Evolution of the Mitochondrial Genome: Protist Connections to Animals, Fungi and Plants Charles E Bullerwell and Michael W Gray

Evolution of the mitochondrial genome: protist connections to animals, fungi and plants Charles E Bullerwell and Michael W Gray

The past decade has seen the determination of complete DNA (mtDNA). For example, the first sequenced mito- mitochondrial genome sequences from a taxonomically chondrial genomes of animals, fungi and plants were diverse set of organisms. These data have allowed an found to have very dissimilar genome organizations that unprecedented understanding of the evolution of the did not immediately suggest common evolutionary ori- mitochondrial genome in terms of gene content and order, as gins. To address issues relating to mtDNA structural well as genome size and structure. In addition, phylogenetic diversity in the context of mitochondrial evolution, infor- reconstructions based on mitochondrial DNA (mtDNA)- mation was needed about mitochondrial genomes from encoded protein sequences have firmly established the representatives spanning the phylogenetic breadth and identities of protistan relatives of the animal, fungal and plant depth of eukaryotes (domain Eucarya). Of particular lineages. Analysis of the mtDNAs of these protists has provided importance in this regard were the protists, mainly uni- insight into the structure of the mitochondrial genome at the cellular organisms that encompass most of the evolution- origin of these three, mainly multicellular, eukaryotic groups. ary diversity of this domain. Further research into mtDNAs of taxa ancestral and intermediate to currently characterized organisms will help to Sequencing efforts over the past decade, particularly refine pathways and modes of mtDNA evolution, as well as those of the Organelle Genome Megasequencing Pro- provide valuable phylogenetic characters to assist in unraveling gram (OGMP; http://megasun.bch.umontreal.ca/ogmp) the deep branching order of all eukaryotes. [4,5] and the Fungal Mitochondrial Genome Project (FMGP; http://megasun.bch.umontreal.ca/People/lang/ Addresses FMGP/FMGP.html) [6,7] have greatly increased the Department of Biochemistry and Molecular Biology, Dalhousie number of complete mtDNA sequences, and also their University, Room 8F-2, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia B3H 1X5, Canada phylogenetic diversity. This research has revealed a vari- e-mail: [email protected] ety of genome structures, from the several hundred small linear pieces found in the mitochondrion of the ichthyosporean Amoebidium parasiticum ([8], discussed below), Current Opinion in Microbiology 2004, 7:528–534 to the gene-rich eubacteria-like mitochondrial genome of This review comes from a themed issue on the jakobid flagellate Reclinomonas americana [9]. These Genomics data, in combination with phylogenies based on mito- Edited by Charles Boone and Philippe Glaser chondrial protein sequences, have strongly supported a

Available online 11th September 2004 monophyletic origin for the mitochondrial genome, specifically from within the a-Proteobacteria [5,10,11,12]. 1369-5274/$ – see front matter Despite enormous variations in genome size, ranging # 2004 Elsevier Ltd. All rights reserved. from the tiny apicomplexan mtDNAs (6 kbp) to the DOI 10.1016/j.mib.2004.08.008 expansive plant mtDNAs (>150 kbp), the coding function of the mitochondrial genome has remained relatively stable. In general, mtDNAs code only for genes involved Abbreviations FMGP Fungal Mitochondrial Genome Project in the mitochondrial translation apparatus, electron trans- mtDNA mitochondrial DNA port and oxidative phosphorylation [12 ]. OGMP Organelle Genome Megasequencing Program A particular goal of the OGMP and the FMGP was to sequence the mitochondrial genomes of protists diverg- Introduction ing basally to the animal, fungal and plant lineages. These The first mitochondrial genome to be completely studies aimed to define the evolution of the mitochondrial sequenced (in 1981) was that of Homo sapiens [1]. Over genome from the presumed protist ancestors of these the following decade, mitochondrial genome sequencing groups by identifying evolutionary intermediates. Candi- focused on other members of the metazoan lineage, as dates for these early diverging groups, based on morpho- well as on ascomycete fungi (such as the yeast Saccharo- logical, ultrastructural and molecular evidence, included myces cerevisiae [2]) and land plants (such as the liverwort the choanoflagellates (believed to represent early diver- Marchantia polymorpha [3]). Although these data consti- ging unicellular ancestors of the animals), chytridiomy- tuted an important starting point in the definition of cetes (believed to be related to fungi), and green algae mitochondrial genomics, they were not sufficient for (believed to be specifically affiliated with the land plant determining the origin and evolution of mitochondrial lineage).

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In this review, we focus on recent advances that have infer the structure of the mitochondrial genome before helped to bridge the gap between the well-established the emergence of multicellularity in this lineage. Of animal, fungal and plant lineages and their unicellular particular interest was the timing of the transition from protistan ancestors. the large, gene-rich mtDNAs seen in some protists to the small, gene-poor mitochondrial genomes seen in Clade Holozoa: choanoflagellates and animals. For example, R. americana contains the most ichthyosporeans are specific relatives ancestral (eubacteria-like) mtDNA identified to date: a of the animals 69 kbp genome that encodes almost 100 genes [9].By Choanocytes, the feeding cells of sponges, bear remark- contrast, animal mitochondrial genomes are much smal- able morphological similarity to members of the choano- ler (with sizes ranging from 13 to 22 kbp) and are highly flagellate protists. This resemblance, first recognized in compact [15], with open reading frames and tRNA the 19th century, prompted the long-standing view that genes often overlapping, and some stop codons created sponges represent an early form of multicellular animal, by the addition of a 30 oligo(A) tail to processed specifically related to choanoflagellates. However, in mRNAs. Metazoan mtDNAs generally encode fewer eukaryotic phylogenies based on molecular sequence than 40 genes, a set that includes no ribosomal protein data, the branching position of the choanoflagellates genes. has not been clear-cut (see [13] for discussion). Thus, it has been argued that choanoflagellates are specific The mitochondrial genome of Monosiga brevicollis, a choa- relatives of either animals or fungi, or that they branch noflagellate, is much larger than metazoan mtDNAs and before the divergence of animals and fungi. The specific encodes many more genes than the latter [8]. M. brevi- relationship of choanoflagellates to the metazoan lineage collis mtDNA is 76 568 bp long, circular mapping, and has only recently been firmly established through phylo- encodes 55 different genes, including 11 specifying ribo- genetic reconstructions based on mitochondrial protein somal proteins. This mtDNA is much more similar to sequence data [13] (Figure 1). This analysis also clearly those of protists such as R. americana than to those of confirms a sister group of the animal-choanoflagellate multicellular animals. Genome content and organization clade: the ichthyosporean protists. Ichthyosporeans, once indicate that the extreme reduction in mtDNA observed referred to as DRIPs (from the first initial of the four in metazoans occurred after the divergence of M. brevi- founding members of the group [14]), were similarly collis (and possibly all choanoflagellates) from the line believed to have diverged near the animal-fungal split. leading to metazoan animals (Figure 2). The mitochon- Based on these new data, Metazoa, Choanoflagellata and drial genome sequence from a member of the sponge Ichthyosporea can be considered a monophyletic group lineage, which is likely to have a branching position (Holozoa [13]) that branches as a sister group to Fungi between those of other multicellular animals and choano- (Figures 1 and 2). flagellates (Figure 1), would undoubtedly help to estab- lish more precisely the evolutionary timing of mtDNA With the identities of some relatively close protistan reduction in the animal lineage. relatives of the animals established, it was possible to In contrast to the mtDNA of M. brevicollis, the mtDNA of Amoebidium parasiticum (an ichthyosporean protist) Figure 1 has possibly the most unusual mitochondrial genome structure ever found [8]. A. parasiticum mtDNA is over animals (>460) 200 kbp in size and consists of hundreds of short (0.3– sponges (0) 8.3 kbp) linear fragments. Of the 80 ‘chromosomes’ that Holozoa have been partially or completely sequenced, three choanoflagellates (1) types have been described: i) small DNA molecules ichthyosporeans (1) without identified coding function, ii) medium-sized ascomycetes (16) DNA species carrying a single gene, and iii) larger basidiomycetes (2) Fungi molecules encoding multiple genes. All chromosomes zygomycetes (3) contain a virtually identical array of short terminal chytridiomycetes (7) repeats. Although other instances of linear mtDNAs land plants (6) and mtDNAs consisting of multiple linear or circular charophytes (2) Viridiplantae components have been described (e.g. [16 ]), no other chlorophytes (10) example is known with such an extensive collection of

Current Opinion in Microbiology genomic fragments. Despite the unusual appearance of A. parasiticum mtDNA, the sequences of the proteins it

Schematic phylogenetic tree illustrating the branching order within speciﬁes and the secondary structures of its encoded Holozoa (red), Fungi (blue) and Viridiplantae (green). Numbers in rRNAs clearly identify it as a unicellular ancestor of the brackets indicate complete mtDNA sequences publicly available. animal lineage [8 ]. www.sciencedirect.com Current Opinion in Microbiology 2004, 7:528–534 530 Genomics

Figure 2

choanoflagellates ichthyosporeans

animals 55 >44 37 M. brevicollis H. sapiens A. parasiticum 35 ascomycetes S. cerevisiae 44 basidiomycetes S. commune 40 zygomycetes R. americana R. stolonifer 98 A. macrogynus 41

S. punctatus 24

R. brooksianum 23 H. curvatum 23 Harpochytrium105 Monoblepharella15 24 Harpochytrium94 25 24 chytridiomycetes

Current Opinion in Microbiology

Branching order of deeply diverging groups within Holozoa (red) and Fungi (blue) based on molecular phylogenetic analyses of mtDNA-encoded protein sequences [13,21]. Chytridiomycete orders are indicated by color: Blastocladiales (pink), Spizellomycetales (purple), Chytridiales (orange), Monoblepharidales (brown). The conformation and relative size of the mtDNA in each species is indicated graphically, with the number of identified genes encoded by the mtDNA (not including introns and unidentified ORFs) indicated. R. americana is included as an outgroup for comparison. Species shown ([references], NCBI acc. no.): Reclinomonas americana ([9], AF007261), Homo sapiens ([1], V00662, X93334, J01415, M12548, M58503, M63932, M63933), Monosiga brevicollis ([8], AF538053), Amoebidium parasiticum ([8], AF538042-AF538052), Saccharomyces cerevisiae ([2], AJ011856), Schizophyllum commune ([20], AF402141), Rhizopus stolonifer ([7], FMGP; unpublished), Allomyces macrogynus ([22], U41288) Spizellomyces punctatus ([20], AF404303-AF404305), Rhizophydium brooksianum ([20], AF404306), Hyaloraphidium curvatum ([20], NC003048), Monoblepharella15 ([21], AY182007), Harpochytrium94 ([21], AY182005), Harpochytrium105 ([21], AY182006).

Clade Fungi: chytridiomycetes are deeply have established with high statistical support the speciﬁc diverging members of the fungi association of chytrids with the rest of the fungi, as well Although the structures of several ascomycete mtDNAs as the branching order of the four fungal divisions (for have been available for some time (for example [2,17– example [6,21]). As expected, the chytrids diverge dee- 19]), efforts over the past decade have provided complete ply with respect to the other three lineages, with zygo- mitochondrial genome sequences from representatives of mycetes separating next relative to the ascomycete and two other lineages long accepted as fungi, namely basi- basidiomycete lineages (Figures 1 and 2). diomycetes and zygomycetes ([7,20], NCBI acc. no. AY376688). A great variety of genome size and structure Complete mtDNA sequences from seven members exists in the fungi, with mtDNAs ranging from 19 kbp of the chytridiomycete lineage have been determined in Schizosaccharomyces pombe [18] to 100 kbp in Podo- [7,20,21,22,23]. These mtDNAs are very similar to spora anserina [17]. By contrast, gene content is quite those of other fungi in terms of gene content and genome consistent across fungal classes, although the distribution size, with the latter ranging from 20 kbp in Harpochy- of several genes is scattered. trium94 to 70 kbp in Rhizophydium brooksianum. The mtDNA of Allomyces macrogynus [22], a deeply branching Chytridiomycetes (chytrids) are of particular evolutionary chytrid, encodes even more genes, including one specify- interest because they have conserved ancestral characters, ing a small subunit ribosomal protein, Rps3 (a gene that such as ﬂagellated spores, that are not present in other has a scattered distribution among fungal mtDNAs), as fungi; accordingly, chytrids are believed to represent well as a full complement of tRNA genes. By contrast, deeply diverging members of the fungal kingdom. Mole- other examined chytrid mtDNAs have markedly reduced cular data including mitochondrial sequence information tRNA gene complements. Thus, we infer that by the time

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chytridiomycetes diverged from the other fungal lineages, Figure 3 the basic form of fungal mtDNA had been established, with features such as genetic code changes (observed in charophytes all divisions except zygomycetes), gene loss and tRNA land plants editing (observed only in chytrid mitochondria [23,24]) 68 emerging later. Because both M. brevicollis and A. para- 67 siticum mtDNAs have gene contents larger than those of 69 C. vulgaris either fungal or animal mtDNAs, and because both fungal C. globosum 65 and animal mitochondrial genomes encode basically the M. polymorpha M. viride 65 same set of genes, we also conclude that independent N. olivacea 61 reduction of gene complements has occurred in the fungal R. americana P. wickerhamii and animal lineages since their divergence from the protistan ancestor of the fungal-animal clade (opistho- 98 P. akinetum 57 konts) (Figure 2). If non-chytrid protists exist that specifically associate with the fungi in molecular phylogenies P. minor 22 to the exclusion of Holozoa, such organisms will yield S. obliquus further insights into when in evolution the common P. parva 42 features of fungal mtDNA structure and gene comple- C. eugametos C. reinhardtii 10 C. elongatum ment took shape. 12 13 12 Clade Viridiplantae: green algae are chlorophytes specifically related to land plants Land plants have long been grouped with green algae on Current Opinion in Microbiology the basis of various biochemical, physiological and ultrastructural data. The organisms in these lineages (compris- Branching order of deeply diverging groups within Viridiplantae ing Viridiplantae) can be further resolved into two phyla: (green plants) based on molecular phylogenetic analyses of mtDNA- encoded protein sequences [26,44] (the position of P. parva is Streptophyta, containing land plants (embryophytes) and based on phylogenetic reconstructions using nuclear [46] and their presumed specific green algal relatives, the charo- chloroplast [47] rRNA sequences). The precise branching position phytes (Charophyceae), and Chlorophyta, which contains of M. viride remains controversial, although recent analyses of both most other green algae (Figures 1 and 3). Chlorophyta mitochondrial [27] and chloroplast [28] gene sequences identify Mesostigma as the earliest diverging plant lineage, emerging consists of three monophyletic classes (Chlorophyceae, before the split between Streptophyta and Chlorophyta. Chlorophyte Ulvophyceae and Trebouxiophyceae) plus a probable lineages are indicated by color: Prasinophyceae (pink), Ulvophyceae paraphyletic assemblage of primitive green algae (Prasi- (brown), Trebouxiophyceae (blue), Chlorophyceae (orange). The nophyceae). Whereas recent phylogenetic analyses based conformation and relative size of the mtDNA in each species is indicated on molecular data support streptophyte monophyly graphically, with the number of genes encoded by the mtDNA (not including introns and unidentified ORFs) indicated. R. americana is [25,26 ], branching order within the chlorophytes and included as an outgroup for comparison. Species shown ([references], the deep relationships of all green plants (e.g. the precise NCBI acc. no.): Reclinomonas americana ([9], AF007261), Marchantia branching position of the primitive green alga, Mesostigma polymorpha ([3], M68929), Chara vulgaris ([26], AY267353), viride [25,27,28]) remain unsettled. This uncertainty is Chaetosphaeridium globosum ([36], AF494279), Mesostigma viride ([27], AF353999), Nephroselmis olivacea ([38], AF110138), Prototheca mostly owing to the high rates of sequence divergence wickerhamii ([37], U02970), Pseudendoclonium akinetum ([44], in some green algal lineages. Despite these limitations, AY359242), Pedinomonas minor ([37], AF116775), Scenedesmus complete sequencing of green plant mtDNAs has consid- obliquus ([42,43], AF204057), Polytomella parva ([16], AY062933, erably deepened our understanding of mitochondrial gen- AY062934), Chlamydomonas reinhardtii ([39,40], U03843), ome evolution in this group, which displays great plasticity Chlorogonium elongatum ([45], Y07814, Y13643, Y13643), Chlamydomonas eugametos ([41], AF008237). in terms of genome size and rate of sequence divergence.

For the six land plant mtDNAs completely sequenced to date ([3,29–32], NCBI acc. No. AY506529) genome size (plastid, nuclear and plasmid). Intriguingly, despite this ranges from 187 kbp in Marchantia polymorpha (liverwort) gross structural variability, plant mitochondrial gene seq- to 570 kbp in Zea mays (corn), with sizes up to 2400 kbp uences are generally considered to exhibit the slowest having been reported for some species of cucurbit [33]. rate of divergence of any genetic system (but see [34]). In Despite their large sizes, land plant mitochondrial gen- addition, although land plant mitochondrial genomes are omes do not encode a proportionately greater number of circular mapping, their native physical structure may be genes than mtDNAs in other lineages (Figure 3); genome considerably more complex [35]. expansion is accounted for primarily by large intergenic regions, repeated segments, introns and intron open read- Recent data from the charophyte algae Chaetosphaeridium ing frames, as well as by incorporation of foreign DNA globosum [36] and Chara vulgaris [26] have placed the www.sciencedirect.com Current Opinion in Microbiology 2004, 7:528–534 532 Genomics

time of origin of the unique genome architecture in land Acknowledgements plants subsequent to the embryophyte–charophyte split, Work in the authors’ laboratory on mitochondrial genome structure and evolution is supported by an operating grant (MOP-4124) to i.e. concurrent with the emergence of land plants M.W.G. from the Canadian Institutes of Health Research. The (Figure 3). The mtDNAs of these two algae, although authors gratefully acknowledge studentship support from the Nova similar in terms of gene content and rate of sequence Scotia Health Research Foundation and Walter C. Sumner Foundation (to C.E.B.) and salary support from the Canada Research Chairs Program divergence to land plant mtDNAs, are much smaller in and Canadian Institute for Advanced Research (to M.W.G.). size (both are less than 70 kbp). This size difference is in marked contrast to the striking resemblance between the References and recommended reading mitochondrial genomes of C. vulgaris and M. polymorpha Papers of particular interest, published within the annual period of in terms of A+T content, codon usage and gene order. A review, have been highlighted as: greater variety of genome architectures in this little- of special interest explored algal lineage is likely, given that ongoing of outstanding interest sequencing of the mtDNA of a basally diverging charo- 1. Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, phyte, Klebsormidium flaccidum, indicates a genome size Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F et al.: close to that of M. polymorpha (OGMP, unpublished). Sequence and organization of the human mitochondrial genome. Nature 1981, 290:457-465. In the more deeply branching chlorophyte algae, a wide 2. 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