5.7: Evolution of Eukaryotes - Biology

5.7: Evolution of Eukaryotes - Biology

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Why can this fish live in these tentacles, but other fish cannot?

Anemones and Clown Fish have a well-known symbiotic relationship. In the ocean, the Clown Fish are protected from predator fish by the stinging tentacles of the anemone, and the anemone receives protection from polyp-eating fish, which the Clown Fish chases away. But what about symbiotic relationships at a much smaller scale? Is it possible for two single-celled organisms to have a symbiotic relationship? As you will find out, yes it is!

Evolution of Eukaryotes

Our own eukaryotic cells protect DNA in chromosomes with a nuclear membrane, make ATP with mitochondria, move with flagella (in the case of sperm cells), and feed on cells which make our food with chloroplasts. All multicellular organisms and the unicellular Protists share this cellular intricacy. Bacterial (prokaryotic) cells are orders of magnitude smaller and have none of this complexity. What quantum leap in evolution created this vast chasm of difference?

The first eukaryotic cells - cells with a nucleus an internal membrane-bound organelles - probably evolved about 2 billion years ago. This is explained by the endosymbiotic theory. As shown in the Figure below, endosymbiosis came about when large cells engulfed small cells. The small cells were not digested by the large cells. Instead, they lived within the large cells and evolved into organelles.

From Independent Cell to Organelle. The endosymbiotic theory explains how eukaryotic cells evolved.

The large and small cells formed a symbiotic relationship in which both cells benefited. Some of the small cells were able to break down the large cell’s wastes for energy. They supplied energy not only to themselves but also to the large cell. They became the mitochondria of eukaryotic cells. Other small cells were able to use sunlight to make food. They shared the food with the large cell. They became the chloroplasts of eukaryotic cells.

Mitochondria and Chloroplasts

What is the evidence for this evolutionary pathway? Biochemistry and electron microscopy provide convincing support. The mitochondria and chloroplasts within our eukaryotic cells share the following features with prokaryotic cells:

  • Their organelle DNA is short and circular, and the DNA sequences do not match DNA sequences found in the nucleus.
  • Molecules that make up organelle membranes resemble those in prokaryotic membranes – and differ from those in eukaryotic membranes.
  • Ribosomes in these organelles are similar to those of bacterial ribosomes, and different from eukaryotic ribosomes.
  • Reproduction is by binary fission, not by mitosis.
  • Biochemical pathways and structures show closer relationships to prokaryotes.
  • Two or more membranes surround these organelles.

The "host" cell membrane and biochemistry are more similar to those of Archaebacteria, so scientists believe eukaryotes descended more directly from that major group (Figure below). The timing of this dramatic evolutionary event (more likely a series of events) is not clear. The oldest fossil clearly related to modern eukaryotes is a red alga dating back to 1.2 billion years ago. However, many scientists place the appearance of eukaryotic cells at about 2 billion years. Some time within Proterozoic Eon, then, all three major groups of life – Bacteria, Archaea, and Eukaryotes – became well established.

What Does it all Mean?

Eukaryotic cells, made possible by endosymbiosis, were powerful and efficient. That power and efficiency gave them the potential to evolve new characteristics: multicellularity, cell specialization, and large size. They were the key to the spectacular diversity of animals, plants, and fungi that populate our world today. Nevertheless, as we close the history of early life, reflect once more on the remarkable but often unsung patterns and processes of early evolution. Often, as humans, we focus our attention on plants and animals, and ignore bacteria. Our human senses cannot directly perceive the unimaginable variety of single cells, the architecture of organic molecules, or the intricacy of biochemical pathways. Let your study of early evolution give you a new perspective – a window into the beauty and diversity of unseen worlds, now and throughout Earth’s history. In addition to the mitochondria that call your 100 trillion cells home, your body contains more bacterial cells than human cells. You, mitochondria, and your resident bacteria share common ancestry – a continuous history of the gift of life.

The three major domains of life had evolved by 1.5 billion years ago. Biochemical similarities show that eukaryotes share more recent common ancestors with the Archaea, but our organelles probably descended from bacteria by endosymbiosis.


  • Eukaryotic cells probably evolved about 2 billion years ago. Their evolution is explained by endosymbiotic theory.
  • Mitochondria and chloroplasts evolved from prokaryotic organisms.
  • Eukaryotic cells would go on to evolve into the diversity of eukaryotes we know today.

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Use the time slider in this resource to answer the questions that follow.

  • Evolution at
  1. When did cells begin to "swallow" other cells?
  2. When did respiration develop?
  3. The rapid rise in atmospheric oxygen favored which cells?
  4. When did eukaryotic cells first form? What distinguished these cells from their predecessors?


  1. When did the first eukaryotic cells evolve?
  2. Describe the endosymbiotic theory.
  3. Discuss the evidence for the evolution of mitochondria and chloroplasts.

5.7: Evolution of Eukaryotes - Biology

Understanding the evolution of eukaryotic cellular complexity is one of the grand challenges of modern biology. It has now been firmly established that mitochondria and plastids, the classical membrane-bound organelles of eukaryotic cells, evolved from bacteria by endosymbiosis. In the case of mitochondria, evidence points very clearly to an endosymbiont of α-proteobacterial ancestry. The precise nature of the host cell that partnered with this endosymbiont is, however, very much an open question. And while the host for the cyanobacterial progenitor of the plastid was undoubtedly a fully-fledged eukaryote, how — and how often — plastids moved from one eukaryote to another during algal diversification is vigorously debated. In this article I frame modern views on endosymbiotic theory in a historical context, highlighting the transformative role DNA sequencing played in solving early problems in eukaryotic cell evolution, and posing key unanswered questions emerging from the age of comparative genomics.

A mitochondrial genomics perspective

Much evidence supports the conclusion that the mitochondrial genome originated from within the (eu)bacterial [8,9,10], not the archaeal [11], domain of life. Specifically, among extant bacterial phyla, the α-proteobacteria are the closest identified relatives of mitochondria, as indicated, for example, by phylogenetic analyses of both protein-coding genes [8,9] and ribosomal RNA (rRNA) genes [12] specified by mitochondrial DNA (mtDNA).

Over the past two decades, many complete mitochondrial genome sequences have been determined, and several recent surveys have summarized various aspects of mitochondrial genome structure, gene content, organization and expression [13,14,15,16]. Two comprehensive mitochondrial genome-sequencing programs have particularly targeted mtDNA in protists [17] and fungi [18]. A number of specific and general insights into mitochondrial genome evolution follow from these data. The first is that ATP production, coupled to electron transport, and translation of mitochondrial proteins represent the essence of mitochondrial function: these functions are common to all mitochondrial genomes and can be traced unambiguously and directly to an α-proteobacterial ancestor. The mitochondrial genome encodes essential components for both of these processes [8,9].

The second insight is that the most ancestral (least derived), most bacterium-like and most gene-rich mitochondrial genome yet described is the 69,034 base pair (bp) mtDNA of the protist Reclinomonas americana, a jakobid flagellate [19] (jakobids are a group of putatively early diverging protozoa that share ultrastructural features with certain amitochondrial protists). By comparison, some other protist mtDNAs, most fungal, and all animal mtDNAs are highly derived, having diverged away from the ancestral pattern exemplified by R. americana mtDNA.

Sequencing has also shown that mitochondrial genomes have, to variable extents, undergone a streamlining process ("reductive evolution" [20]), leading to a marked loss of coding capacity compared to that of their closest eubacterial relatives. Mitochondrial gene content varies widely, from a high of 67 protein-coding genes in R. americana mtDNA to only three in the mitochondrial genome of apicomplexans [8,9], a group of strictly parasitic protists (specific relatives of dinoflagellates) including such organisms as Plasmodium falciparum, the causative agent of malaria. Differential gene content in mtDNAs is attributable primarily to mitochondrion-to-nucleus gene transfer [8,9,10,21,22] (which is demonstrably an on-going process in certain lineages, notably flowering plants [23]). Mitochondrial DNA may also lose genes whose functions are substituted for by unrelated genes encoded in the nucleus. A notable example is the replacement of an original multi-subunit bacteria-like RNA polymerase (inherited from the proto-mitochondrial ancestor and still encoded in certain jakobid - but no other - mitochondrial genomes) by a single-subunit bacteriophage T3/T7-like RNA polymerase, which directs mitochondrial transcription in virtually all eukaryotes [24]. Conversely, there may be complete loss of particular mitochondrial genes (and hence the corresponding functions) without functional complementation by nuclear genes. The complex I (nad) genes of the respiratory chain are one example of such loss. In the yeast Saccharomyces cerevisiae, neither the mitochondrial nor the nuclear genome contains classical complex I genes [25] their disappearance from yeast mtDNA results in the absence of the first coupling site in the yeast electron-transport chain.

Furthermore, genome sequencing shows that the mitochondrial genome (and therefore mitochondria per se) arose only once in evolution. Several observations support this contention [8,9,10]. First, in any particular mitochondrial genome (with few exceptions [26]), genes that have an assigned function are a subset of those found in R. americana mtDNA. Second, in a number of cases, mitochondrial protein-coding clusters retain the gene order of their bacterial homologs, but these clusters exhibit mitochondrion-specific deletions that are most parsimoniously explained as having occurred in a common ancestor of mitochondrial genomes, subsequent to its divergence from the bacterial ancestor. Third, mitochondria form a monophyletic assemblage to the exclusion of bacterial species in phylogenetic reconstructions using concatenated protein sequences [8,9,25,27,28] as well in small-subunit rRNA trees [12].

A final insight from mitochondrial genome sequencing is the emergence of striking parallels in phylogenetic trees separately reconstructed from genes encoded by nuclear DNA [7] and mtDNA [8,9]. In both cases, certain clades (such as animals plus fungi or red plus green algae) have become robust, although connections among these clades and other eukaryotic species or groups cannot yet be precisely resolved. These emerging parallels support the view that mitochondrial and nuclear genomes have evolved in concert throughout much, if not most, of the evolutionary history of the domain Eukarya.


Classification of PufSF proteins

We initially classified the PufSF proteins across eukaryotic diversity. First, we performed a clustering analysis based on sequence similarity. This unbiased approach is based on mutual pairwise BLAST comparisons, and it is especially useful for the analysis of large protein datasets [38]. The initial dataset contained 4469 unique eukaryotic proteins carrying Puf repeat domain(s) (PF00806). No PufSF proteins were identified in Archaea or Bacteria, confirming the eukaryotic origin of the family. The clustering analysis revealed three major groups of PufSF proteins (Fig. 1, Additional file 1: Table S1): the Puf cluster, consisting of 2955 proteins containing eight Puf repeats in the C-terminal part of the protein the PUM3 cluster, consisting of 674 PUM3 orthologues, including plant PUM24 proteins, with up to eleven Puf repeats and the Nop9 cluster, consisting of 675 Nop9 proteins including the plant PUM23 proteins.

Clustering analysis of Puf family proteins. PufSF proteins were analysed by CLANS. A total of 4469 proteins formed three major clusters of proteins corresponding to Nop9 homologues (Nop9 cluster), ‘classical’ Puf proteins (Puf cluster), and PUM3 orthologues (PUM3 cluster). The protein sequences were colour-coded according to the taxonomic affiliation to the eukaryotic supergroup. The details of each large cluster show mostly lineage-specific subclusters, except two large subclusters of Puf proteins. The diagrams above the cluster details depict the general domain arrangement of PufSF proteins PF00806 corresponds to Puf RNA-binding repeat. The x and y axes represent relative positions of the protein sequence in 2D CLANS

The proximity of the Puf and PUM3 proteins in our clustering analysis suggests that these proteins are more similar at the sequence level when compared to Nop9s, despite carrying different numbers of Puf repeats. Given the conservation of 11 Puf repeats in both the rRNA-binding proteins PUM3 and Nop9 proteins, we suspect that this was the ancestral domain arrangement of all members of the PufSF. Following adaptation to interaction with mRNA molecules, the Puf proteins adopted a domain arrangement of only eight Puf domains [2, 21, 22]. The clustering approach was not sensitive enough to reveal detailed taxon-specific grouping of PufSF proteins except for the most represented eukaryotic groups of animals, plants, and fungi. The only exception was the formation of two clear large subclusters within the Puf cluster (Fig. 1) containing proteins of mixed taxonomic affiliation, which indicated the existence of at least two different Puf orthologues in the last common ancestor of eukaryotes. The position of eukaryotic supergroups encompassing protist lineages was rather dispersed across the subclusters. Metamonad proteins including the G. intestinalis sequences represented one of the most diverge proteins of the family.

Taxonomic distribution of PufSF proteins

The number of PufSF proteins encoded in a given genome differs significantly across the eukaryotic diversity (e.g. [4, 25]). Hence, we surveyed the distribution of PufSF of proteins on a species level across the tree of eukaryotes. To this aim, we retrieved all eukaryotic 1180 reference proteomes from UniProtKB and classified them as Puf, PUM3, or Nop9 orthologues. While the dataset is biassed towards the proteomes from Opisthokonta and plants, it also contains curated proteomes of species from other eukaryotic supergroups. We used combination of InterPro precomputed protein families as a final determiner for the affiliation to one of the PufSF members (IPR001313—Puf repeat, IPR040000—Nop9, IPR040059—PUM3). In total, 7762 proteins were identified, and the proteins were classified according to hierarchic taxonomic groups (Fig. 2, Additional file 2: Table S2). Plotting the taxonomic distribution of the proteins showed that the highest number of PufSF proteins can be found in most of the plant species (Streptophyta), where the number of proteins ranged from 10 to 50. Extremely high number of proteins was also identified in ciliate Paramecium tetraurelia (43) but not in other ciliates or alveolates. In addition, all organism of analysed Euglenozoa group such as parasitic Trypanosoma and Leishmania species showed higher number of proteins (12–22). On the opposite end of the spectrum were parasites with reduced genomes, especially microsporidia or Cryptosporidium species with only one or two PufSF proteins. However, many animal taxa including insect and nematodes were also found to have only two or three proteins. Hence, at this point, we observed no clear relationship between the number of PufSF proteins and the biology or complexity of the surveyed organisms.

PufSF proteins in major groups of eukaryotes. Puf family proteins identified in all UniProt reference proteomes were grouped according to taxonomic affiliation as specified in Additional file 2: Table S2 and showed in circle packing plot. Each dot corresponds to a genome of particular eukaryotic species, and the size of the dot represents a number of Puf family proteins in the predicted proteome and the colour-codes to the taxonomic affiliation to the eukaryotic supergroup. Grey circles depict taxonomic groups

Three types of PufSF proteins have undergone different evolution in eukaryotes

Of all 7762 proteins from the reference proteomes, 1135 Nop9s, 5423 Pufs, and 1204 PUM3 proteins were identified (Additional file 2: Table S2). Interestingly, on average, each eukaryotic species contains a single Nop9 and PUM3 homologue and five Pufs (Fig. 3a). Moreover, while the number of Pufs is highly variable among lineages, the occurrence of single Nop9 and PUM3 proteins seems to be retained across eukaryotic diversity. The high number of PufSF proteins found in plants, Euglenozoa, and other species reflects lineage-specific amplification of the Puf proteins and not the ancestral state of early eukaryotes (Fig. 3a). The discrepancy observed between Nop9 and PUM3 gene copy number compared to Puf copy number might be related to different selective pressures experienced by these proteins owing to their role in the biogenesis of ribosomal RNA. On the other hand, given the Pufs’ role in controlling the translation of multiple mRNAs—which will vary between organisms—it is possible that the number of Pufs will differ and could instead relate to the total number of the protein-coding genes in the cell. In order to test this hypothesis, we normalized the number of Pufs with respect to the total number of the protein-coding genes in the corresponding species (Fig. 3b) (Additional file 2: Table S2). Interestingly, the resulting ratio between Pufs and the pool of putative target transcripts seem to be very similar across eukaryotes (Fig. 3b) with the average number of one Puf for every 3.47 × 10 4 protein-coding genes.

Three types of PufSF proteins in major groups of eukaryotes. a The number of Nop9, Puf, and PUM3 orthologues was identified for each proteome in the dataset, and the values were averaged for the taxonomic group of eukaryotes. The error bars correspond to the standard deviation of variance of values within the particular taxonomic group. Error bars for Streptophyta and Ciliophora were cut for better visualization. b The number of Pufs normalized with respect to the total number of the protein coding colour-codes correspond to the taxonomic affiliation to the eukaryotic supergroups

Phylogenetic reconstruction of PufSF proteins

In order to get insight into the evolution of PufSF proteins, we performed the phylogenetic analyses on a dataset containing all three types of the proteins or the just a particular subset of either Puf, PUM3, or Nop9 orthologues (see the ‘Materials and methods’ section for more details). While the phylogenetic reconstructions proved to be problematic due to the repetitive structure of PufSF proteins, the overall tree (Fig. 4, Additional File 3: Fig. S1) shows three distinct clades corresponding to Puf, PUM3, and Nop9. Subsequent subtrees of PUM3 and Nop9 proteins, which are present as single proteins, resolved all major eukaryotic groups with some unexpected position of orthologues mainly from the Metamonada supergroup (Additional File 3: Fig. S1), most likely caused by their high sequence divergence.

Phylogenetic analysis of PufSF proteins. Maximum likelihood phylogenetic inference of PufSF proteins with sequences from H. sapiens, C. elegans, S. cerevisiae, and A. thaliana is shown as triangles with the indicated colours. For visualization purposes, support values were removed. Full phylogenies of the PUM3, Nop9, and Puf proteins can be found in Additional file 3: Fig. S1

Given the presence of several Pufs in most eukaryotes, we endeavoured to resolve their evolutionary relationships and specifically if the presence of multiple Pufs reflects the ancestral state of LECA or rather they are independent paralogues arisen by linage-specific gene duplication(s). The phylogenetic reconstructions of Pufs remained very problematic, and despite using distinct alignment strategies, they did not return clear separation among individual Pufs and the eukaryotic taxa (Additional File 3: Fig. S1). However, we could identify two clear groups (labelled ‘I’ and ‘II’) (Fig. 4) that encompass the vast majority of eukaryotic taxa. This possibly reflects the presence of only two Pufs in LECA. Within group I and group II, there have been a number of lineage-specific duplications giving rise to the multitude of Pufs seen in different genomes.

PufSF proteins in the metamonad G. intestinalis

To test whether PufSF proteins are conserved in some of the most divergent eukaryotes, we specifically investigated the presence of PufSF proteins in G. intestinalis. Using a variety of sensitive sequence searching strategies (see the ‘Materials and methods’ section), we identified six proteins in G. intestinalis. The position and number of Puf repeats within the domain were predicted using HHpred and the alignments with the structurally characterized classical Pufs, Nop9, or PUM3 proteins, respectively. The classification of the proteins into the three types was confirmed by comparison with the domains defined at InterPro. Based on these classifications, we identified four G. intestinalis Puf homologues (GiPuf1–GiPuf4), one Nop9 (GiNop9), and PUM3 (GiPUM3) homologue (Fig. 5a). All four G. intestinalis Pufs were predicted to contain eight Puf repeats corresponding to eight TRMs (Fig. 5b), while both Nop9 and PUM3 homologues contained 11 Puf repeats similar to their homologues in Saccharomyces cerevisiae (Additional File 4: Fig. S2 and S3). The prediction of TRMs was performed by HHpred against the structurally characterized orthologues from S. cerevisiae and D. melanogaster (PDB ACNO. 5BZ1and 5KLA). However, the obtained TRMs for more divergent GiPuf1 and GiPuf2 were not in full agreement with the protein sequence alignment containing other G. intestinalis Puf proteins (Fig. 5b) as their Puf domain appeared shifted by two Puf repeats towards the C-terminus. At present, it is difficult to resolve if just the two Puf repeats were re-arranged or the entire domain was modified in these two proteins.

Domain structure of G. intestinalis PufSF proteins. a The Pumilio homology domain containing Puf repeats was predicted using HHPred against Pfam database and denoted as oval for each of the Giardia intestinalis proteins (shades of brown). The numbers in the ovals represent the position of the domain within the protein (black lines). The expectation value (E value) of the domain detection is shown in brackets. The fruit fly, human, and fungal orthologues are shown for comparison. Gi, Giardia intestinalis Dm, Drosophila melanogaster Hs, Homo sapiens Sc, Saccharomyces cerevisiae. b Protein sequence alignment of G. intestinalis Pufs with selected proteins from S. cerevisiae, D. melanogaster, and H. sapiens. Open red rectangles highlight TRMs. Light and dark grey rectangles depict Puf repeats. Fraction of identical amino acids at the particular position is coloured: dark blue > 80%, blue > 60%, light blue > 40%, white < 40%

According to the phylogenetic reconstruction, the four G. intestinalis Pufs grouped together with the orthologues from closely related diplomonad species Spironucleus salmonicida and Trepomonas sp. distributed in group I and group II (Additional File 3: Fig. S1). GiPuf3, the most conserved Puf homologue of G. intestinalis, branched with group I Puf proteins while GiPuf1, GiPuf2, and GiPuf4 affiliated with the proteins from group II (Additional File 3: Fig. S1). The latter proteins thus likely represent lineage-specific gene duplications.

Cellular localization of G. intestinalis PufSF proteins

In general, PufSF proteins localize to the nucleus or cytosol. In the cytosol, Puf proteins often associate with the cytoplasmic face of cellular compartments [1].

To test the cellular localizations of each PufSF protein in G. intestinalis, we explored bioinformatic and experimental strategies. For bioinformatic predictions, we used DeepLoc [39], which uses neural networks to assess the localization of proteins based on a training set of experimentally localized proteins on UniProt. This algorithm predicted a cytoplasmic localization for all four G. intestinalis Pufs and Nop9 homologue and nuclear localization for only GiPUM3 (Fig. 6a). The cytosolic localization of the Pufs and the nuclear localization of GiPUM3 are in agreement with the expected roles of PufSF proteins, which control the stability and the localization of mRNAs in the cytosol and the nucleolar processing of 7S rRNA, respectively [2, 3]. However, given the role of Nop9 proteins in the maturation of pre-18S rRNA, the protein is expected to be in the nuclear compartment.

Cellular localization of G. intestinalis Puf and Nop9 proteins. a The scores obtained by DeepLoc prediction indicate cytosolic localization of all but GiPUM3 protein, which is predicted as nuclear protein. Colouring gradient represents the values from 0 (red) to 1 (green). Cyt, cytosol Nuc, nucleus CM, cytoplasmic membrane ER, endoplasmic reticulum Mit, mitochondrion Extra, extracellular. b Western blot analysis of G. intestinalis expressing BAP-tagged PufSF proteins (one of at least three independent cell experiments is show). SDS-PAGE and immunoblots show total lysate (Lys), cytosolic (Cyt), and high-speed pellet (HSP) fraction. c Immunofluorescence analysis of the same cell lines shows cellular localization of the proteins. PufSF proteins in green, Sec20-endomembrane system marker in red. DIC, Differential Interference Contrast. d Detailed imaging of GiPUM3 by confocal and 2D STED (GiPUM3 in green, DNA in blue). 3D STED of GiPUM3 with the orthogonal projections (GiPUM3 in red)

To test these subcellular localization predictions, we expressed all the G. intestinalis PufSF proteins with a C-terminal BAP (biotin acceptor peptide) tag in G. intestinalis and analysed their localizations with cell fractionation and microscopy using antibodies directed at the BAP tag. Expression of all constructs but one (GiPuf4) was highly unstable and diminished quickly after establishing stable cell lines. Therefore, G. intestinalis cell lysates were obtained as soon as possible and separated into two fractions: (i) the high-speed pellet, containing sedimentable membrane-bound organelles such as the nucleus, endoplasmic reticulum, peripheral vacuoles, or mitosomes, and (ii) the cytoplasmic fraction [40] (Fig. 6b). In general, the Puf proteins showed three different types of distribution: GiPuf3 and GiNop9 were found specifically in the cytosolic fraction, while GiPuf1 and GiPUM3 were present predominantly in high-speed pellet fraction. Finally, GiPuf2 and GiPuf4 showed the presence in both cytosolic and high-speed pellet fractions indicating their partial association with the cellular membranes.

In agreement with the western blot analyses, the immunofluorescence confocal microscopy of GiPuf3 and GiNop9 showed mainly cytosolic localization of the proteins with some punctate distribution in the cell that do not co-localize with the endomembrane marker Sec20 (Fig. 6c). While the data are in agreement with the bioinformatic prediction, the cytosolic presence of G. intestinalis Nop9 homologue remains puzzling. GiPuf1, GiPuf2, and GiPuf4 were present in different kinds of vesicular structures likely corresponding to specific regions of the endomembrane system, which however did not co-localize, with our endomembrane marker protein Sec20 (Fig. 6c). In addition, GiPuf2 and GiPuf4 showed also a perinuclear staining, which indicated that the protein is associated with the nuclear membrane. Conversely, a very specific labelling of two G. intestinalis nuclei was observed for GiPUM3. In other eukaryotes, PUM3 localizes to discrete nucleolar spots in the nuclear matrix [3, 10]. To determine the subnuclear localization of GiPUM3, we performed high-resolution STED microscopy. In both 2D and 3D STED microscopy, we observed GiPUM3 localizing to the periphery of the nucleus (Fig. 6d).

To determine potential Puf-interacting proteins in G. intestinalis, we explored the Puf-interactome using a high-resolution proximity labelling coupled to mass spectrometry. By determining potential interaction partners of the G. intestinalis Pufs, we could better predict their involvement in gene expression control. Unfortunately, the expression of tagged Pufs was highly unstable and diminished quickly after the cell transformation and we thus could not perform larger scale experiments required for protein- or RNA-pull down experiments. We were, however, able to generate a cell line weakly expressing BAP-tagged GiPuf4 in the presence of cytosolic biotin ligase BirA [41]. Upon crosslinking and purification of GiPuf4 on streptavidin-coupled Dynabeads, the triplicate samples were analysed by mass spectrometry. The purified GiPuf4 was found to be specifically enriched in our sample, although the experiment did not reveal any specific interacting partner protein above the statistical threshold (Additional file 5: Fig. S4, Additional file 6: Table S3). Thus, any functional predictions could not be drawn at this stage.

Prediction of binding motifs of G. intestinalis Pufs and their target mRNA

While we could not identify the interacting factors for G. intestinalis Pufs by mass spectrometry, we decided to predict the sets of recognized mRNAs for each homologue. The RNA sequence motif recognized by Puf/Nop9 proteins is determined by the combination of three amino acid residues, referred to as tripartite recognition motif (TRM). TRM is part of five residues in the second α-helix of each Puf repeat represented as 1-2-X-X-5 (where X is any hydrophobic residues). Within the TRM, positions 1 and 5 bind the edge of the RNA base, while the position 2 makes a stacking interaction with RNA molecule [42]. Some TRMs have been shown to be specific for particular base [11]. Hence, upon the identification of the TRMs in each Puf repeat, it is possible to predict its sequence-specific binding properties [9]. However, it should be noted that for some naturally occurring TRMs, the specificity has not been determined. By comparing the most closely related sequences to each G. intestinalis Puf identified with HHpred, we predicted the putative binding motif by manually checking the position of individual Puf repeats (Fig. 5b), and we could predict putative RNA-binding motifs for all G. intestinalis Pufs (Fig. 7a). Several predicted TRMs located in GiPuf1, GiPuf2, and GiPuf4 contained experimentally unidentified amino acid combinations which left these putative binding motifs incomplete. Interestingly, a complete binding motif predicted for GiPuf3 (5′-UGUAUUUA-3′) was found to be highly similar to 5′-UGUAUAUA-3′motif of prototypical members of the protein family such as human PUM1 or yeast Puf3 [43].

Predicted binding motifs of G.intestinalis PufSF proteins. a The tripartite recognition motifs (TRMs) of each Puf repeats of G.intestinalis Pufs were predicted, and the resulting sequence was used to search the conceptual transcriptome. The number of putative mRNA targets, which contain the motif in the 3′-UTR, is shown in bold. Asterisk denotes the same putative mRNA recognized by two different Pufs. b Sequence alignment of 18S rRNA shows the conservation of the sequence recognized by Nop9 as it was experimentally identified for S. cerevisae Nop9. Fraction of identical nucleotides at the particular position is coloured: dark blue > 80%, blue > 60%, light blue > 40%, white < 40%

The predicted motifs of G. intestinalis Pufs were then used to search the dataset of theoretical 3′-UTR of all 9747 G. intestinalis genes retrieved from GiardiaDB. Given that the 3′-UTRs of G. intestinalis mRNAs are very short [30, 32, 44], the length of the UTRs was limited to 50 bases only.

Using the FIMO (Find Individual Motif Occurrence) algorithm [45], a specific set of possible cognate mRNAs for GiPuf1–GiPuf4 was retrieved (Fig. 7, Additional file 8: Table S5). Each of the G. intestinalis Puf proteins was predicted to interact with the different number of transcripts, and this number was also inversely proportional to the G. intestinalis length of the predicted binding motif: where GiPuf3, GiPuf1, GiPuf2, and GiPuf4 were predicted to interact with 7, 9, 24, and 44 transcripts, respectively. These numbers are substantially lower than other Puf proteins that are predicted to interact with hundreds of RNA targets [19, 46]. We next explored the putative function and subcellular localization of the protein products of the predicted target transcripts (Additional file 8: Table S5). Interestingly, the target transcripts included other RNA-processing proteins (e.g. fibrillarin) and a component of the ERAD (endoplasmic reticulum-associated degradation) pathway (e.g. Derlin 1) (Additional file 8: Table S5).

Unlike Pufs, Nop9s use their 11 Puf repeats [21, 22] to specifically bind to the pre-18S rRNA at the central pseudoknot region [22] and regulate its processing possibly by competing with Nob1 nuclease [21]. However, how Nop9 TRMs interact with the rRNA was unknown until recently where it was shown that yeast Nop9 binds to a specific 11-nucleotide region of the pre-18S rRNA [22]. The alignment of 18S rRNA sequences showed that this region is very well conserved across eukaryotes including species from the Metamonada group (Fig. 6b) suggesting that Nop9 might also recognize this region. However, in G. intestinalis, the cytoplasmic (and not nuclear) localization of this protein challenges whether GiNop9 and rRNA processing occurs in the nucleus.

Finally, when GiPUM3 was aligned with its characterized human and yeast counterparts, no previously identified TRMs could be identified within the sequence. This supported the absence of recognizable binding motif for this type of PufSF proteins (Additional File 4: Fig. S3) [2, 23].


The present analysis of KOGs provides quantitative backing for many trends in the evolution of eukaryotic genomes that previously have been noticed on the general, qualitative level. The important quantities reported here include the size of the conserved core of eukaryotic genes, the conservative reconstructions of ancestral gene sets, the numbers of genes that appear to have been lost and gained in individual eukaryotic lineages, and the extent of correlation between gene dispensability and evolutionary conservation, which is reflected in phyletic patterns. In addition, we evaluated the range of variation of evolutionary rates of genes in different functional categories and obtained statistical support for the important evolutionary phenomenon of domain accretion. Furthermore, we observed that only a minority of eukaryotic KOGs have readily detectable prokaryotic counterparts, which emphasizes the extent of innovation linked to the origin of eukaryotes and subsequent major transitions in eukaryotic evolution, such as the origin of multicellularity and the origin of animals.

The case study of the KOGs that are represented by just one member in all eukaryotic genomes compared shows the potential of KOGs for functional prediction by inferring the probable functions for almost all KOGs in this set that had remained uncharacterized. This analysis also revealed unexpected facets of evolution of widespread and essential eukaryotic proteins, such as the counterintutitive preponderance of WD40-repeat proteins among the single-member pan-eukaryotic KOGs.

The current KOG set includes proteins from seven genomes whose sequences were available as of 1 July, 2002. The genomes of the mouse [87], the fugu fish [88], the Anopheles mosquito [89], the urochordate Ciona instestinalis [90] and the malarial parasite Plasmodium falciparum [91] have become available since then but were not included, partly because of problems with protein annotation for some of these genomes, and partly due to the time-consuming and labor-intensive nature of KOG analysis. Inclusion of these and other newly sequenced genomes should proceed at a faster rate once the system itself is established, and will enable further, deeper studies into the functional and evolutionary patterns of eukaryotic life.

The Origin and Evolution of Eukaryotes

The timing of mitochondrial acquisition during eukaryogenesis is widely debated. There are several plausible scenarios that account for the cell biological mechanisms by which acquisition may have occurred.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

Many features of eukaryotic genomes (e.g., the modular rearrangement of genes) may have their origins in an RNA or RNA/protein world.

Bacterial Influences on Animal Origins

Animals evolved in seas filled with bacteria. Animal–bacterial interactions (e.g., bacterivory, commensalism, and infection) likely influenced aspects of animal origins, including multicellularity, development, and immunity.

Green Algae and the Origins of Multicellularity in the Plant Kingdom

Multicellularity requires several key innovations (e.g., cell–cell communication and adhesion). Two groups of green algae, charophytes and volvocines, provide unprecedented insights into the evolution of these innovations.

The Archaeal Legacy of Eukaryotes: A Phylogenomic Perspective

Phylogenomic studies suggest that eukaryotes may have emerged from the archaeal TACK superphylum. Orthologs of several eukaryotic signature proteins (e.g., actin) have recently been identified in genomes of TACK Archaea.

Missing Pieces of an Ancient Puzzle: Evolution of the Eukaryotic Membrane-Trafficking System

  • Alexander Schlacht ,
  • Emily K. Herman ,
  • Mary J. Klute ,
  • Mark C. Field ,
  • and Joel B. Dacks

The eukaryotic membrane-trafficking system affects virtually every cellular component. Genome sequencing, molecular phylogenetics, and cell biology have advanced our understanding of its evolution.

The Neomuran Revolution and Phagotrophic Origin of Eukaryotes and Cilia in the Light of Intracellular Coevolution and a Revised Tree of Life

A eubacterium that underwent major cell wall changes 1.2 Gy ago gave rise to the clade neomura, composed of eukaryotes and archaebacteria. Phagotrophy was soon afterward the major driver of eukaryogenesis.

Origin and Evolution of the Self-Organizing Cytoskeleton in the Network of Eukaryotic Organelles

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. But it has additional features (e.g., motor proteins) not found in prokaryotes.

Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

The progressive compartmentalization of eukaryotes required the coevolution of intracellular protein transport systems. These were most likely built on preexisting structures and mechanisms from bacterial ancestors.

On the Age of Eukaryotes: Evaluating Evidence from Fossils and Molecular Clocks

Fossil-calibrated molecular clock-based approaches are being used to estimate the age of major evolutionary events in eukaryotes. The estimates are heavily influenced by the methods and models used, but are steadily improving.

The Archaeal Legacy of Eukaryotes: A Phylogenomic Perspective

Phylogenomic studies suggest that eukaryotes may have emerged from the archaeal TACK superphylum. Orthologs of several eukaryotic signature proteins (e.g., actin) have recently been identified in genomes of TACK Archaea.

What Was the Real Contribution of Endosymbionts to the Eukaryotic Nucleus? Insights from Photosynthetic Eukaryotes

The genomes of photosynthetic eukaryotes are derived from many sources (e.g., mitochondria, primary plastids, and algal endosymbionts). But in many cases, their phylogenetic signals have been obscured over time.

Protein and DNA Modifications: Evolutionary Imprints of Bacterial Biochemical Diversification and Geochemistry on the Provenance of Eukaryotic Epigenetics

  • L. Aravind ,
  • A. Maxwell Burroughs ,
  • Dapeng Zhang ,
  • and Lakshminarayan M. Iyer

Accompanying the origin of eukaryotes was the emergence of epigenetic marks in DNA and proteins (e.g., histones). Precursors of these epigenetic systems may have first evolved in the context of bacterial conflict systems.

How Natural a Kind Is "Eukaryote?"

Most biologists feel that "eukaryote" is a natural kind (discovered rather than invented). Naturalness of kinds comes in degrees, and "eukaryote" fares well on several counts.

Origin of Spliceosomal Introns and Alternative Splicing

Spliceosomal introns are ubiquitous in nearly every eukaryotic genome. The α-proteobacterial ancestor of mitochondria may have possessed group II self-splicing introns that gave rise to these spliceosomal introns.

Bioenergetic Constraints on the Evolution of Complex Life

Bacteria respire across their cell membranes and show little tendency to evolve complex traits. In eukaryotes, mitochondria energetically support the nuclear genome this may have enabled the evolution of complex traits.

The Eukaryotic Tree of Life from a Global Phylogenomic Perspective

Thanks to next-generation sequencing, genome-scale data for taxonomically diverse species are rapidly being produced. Comparisons of these data have permitted the resolution of evolutionary relationships among eukaryotic supergroups.

Origin and Evolution of Plastids and Photosynthesis in Eukaryotes

Although a single endosymbiotic cyanobacterium gave rise to chloroplasts and most other plastids in photosynthetic eukaryotes, a second lineage of primary plastids in Paulinella chromatophora was recently confirmed.

The Dispersed Archaeal Eukaryome and the Complex Archaeal Ancestor of Eukaryotes

The apparent ancestors of key eukaryotic features (e.g., ubiquitin signaling, RNA interference, and cytoskeletal structures) are identifiable in different Archaea. But the specific archaeal ancestor of eukaryotes remains elusive.

The Pre-Endosymbiont Hypothesis: A New Perspective on the Origin and Evolution of Mitochondria

Only 10%–20% of mitochondrial proteins are α-proteobacterial in origin. The remainder may be from a premitochondrion, a metabolic but non-energy-generating organelle that existed before the α-proteobacterial symbiont arrived.

Origins of Eukaryotic Sexual Reproduction

The evolution of sex—including meiosis, fertilization, sex determination, uniparental inheritance of organelle genomes, and speciation—may have involved several major genetic and cellular innovations occurring in parallel.

Symbiosis as a General Principle in Eukaryotic Evolution

Resident microorganisms have influenced the evolutionary history of eukaryotes. They serve as a source of novel capabilities (e.g., metabolic pathways) and improve the fitness of their host (e.g., by modulating cell signaling).

The Impact of History on Our Perception of Evolutionary Events: Endosymbiosis and the Origin of Eukaryotic Complexity

Preceding thought influences how we interpret information. The acceptance of the endosymbiotic origin of mitochondria and plastids may have unduly affected how other aspects of the eukaryotic cell are explained.

Paleobiological Perspectives on Early Eukaryotic Evolution

The geologic record has provided clues to the history of eukaryotes: their origins, diversification, and the environmental context in which these events took place.

5.7: Evolution of Eukaryotes - Biology

Which of the following questions can be asked about organisms that live in fresh water?

  1. Will their bodies take in too much water?
  2. Can they control their tonicity?
  3. Can they survive in salt water?
  4. Will their bodies lose too much water to their environment?

Which of the following explains why active movement of molecules across membranes must function continuously?

  1. Diffusion cannot occur in certain cells.
  2. Diffusion is constantly moving solutes in opposite directions.
  3. Facilitated diffusion works in the same direction as active transport.
  4. Not all membranes are amphiphilic.
  1. by expelling anions
  2. by pulling in anions
  3. by expelling more cations than it takes in
  4. By taking in and expelling an equal number of cations.
  1. Primary active transport is indirectly dependent on ATP, while secondary active transport is directly dependent on ATP.
  2. Primary active transport is directly dependent on ATP, while secondary active transport is indirectly dependent on ATP.
  3. Primary active transport does not require ATP, while secondary active transport is indirectly dependent on ATP.
  4. Primary active transport is indirectly dependent on ATP, while secondary active transport does not require ATP
  1. It leaves the cell.
  2. It is disassembled by the cell.
  3. It fuses with and becomes part of the plasma membrane.
  4. It is used again in another exocytosis event.
  1. It transports only small amounts of fluid.
  2. It does not involve the pinching off of membrane.
  3. It brings in only a specifically targeted substance.
  4. It brings substances into the cell, while phagocytosis removes substances.
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  • 5.7 Review Questions

This text is based on Openstax Biology for AP Courses, Senior Contributing Authors Julianne Zedalis, The Bishop's School in La Jolla, CA, John Eggebrecht, Cornell University Contributing Authors Yael Avissar, Rhode Island College, Jung Choi, Georgia Institute of Technology, Jean DeSaix, University of North Carolina at Chapel Hill, Vladimir Jurukovski, Suffolk County Community College, Connie Rye, East Mississippi Community College, Robert Wise, University of Wisconsin, Oshkosh

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 Unported License, with no additional restrictions

3.7 Cells

All living things are composed of one or more cells (viruses are an exception to this, but the jury is still out on whether or not they are actually alive…). Some organisms are one cell, while others are made up of dozens to quadrillions of cells (the estimated number of cells in a blue whale is greater than 100 quadrillion!)

So what actually defines a cell? A cell is a phospholipid-membrane-enclosed living structure that can replicate and uses biomolecules (often sugars) for energy.

There are two main categories of cells: prokaryotes and eukaryotes. Prokaryotes are visually simpler, in that they have cytoplasm (the inside stuff), are enclosed by a membrane, and do not have smaller enclosed structures (called organelles) inside.

Prokaryotes are generally single-celled organisms (there are a few exceptions that we will not discuss here). Two classifications of prokaryotes are Bacteria and Archaea. While these two types of prokaryotes look similar under a microscope, they are actually only very distantly related to one another (they branched off from a common ancestor over three billion years ago!).

Figure 3.16 Scientists’ most current understanding of the relatedness of organisms. Note that archaea and eukaryotes are closer together than archaea and bacteria. Figure from Hug et al. 2016.

Eukaryotes have a more complicated cell structure. Eukaryotes contain organelles within the cell including the nucleus, mitochondria, chloroplasts (in plants and algae) and others.

Figure 3.17 Schematic of an animal cell.

Eukaryotes can be single-celled organisms or multicellular organisms (including all plants and animals).

/>Figure 3.18 Examples of single-celled and multicellular organisms. a) a eukaryotic single-celled organism called a paramecium. b) a microscopic multicellular animal called a daphnid. c) a chicken (multicellular animal) and d) a larche (multicellular plant)

All sexually reproducing organisms are eukaryotes. For that reason, we will focus on eukaryotes. Inside eukaryotic cells is a membrane-bound organelle called a nucleus, which is surrounded by other membrane-bound organelles called mitochondria. See the video in section 3.8 for a description of all the organelles and their functions. This text will focus mainly on the nucleus and the mitochondria. The nucleus contains the vast majority of the cell’s DNA. The mitochondria act as the cell’s power-supply centers. Mitochondria take the energy from sugar and create high-energy molecules that the rest of the cell can use to do work and to replicate themselves.


Chloroplasts are one type of plastid , a group of related organelles in plant cells that are involved in the storage of starches, fats, proteins, and pigments. Chloroplasts contain the green pigment chlorophyll and play a role in photosynthesis. Genetic and morphological studies suggest that plastids evolved from the endosymbiosis of an ancestral cell that engulfed a photosynthetic cyanobacterium. Plastids are similar in size and shape to cyanobacteria and are enveloped by two or more membranes, corresponding to the inner and outer membranes of cyanobacteria. Like mitochondria, plastids also contain circular genomes and divide by a process reminiscent of prokaryotic cell division. The chloroplasts of red and green algae exhibit DNA sequences that are closely related to photosynthetic cyanobacteria, suggesting that red and green algae are direct descendants of this endosymbiotic event.

Mitochondria likely evolved before plastids because all eukaryotes have either functional mitochondria or mitochondria-like organelles. In contrast, plastids are only found in a subset of eukaryotes, such as terrestrial plants and algae. One hypothesis of the evolutionary steps leading to the first eukaryote is summarized in [Figure 2].

Figure 2: The first eukaryote may have originated from an ancestral prokaryote that had undergone membrane proliferation, compartmentalization of cellular function (into a nucleus, lysosomes, and an endoplasmic reticulum), and the establishment of endosymbiotic relationships with an aerobic prokaryote and, in some cases, a photosynthetic prokaryote to form mitochondria and chloroplasts, respectively.

The exact steps leading to the first eukaryotic cell can only be hypothesized, and some controversy exists regarding which events actually took place and in what order. Spirochete bacteria have been hypothesized to have given rise to microtubules, and a flagellated prokaryote may have contributed the raw materials for eukaryotic flagella and cilia. Other scientists suggest that membrane proliferation and compartmentalization, not endosymbiotic events, led to the development of mitochondria and plastids. However, the vast majority of studies support the endosymbiotic hypothesis of eukaryotic evolution.

The early eukaryotes were unicellular like most protists are today, but as eukaryotes became more complex, the evolution of multicellularity allowed cells to remain small while still exhibiting specialized functions. The ancestors of today’s multicellular eukaryotes are thought to have evolved about 1.5 billion years ago.

Biology: A Tree of Eukaryotes

I spend my days teaching about strangers: blobs of slime, walkers on legs that aren’t legs, seers with eyes that aren’t eyes. Most days I walk through biology in the dark, my hands outstretched, calling to those who walk behind me, holding on to the hem of my shirt: “Watch out, there’s a bump here,” “give me your hand: feel how smooth this is,” “taste this,” “now: duck.”

Teaching equals storytelling about shadows on the wall the slide projector, my bonfire in Plato’s cave. Most days it feels like singing in the dark.

The tree of the eukaryotes was like that. For decades, I taught about forks in the road of evolution of the nucleated cells: left to the fungi, right to the animals. Remember, we lost the plants way back, one point five billion years ago, though by then we had stuck it out together, through hell and high water, for a half a billion years. And, oh yes, finally, don’t forget to look over there, far off to the left: see that big bag full of beings we don’t know what to do with? The amoebae, protozoans, flagellates? The algae, water molds, and parameciums? We call them protists. And then we usually just leave them there, rattling around together, like the scissors, string, and thumbtacks the postcard from Aunt Mary we never got around to answering the wine bottle corks we always meant to make into something else, the potholders and the shipping tape. That most important drawer in our kitchen where we keep things joined only by our inability to figure out where they belong.

But then: someone cared enough! Many people did. For decades, they deep-read the DNA. They scratched their heads. They sorted. They debated. They tried again. And then they said: “This isn’t quite right, and yes, we’re going to mess with this some more, and probably we’ll keep on messing for many years to come. We never may be done. But meanwhile: here’s a map.”

A map of sorts. Not like the maps of yore, with large white swaths—here be dragons—but one with way too many names and lines. Like barcodes bristling out in all directions, forming a frightened porcupine. Like the world’s most complicated game of pick-up sticks. The tree, the river delta, the forks in the road, had burst into a sun, a wild wheel, orange, yellow, red. A solar explosion, with many roads erupting from the center all at once.

Well, you know, we just don’t know. Right here, at the heart of things? Is mystery. Yes, we know there ought to be a single root, but for right now, life’s not a tree and not a bush—it is a tumbleweed.

Thank you, I sighed. For being honest. Thank you for the fierce orange glow.

The slime molds had come home, snuggling up against fungi and animals, asking “will you be my neighbor?” The amoebae, like splattered raw egg white, had fragmented all over everything, crawling along branches far apart. Adoption of chloroplasts by nucleated cells happened not once, but here, and here, and here, leaving hope for the future. (Really, you have never wanted to be green?)

“But, in the end, the point is,” I tell my students, waving my hands, “the point is that, right now, some of the cells that are your blood hunt, blob-like, for bacteria like amoebae crawling through a bog. Along your airways your ciliated cells beat in unison, like a blanket woven from a thousand docile parameciums. Bone precipitates in tiny spaces made by cells pretending to be foraminifers. And they all talk, sing symphonies of molecules, hum melodies of chemistry they picked up in archaeal soup: We’re in this together, sharing wild inventions. Your body came from a flagellate swimming towards an egg’s scent with the same whiplash strokes used by any microscopic alga making a red tide.”

I still lie to my students, all the way through years one and two. I still teach them to count: one—animals, two—fungi, three—plants, four—protists, five—bacteria. A lie of separation, as blatant as it is safe a rooted story branching towards a familiar present tense. I lull them, so they’ll follow me into the dark. And then I let them hate me when the sun bursts out: I yank the root and watch them flail, initiate them into the tribe of tumbleweeds.

Provenance: Submission

Catharina Coenen is a plant biologist and first-generation German immigrant to Northwestern Pennsylvania, where she teaches biology at Allegheny College. Her academic research has been published in Plant Physiology, New Phytologist and other plant biology journals. Her creative nonfiction pieces are forthcoming in Appalachian Heritage, Christian Science Monitor, and elsewhere.

Watch the video: Evolution of Eukaryotes - Biology Tutorial (June 2022).


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