Convergent Evolution of Hydrogenosomes from Mitochondria by Gene Transfer and Loss William H
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Convergent Evolution of Hydrogenosomes from Mitochondria by Gene Transfer and Loss William H. Lewis,*,†,1,2,3 Anders E. Lind,†,2 Kacper M. Sendra,1 Henning Onsbring,2,3 Tom A. Williams,4 Genoveva F. Esteban,5 Robert P. Hirt,1 Thijs J.G. Ettema,2,3 and T. Martin Embley*,1 1Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-Upon-Tyne, United Kingdom 2Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden 3Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands 4School of Biological Sciences, University of Bristol, Bristol, United Kingdom 5Department of Life and Environmental Sciences, Bournemouth University, Poole, United Kingdom †These authors contributed equally to this work. *Corresponding authors: E-mails: [email protected]; [email protected]. Associate editor: Fabia Ursula Battistuzzi All sequencing data generated in the present study have been deposited in an NCBI BioProject (accession no. PRJNA542330). Abstract Hydrogenosomes are H2-producing mitochondrial homologs found in some anaerobic microbial eukaryotes that provide a rare intracellular niche for H2-utilizing endosymbiotic archaea. Among ciliates, anaerobic and aerobic lineages are interspersed, demonstrating that the switch to an anaerobic lifestyle with hydrogenosomes has occurred repeatedly and independently. To investigate the molecular details of this transition, we generated genomic and transcriptomic data sets from anaerobic ciliates representing three distinct lineages. Our data demonstrate that hydrogenosomes have evolved from ancestral mitochondria in each case and reveal different degrees of independent mitochondrial genome and proteome reductive evolution, including the first example of complete mitochondrial genome loss in ciliates. Intriguingly, the FeFe-hydrogenase used for generating H2 has a unique domain structure among eukaryotes and appears to have been present, potentially through a single lateral gene transfer from an unknown donor, in the common aerobic ancestor of all three lineages. The early acquisition and retention of FeFe-hydrogenase helps to explain the facility whereby mitochondrial function can be so radically modified within this diverse and ecologically important group of microbial eukaryotes. Key words: evolution, genomics, hydrogenosomes, mitochondria, microbial eukaryotes, anaerobic metabolism. Article Introduction Ciliates provide an excellent system for studying the evo- Mitochondria are an ancestral feature of eukaryotic cells that lutionary transition from mitochondria to hydrogenosomes have diversified in form and function during their separate because anaerobic, hydrogenosome-containing ciliates are in- evolution in eukaryotes under different living conditions, pro- terleaved among aerobic, mitochondria-bearing forms in the ducing a spectrum of homologous organelles with different ciliate tree (Embley et al. 1995; Fenchel and Finlay 1995). proteomes and phenotypes (Embley and Martin 2006; Muller Previous work has also shown that the hydrogenosomes of et al. 2012; Stairs et al. 2015). Among the most interesting of the anaerobic ciliate Nyctotherus ovalis haveamitochondrial these mitochondrial homologs are the hydrogenosomes genome, providing direct molecular evidence of their mito- (Mu¨ller 1993) found in anaerobic free-living and parasitic chondrial ancestry (Akhmanova et al. 1998; Boxma et al. 2005; microbial eukaryotes. Hydrogenosomes produce H2 using de Graaf et al. 2011). The retention of a mitochondrial ge- the enzyme FeFe-hydrogenase, a type of metabolism that nome contrasts with better-studied hydrogenosome-con- has been typically associated with bacteria rather than eukar- taining protists like Trichomonas, where the organellar yotes (Mu¨ller 1993; Embley et al. 1997; Horner et al. 2000; genome has been entirely lost, and where close metamonad Muller et al. 2012; Stairs et al. 2015). The evolution of hydro- relatives lack classical aerobic mitochondria for comparison genosomes and the origins of their anaerobic metabolism are (Stairs et al. 2015; Leger et al. 2017). Anaerobic ciliates thus actively debated (Martin and Mu¨ller 1998; Martin et al. 2015; provide a rare opportunity to investigate the plasticity of Stairs et al. 2015; Spang et al. 2019). Here, we have addressed mitochondrial function and the repeated convergent evolu- these questions by investigating the repeated convergent evo- tion of hydrogenosomes within a phylogenetically coherent lution of hydrogenosomes from mitochondria among free- and diverse lineage (Embley et al. 1995, 1997; Akhmanova living anaerobic ciliates. et al. 1998; Boxma et al. 2005; de Graaf et al. 2011). ß The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access 524 Mol. Biol. Evol. 37(2):524–539 doi:10.1093/molbev/msz239 Advance Access publication October 24, 2019 Convergent Evolution of Hydrogenosomes . doi:10.1093/molbev/msz239 MBE Anaerobic ciliates with hydrogenosomes are unusual genome (mtDNA), we generated new molecular data among eukaryotes because they typically harbor endosymbi- sets for M. contortus, M. es, M. striatus, P. frontata, otic archaea, which in some cases form intricate physical T. finlayi (Lewis et al. 2018), and C. porcatum using multi- interactions with ciliate hydrogenosomes (van Bruggen ple displacement amplification and genomic sequencing et al. 1983; Finlay and Fenchel 1991; Embley et al. 1992; of DNA from hydrogenosome-enriched samples, and Fenchel and Finlay 1995). The endosymbionts are methano- complemented these data using single-cell RNAseq (sup- gens that use the H2 produced by hydrogenosomes as an plementary table 1, Supplementary Material online). We electron donor for ATP-producing methanogenesis also produced new data for N. ovalis to complement the (Fenchel and Finlay 1995). Physiological studies (Fenchel partial mtDNA sequence already available for this species and Finlay 1991, 1995) have suggested that the endosym- (Akhmanova et al. 1998; de Graaf et al. 2011). We found bionts provide an electron sink that can compensate for evidence for mtDNA in samples from M. contortus, M. es, the reduced oxidative capacity of ciliate hydrogenosomes. M. striatus,andN. ovalis, including ribosomal RNA genes Consistent with this, published data (Fenchel and Finlay that cluster strongly with mitochondrial sequences from 1991) have demonstrated that large ciliates like Metopus con- related aerobic mitochondria-containing ciliates (fig. 1b). tortus and Plagiopyla frontata grow better when they harbor These data suggest that retention of mtDNA may be a methanogens. However, there are currently no published conserved feature of the exclusively anaerobic class data describing the proteomes and electron transport chains Armophorea (Fenchel and Finlay 1995; Lynn 2008) that (ETCs) of the hydrogenosomes of either species to identify contains Metopus and Nyctotherus. We did not detect any the molecular details underpinning their symbioses. mtDNA in the samples for C. porcatum, but we did detect In the present study, we have investigated the repeated transcripts in the C. porcatum RNAseq data for mitochon- convergent evolution of ciliate hydrogenosomes (Embley drial protein-coding genes (fig. 2) and for mitochondrial et al. 1995), in representative species from three taxonomi- LSU and SSU rRNA (fig. 1b), which suggests that cally distinct anaerobic lineages. Small-scale genomic amplifi- C. porcatum has also retained a mitochondrial genome. cation after hydrogenosome enrichment was used to recover By contrast, we found no molecular evidence for mtDNA organellar genome sequences for individual species, and these in the hydrogenosomes of P. frontata or T. finlayi.This data were complemented by nuclear and organellar tran- suggests that species in this clade have, so far uniquely scriptomics data generated by single-cell RNAseq. The mo- among ciliates, completely lost the mitochondrial genome lecular data sets generated were used to reconstruct during hypoxia-driven reductive evolution of their hydrogenosome metabolism for Cyclidium porcatum, M. hydrogenosomes. contortus and P. frontata, and phylogenetics was used to in- We assembled a single 48,118-bp contig of mtDNA for vestigate the evolutionary history and origins of the key an- N. ovalis (fig. 2a and b) which is similar to the size (48 kb) aerobic enzymes for H2 generation. of its mtDNA as previously estimated from Southern blots (de Graaf et al. 2011). Based on the size of the mtDNA of Results and Discussion N. ovalis,weestimatethatwealsoobtainednear-complete mtDNA data for M. contortus (48,599 bp) and M. es Phylogenetic Analysis Supports the Independent (48,877 bp), assembled in several short contigs and transcripts Origins of Hydrogenosomes in Different Anaerobic (fig. 2). One end of the new contig from N. ovalis was found to Ciliate Lineages contain a 38-bp sequence (TATTGTAATACTAATAATA Selective enrichment culturing was used to isolate M. contortus TGTGTGTTAATGCGCGTAC) that is repeated in tandem and P. frontata from marine sediments and C. porcatum, three times, resembling the structure of telomeres from the Metopus es, Metopus