martínez, R. et al. Unexpected mitochondrial genome diversity revealed by targeted single-cell genomics of heterotrophic flagellated protists. Nat Microbiol 5, 154–165 (2020). https://doi.org/10.1038/s41564-019-0605-4

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