Membrane Biochemistry Challenges Route to Evolutionary Origin of Complex Cells


By Fazale Rana – July 10, 2019

Unfortunately, the same thing could be said to biologists trying to discover the evolutionary route that led to the emergence of complex, eukaryotic cells. No matter the starting point, it seems as if you just can’t get there from here.

This frustration becomes most evident as evolutionary biologists try to account for the biochemical makeup of the membranes found in eukaryotic cells. In my opinion, this struggle is not just an inconvenient detour. As the following paragraphs show, obstacles line the roadway, ultimately leading to a dead end that exposes the shortcomings of the endosymbiont hypothesis—a cornerstone idea in evolutionary biology.

Endosymbiont Hypothesis

Most biologists believe that the endosymbiont hypothesis stands as the best explanation for the origin of complex cells. According to this hypothesis, complex cells originated when symbiotic relationships formed among single-celled microbes after free-living bacterial and/or archaeal cells were engulfed by a “host” microbe.

The mitochondrion represents the “poster child” of the endosymbiont hypothesis. Presumably, this organelle started as an endosymbiont. Evolutionary biologists believe that once engulfed by the host cell, the microbe took up permanent residency, growing and dividing inside the host. Over time, the endosymbiont and host became mutually interdependent, with the endosymbiont providing a metabolic benefit—such as a source of ATP—for the host cell. In turn, the host cell provided nutrients to the endosymbiont. Presumably, the endosymbiont gradually evolved into an organelle through a process referred to as genome reduction. This reduction resulted when genes from the endosymbiont’s genome were transferred into the genome of the host organism.

Evidence for the Endosymbiont Hypothesis
1. Most of the evidence for the endosymbiont hypothesis centers around mitochondria and their similarity to bacteria. Mitochondria are about the same size and shape as a typical bacterium and have a double membrane structure like gram-negative cells. These organelles also divide in a way that is reminiscent of bacterial cells.

2. Biochemical evidence also exists for the endosymbiont hypothesis. Evolutionary biologists view the presence of the diminutive mitochondrial genome as a vestige of this organelle’s evolutionary history. They see the biochemical similarities between mitochondrial and bacterial genomes as further evidence for the evolutionary origin of these organelles.

3. The presence of the unique lipid, cardiolipin, in the mitochondrial inner membrane also serves as evidence for the endosymbiont hypothesis. This important lipid component of bacterial inner membranes is not found in the membranes of eukaryotic cells—except for the inner membranes of mitochondria. In fact, biochemists consider it a signature lipid for mitochondria and a vestige of the organelle’s evolutionary history. So far, the evolutionary route looks well-paved and clear.

Discovery of Lokiarchaeota

Evolutionary biologists have also developed other lines of evidence in support of the endosymbiont hypothesis. For example, biochemists have discovered that the genetic core (DNA replication and the transcription and translation of genetic information) of eukaryotic cells resembles that of the archaea. This similarity suggests to many biologists that a microbe belonging to the archaeal domain served as the host cell that gave rise to eukaryotic cells.

Life scientists think they may have determined the identity of that archaeal host. In 2015, a large international team of collaborators reported the discovery of Lokiarchaeota, a new phylum belonging to the archaea. This phylum clusters with eukaryotes on the evolutionary tree. Analysis of the genomes of Lokiarchaeota identifies a number of genes involved in membrane-related activities, suggesting that this microbe may well have possessed the ability to engulf other microbes.1 At this point, it looks like “you can get there from here.”

Challenges to the Endosymbiont Hypothesis

Despite this seemingly compelling evidence, the evolutionary route to the first eukaryotic cells is littered with potholes. I have written several articles detailing some of the obstacles. (See Challenges to the Endosymbiont Hypothesis in the Resources section.) Also, a divide on the evolutionary roadway called the lipid divide compounds the problem for the endosymbiont hypothesis.

Lipid Divide

The lipid divide refers to the difference in the chemical composition of the cell membranes found in bacteria and archaea. Phospholipids comprise the cell membranes of both sorts of microbes. But the similarity ends there. The chemical makeup of the phospholipids is distinct in bacteria and archaea.

Bacterial phospholipids are built around a d-glycerol backbone, which has a phosphate moiety bound to the glycerol in the sn-3 position. Two fatty acids are bound to the d-glycerol backbone at the sn-1 and sn-2 positions. In water, these phospholipids assemble into bilayer structures.


Figure: Difference between archaeal (top) and bacterial (middle and bottom) phospholipids. Features include 1: isoprene chains, 2: ether linkage, 3: l-glycerol, 4 and 8: phosphate group, 5: fatty acid chains, 6: ester linkages, 7: d-glycerol, 9: lipid bilayer of bacterial membranes, 10: lipid monolayer found in some archaea. Image credit: Wikipedia

Archaeal phospholipids are constructed around an l-glycerol backbone (which produces membrane lipids with different stereochemistry than bacterial phospholipids). The phosphate moiety is attached to the sn-1 position of glycerol. Two isoprene chains are bound to the sn-2 and sn-3 positions of l-glycerol via ether linkages. Some archaeal membranes are formed from phospholipid bilayers, while others are formed from phospholipid monolayers.

Presumably, the structural features of the archaeal phospholipids serve as an adaptation that renders them ideally suited to form stable membranes in the physically and chemically harsh environments in which many archaea find themselves.

Lipid Divide Frustrates the Origin of Eukaryotic Cell Membranes

In light of the lipid divide and the evidence that seemingly indicates that the endosymbiotic host cell likely belonged to Lokiarchaeota, it logically follows that the membrane composition of eukaryotic cells should be archaeal-like. But, this expectation is not met and the evolutionary route encounters another pothole. Instead, the cell membranes of eukaryotic cells closely resemble bacterial membranes.

One way to repair the roadway is to posit that during the evolutionary process that led to the emergence of eukaryotic cells, a transition from archaeal-like membranes to bacterial-like membranes took place. In fact, supporting evidence comes from laboratory studies demonstrating that stable bilayers can form from a mixture of bacterial and archaeal phospholipids, even though the lipids from the two sources have opposite stereochemistry.

Evolutionary biologists Purificación López-García and David Moreira question if evidence can be marshaled in support of this scenario for two reasons.2 First, mixing of phospholipids in the lab is a poor model for cell membranes that function as a “dynamic cell-environment interface.”3

Second, they question if this transition is feasible given how exquisitely optimized membrane proteins must be to fit into cell membranes. The nature of protein optimization is radically different for bacterial and archaeal membranes. Because cell membrane systems are optimized, the researchers question if an adequate driving force for this transition exists.

In other words, these two scientists express serious doubts about the biochemical viability of a transitional stage between archaeal membranes. In light of these obstacles, López-García and Moreira write, “The archaea-to-bacteria membrane shift remains the Achilles’ heel for these models [that propose an archaeal host for endosymbionts].”4

In other words, you can’t get there from here.

Can Lokiarchaeota Traverse the Lipid Divide?

In the midst of this uncertain evolutionary route, a recent study by investigators from the Netherlands seems to point the way toward the evolutionary origin of eukaryotic membranes.5 Researchers screened the Lokiarchaeota genome for enzymes that would take part in phospholipid synthesis with the hope of finding clues about how this transition may have occurred. They conclude that this group of microbes could not make l-glycerol-1-phosphate (a key metabolic intermediate in the production of archaeal phospholipids) because it lacked the enzyme glycerol-1-phosphate dehydrogenase (G1PDH). They also discovered evidence that suggests that this group of microbes could make fatty acids and chemically attach them to sugars. The researchers argue that Lokiarchaeota could make some type of hybrid phospholipid with features of both archaeal and bacterial phospholipids.

The team’s approach to understanding how evolutionary processes could bridge the lipid divide and account for the origin of eukaryotic membranes is clever and inventive, to be sure. But it is far from convincing for at least four reasons.

1. Absence of evidence is not evidence of absence, as the old saying goes. Just because the research team didn’t find the gene for G1PDH in the Lokiarchaeota genetic material doesn’t mean this microbe didn’t have the capacity to make archaeal-type phospholipids. Toward this end, it is important to note that researchers have not cultured any microbe that belongs to this group organisms. The group’s existence is inferred from metagenomic analysis, which involves isolating small fragments of DNA from the environment (in this case a hydrothermal vent system in the Atlantic Ocean, called Loki’s Castle) and stitching them together into a genome. The Lokiarchaeota “genome” is low quality (1.4-fold coverage) and incomplete (8 percent of the genome is missing). Around one-third (32 percent) of the genome codes for proteins with unknown function. Could it be that an enzyme capable of generating l-glycerol-1-phosphate exists in the mysterious third of the genome? Or in the missing 8 percent?

2. While the researchers discovered that genes could conceivably work together to make d-glycerol-3-phosphate (though the enzymes encoded by these genes perform different metabolic functions), they found no direct evidence that Lokiarchaeota produces d-glycerol-3-phosphate. Nor did they find evidence for glycerol-3-phosphate dehydrogenase (G3PDH) in the Lokiarchaeota genetic material. This enzyme plays a key role in the synthesis of phospholipids in bacteria.

3. Though the researchers found evidence that Lokiarchaeota had the capacity to make fatty acids, some of the genes required for the process seem to have been acquired by these microbes via horizontal gene transfer with genetic material from bacteria. (It should be noted that 29 percent of the Lokiarchaeota genome comes from the bacteria.) It is not clear when Lokiarchaeota acquired these genes and, therefore, if this metabolic capability has any bearing on the origin of eukaryotes.

4. The researchers present no evidence that Lokiarchaeota possessed the protein machinery that would chemically attach isoprenoid lipids to d-glycerol-3-phosphate via ether linkages.

Thus, the only way to establish Lokiarchaeota membranes as a transitional evolutionary pathway between those found in Archaea and Bacteria is to perform chemical analysis of its membranes. At this juncture, such analysis is impossible to perform because no one has been able to culture Lokiarchaeota. In fact, other evidence suggests that this group of microbes possessed archaeal-type membranes. Researchers have recovered archaeal lipids in the sediments surrounding Loki’s Castle, but they have not recovered bacterial-like lipids.

More Lipid Divide Frustration

Given these problems, could it be that the host microbe for the endosymbiont was a member of Bacteria, not Archaea? While this model would solve the problem of the lipid divide, it leaves unexplained the similarity between the genetic core of eukaryotes and the Archaea. Nor does it account for the grouping of eukaryotes with the Archaea.

It doesn’t look like you can get there from here, either.

Evolutionary biologists Jonathan Lombard, Purificación López-García and David Moreira sum things up when they write, “The origin of eukaryotic membranes is a problem that is rarely addressed by the different hypotheses that have been proposed to explain the emergence of eukaryotes.”6 Yet, until this problem is adequately addressed, the evolutionary route to eukaryotes will remain obscure and the endosymbiont hypothesis noncompelling.

In light of this challenge and others, maybe a better way to make sense of the origin of eukaryotic cells is to view them as the Creator’s handiwork. For many scientists, it is a road less traveled, but it accounts for all of the data. You can get there from here.


Challenges to the Endosymbiont Hypothesis

Support for a Creation Model for the Origin of Eukaryotic Cells

  1. Anja Spang et al., “Complex Archaea that Bridge the Gap between Prokaryotes and Eukaryotes,” Nature 521 (May 14, 2015): 173–79, doi:10.1038/nature14447; Katarzyna Zaremba-Niedzwiedzka et al., “Asgard Archaea Illuminate the Origin of Eukaryotic Cellular Complexity,” Nature 541 (January 19, 2017): 353–58, doi:10.1038/nature21031.
  2. Purificación López-García and David Moreira, “Open Questions on the Origin of Eukaryotes,” Trends in Ecology and Evolution 30, no. 11 (November 2015): 697–708, doi:10.1016/j.tree.2015.09.005.
  3. López-García and Moreira, “Open Questions.”
  4. López-García and Moreira, “Open Questions.”
  5. Laura Villanueva, Stefan Schouten, and Jaap S. Sinninghe Damsté, “Phylogenomic Analysis of Lipid Biosynthetic Genes of Archaea Shed Light on the ‘Lipid Divide,’” Environmental Microbiology 19, no. 1 (January 2017): 54–69, doi:10.1111/1462-2920.13361.
  6. Jonathan Lombard, Purificación López-García, and David Moreira, “The Early Evolution of Lipid Membranes and the Three Domains of Life,” Nature Reviews Microbiology 10 (June 11, 2012): 507–15, doi:10.1038/nrmicro2815.

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Endosymbiont Hypothesis and the Ironic Case for a Creator



i ·ro ·ny

The use of words to express something different from and often opposite to their literal meaning.
Incongruity between what might be expected and what actually occurs.

—The Free Dictionary

People often use irony in humor, rhetoric, and literature, but few would think it has a place in science. But wryly, this has become the case. Recent work in synthetic biology has created a real sense of irony among the scientific community—particularly for those who view life’s origin and design from an evolutionary framework.

Increasingly, life scientists are turning to synthetic biology to help them understand how life could have originated and evolved. But, they have achieved the opposite of what they intended. Instead of developing insights into key evolutionary transitions in life’s history, they have, ironically, demonstrated the central role intelligent agency must play in any scientific explanation for the origin, design, and history of life.

This paradoxical situation is nicely illustrated by recent work undertaken by researchers from Scripps Research (La Jolla, CA). Through genetic engineering, the scientific investigators created a non-natural version of the bacterium E. coli. This microbe is designed to take up permanent residence in yeast cells. (Cells that take up permanent residence within other cells are referred to as endosymbionts.) They hope that by studying these genetically engineered endosymbionts, they can gain a better understanding of how the first eukaryotic cells evolved. Along the way, they hope to find added support for the endosymbiont hypothesis.1

The Endosymbiont Hypothesis

Most biologists believe that the endosymbiont hypothesis (symbiogenesis) best explains one of the key transitions in life’s history; namely, the origin of complex cells from bacteria and archaea. Building on the ideas of Russian botanist Konstantin Mereschkowski, Lynn Margulis(1938–2011) advanced the endosymbiont hypothesis in the 1960s to explain the origin of eukaryotic cells.

Margulis’s work has become an integral part of the evolutionary paradigm. Many life scientists find the evidence for this idea compelling and consequently view it as providing broad support for an evolutionary explanation for the history and design of life.

According to this hypothesis, complex cells originated when symbiotic relationships formed among single-celled microbes after free-living bacterial and/or archaeal cells were engulfed by a “host” microbe. Presumably, organelles such as mitochondria were once endosymbionts. Evolutionary biologists believe that once engulfed by the host cell, the endosymbionts took up permanent residency, with the endosymbiont growing and dividing inside the host.

Over time, the endosymbionts and the host became mutually interdependent. Endosymbionts provided a metabolic benefit for the host cell—such as an added source of ATP—while the host cell provided nutrients to the endosymbionts. Presumably, the endosymbionts gradually evolved into organelles through a process referred to as genome reduction. This reduction resulted when genes from the endosymbionts’ genomes were transferred into the genome of the host organism.


Figure 1: Endosymbiont hypothesis. Image credit: Wikipedia.

Life scientists point to a number of similarities between mitochondria and alphaproteobacteria as evidence for the endosymbiont hypothesis. (For a description of the evidence, see the articles listed in the Resources section.) Nevertheless, they don’t understand how symbiogenesis actually occurred. To gain this insight, scientists from Scripps Research sought to experimentally replicate the earliest stages of mitochondrial evolution by engineering E. coli and brewer’s yeast (S. cerevisiae) to yield an endosymbiotic relationship.

Engineering Endosymbiosis

First, the research team generated a strain of E. coli that no longer has the capacity to produce the essential cofactor thiamin. They achieved this by disabling one of the genes involved in the biosynthesis of the compound. Without this metabolic capacity, this strain becomes dependent on an exogenous source of thiamin in order to survive. (Because the E. coli genome encodes for a transporter protein that can pump thiamin into the cell from the exterior environment, it can grow if an external supply of thiamin is available.) When incorporated into yeast cells, the thiamin in the yeast cytoplasm becomes the source of the exogenous thiamin, rendering E. coli dependent on the yeast cell’s metabolic processes.

Next, they transferred the gene that encodes a protein called ADP/ATP translocase into the E. coli strain. This gene was harbored on a plasmid (which is a small circular piece of DNA). Normally, the gene is found in the genome of an endosymbiotic bacterium that infects amoeba. This protein pumps ATP from the interior of the bacterial cell to the exterior environment.2

The team then exposed yeast cells (that were deficient in ATP production) to polyethylene glycol, which creates a passageway for E. coli cells to make their way into the yeast cells. In doing so, E. coli becomes established as endosymbionts within the yeast cells’ interior, with the E. coli providing ATP to the yeast cell and the yeast cell providing thiamin to the bacterial cell.

Researchers discovered that once taken up by the yeast cells, the E. coli did not persist inside the cell’s interior. They reasoned that the bacterial cells were being destroyed by the lysosomal degradation pathway. To prevent their destruction, the research team had to introduce three additional genes into the E. coli from three separate endosymbiotic bacteria. Each of these genes encodes proteins—called SNARE-like proteins—that interfere with the lysosomal destruction pathway.

Finally, to establish a mutualistic relationship between the genetically-engineered strain of E. coli and the yeast cell, the researchers used a yeast strain with defective mitochondria. This defect prevented the yeast cells from producing an adequate supply of ATP. Because of this limitation, the yeast cells grow slowly and would benefit from the E. coli endosymbionts, with the engineered capacity to transport ATP from their cellular interior to the exterior environment (the yeast cytoplasm.)

The researchers observed that the yeast cells with E. coli endosymbionts appeared to be stable for 40 rounds of cell doublings. To demonstrate the potential utility of this system to study symbiogenesis, the research team then began the process of genome reduction for the E. coli endosymbionts. They successively eliminated the capacity of the bacterial endosymbiont to make the key metabolic intermediate NAD and the amino acid serine. These triply-deficient E. coli strains survived in the yeast cells by taking up these nutrients from the yeast cytoplasm.

Evolution or Intentional Design?

The Scripps Research scientific team’s work is impressive, exemplifying science at its very best. They hope that their landmark accomplishment will lead to a better understanding of how eukaryotic cells appeared on Earth by providing the research community with a model system that allows them to probe the process of symbiogenesis. It will also allow them to test the various facets of the endosymbiont hypothesis.

In fact, I would argue that this study already has made important strides in explaining the genesis of eukaryotic cells. But ironically, instead of proffering support for an evolutionary origin of eukaryotic cells (even though the investigators operated within the confines of the evolutionary paradigm), their work points to the necessary role intelligent agency must have played in one of the most important events in life’s history.

This research was executed by some of the best minds in the world, who relied on a detailed and comprehensive understanding of biochemical and cellular systems. Such knowledge took a couple of centuries to accumulate. Furthermore, establishing mutualistic interactions between the two organisms required a significant amount of ingenuity—genius that is reflected in the experimental strategy and design of their study. And even at that point, execution of their experimental protocols necessitated the use of sophisticated laboratory techniques carried out under highly controlled, carefully orchestrated conditions. To sum it up: intelligent agency was required to establish the endosymbiotic relationship between the two microbes.


Figure 2: Lab researcher. Image credit: Shutterstock.

Or, to put it differently, the endosymbiotic relationship between these two organisms was intelligently designed. (All this work was necessary to recapitulate only the presumed first step in the process of symbiogenesis.) This conclusion gains added support given some of the significant problems confronting the endosymbiotic hypothesis. (For more details, see the Resources section.) By analogy, it seems reasonable to conclude that eukaryotic cells, too, must reflect the handiwork of a Divine Mind—a Creator.



  1. Angad P. Mehta et al., “Engineering Yeast Endosymbionts as a Step toward the Evolution of Mitochondria,” Proceedings of the National Academy of Sciences, USA 115 (November 13, 2018): doi:10.1073/pnas.1813143115.
  2. ATP is a biochemical that stores energy used to power the cell’s operation. Produced by mitochondria, ATP is one of the end products of energy harvesting pathways in the cell. The ATP produced in mitochondria is pumped into the cell’s cytoplasm from within the interior of this organelle by an ADP/ATP transporter.
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