Book Review The Tangled Tree by David Quammen: How Studying Bacteria Revolutionized the Understanding of Evolution

Introduction

David Quammen, the detective-science writer, discovered Ford Doolittle’s articles on evolution in 2013 and was immediately struck by their implications.?Horizontal Gene Transfer (HGT)?and microbial dynamics proved that the Tree of Life was wrong. The pattern by which species evolve is not simply by divergence from a common trunk, but recombinations down the line, exchanges of plasmids, organelles, and viruses made for a richer, more complex, and more satisfying Web of Life. Here is a 12 minutes summary of a 14-hour book covering over 200 years of scientific investigation of where we come from.

Lamarck: the first evolutionary tree

The evolutionary theory is usually traced back to Darwin, but French Botanist Augier already made an Arbre Botanique in 1801, grouping plants along a tree. However, it was not clear if he meant it as a measure of origin and descent or a showcase of plant similarities. Lamarck, in 1809, made a tree more explicitly titled “to show the origin of different animals”, known as the earliest evolutionary tree.

Darwin: species are not?fixed

Darwin’s grandfather had evoked the idea that species diverge from one another long before his grandson. Charles Darwin’s observations while traveling on the Beagle confirmed these intuitions. In 1837, young Darwin drew a little sketch in his leather book with diverging branches, and further, he commented “Organized beings represent a tree”.

Darwin was a thinker, and he spoke of his theory in close circles, matured it, and thought of writing a book, but went on without publishing it for 20 years.?

Wallace: another traveler with the same?ideas

Alfred Russel Wallace did a similar journey as Darwin and came up with similar ideas in 1858. He reached out to Darwin to ask for help in getting the theory published. You can imagine Darwin’s shock and dilemma.

They decided to publish conjointly, and the year after, Darwin released On the Origin of Species. The double announcement made a lot of noise and controversy: these were dangerous ideas that questioned the Christianity of the Biologists. Maybe because Darwin was recognized as the father of this idea, or maybe because he popularized it in his book, history remembers him over Wallace.

This theory changed the world’s story from being created as men were seeing it, to a dynamic path of species emerging, thriving, and going extinct. This narrative in the shape of a Great Tree, like Haeckel’s oak, would last for two centuries.

Crick, Pauling, and Zuckerkandl: the advent of molecular phylogenetics

Fast-forward to the 1950s, when DNA was known but considered a boring molecule. “Genes” was a word used to refer to transmissible traits, but what molecules genes were exactly was unclear. Then Crick published “On Protein Synthesis” where he showed the importance of DNA instructions, and advised that these long chains of nucleic acids could provide evidence for evolutionary trees. In 1963, Pauling and Zuckerkandl went further and offered the metaphor of DNA as the “molecular evolutionary clock”.

“Scrutiny of such molecules can tell us three things: how much time has passed since the lineages split, what the ancestral molecules must have looked like, and what were the lines of descent” — Pauling & Zuckerkandl.

Woese: using RNA as an evolutionary clock

Carl Woese was fascinated by what happened 4 billion years ago when life first emerged. At 36 years, he had the brilliant idea of using RNA to decipher the story back to the very first cells.

All living cells contain ribosomes (the organelles that translate RNA into protein), and the small unit of ribosomes, called 16s, is both shared enough and variable enough across all living beings that it offers a wealth of information.

Woese trained as a biophysicist but was not much of an experimentalist. His team cultured microbes, extracted RNA, cut it into pieces with specific enzymes, and worked electrophoresis. And Woese? Woese stared at the spots, training his eyes on the alphabet of fragments. He learned to recognize patterns and to use similarities and differences to classify microorganisms.

At this time, the Tree of Life was composed of two main branches: Eukaryotes, meaning organisms whose cells have a real nucleus, including all animals, fungi, and plants, and Prokaryotes, meaning those without nuclei - basically bacteria.

Wolfe & Woese: what are really methanogens?

In 1976, Ralph Wolfe sent Woese a sample of an “unusual bacterium”, a methanogen, for an RNA analysis. The result of the fingerprinting was very strange. Prokaryote signatures were missing, and some Eukaryotic fragments appeared, that shouldn’t have been there. A puzzling, incomprehensible outcome.

“It dawned on me, Woese wrote. There was something out there other than prokaryotes and eukaryotes. A third form of life, separate” — Carl Woese, upon discovering the RNA of Delta H.

Woese called this revelation his “out-of-biology experience”. Wolfe recalled their discussions: “Carl’s voice was full of disbelief when he told me, “Wolfe, these things aren’t even bacteria”.?

“Wolfe, these things aren’t even bacteria” — Woese to Wolfe upon discovering the fingerprinting of the methanogen Delta?H.

The research team spent the rest of the year describing other methanogens and confirming their hypothesis, that methanogens were an entirely new form of life. They expected this new domain to be drastically distinct from the rest of the living world, and yet to encompass great diversity. They called it “Archaebacteria”.

It was a poor decision that caused a lot of confusion and didn’t help Woese get the credit he deserved for the discovery: no one understood why it was so important, especially if these things were just another form of bacteria. Which they weren’t. Their structure, metabolism, and genes were too distinct. 12 years later, the term was corrected to Archaea.?

Margulis: Eukaryotic cells as compound individuals

Lynn Margulis published in 1967 On the Origin of Mitosing Cells, substantiating eukaryotic cells as “compound individuals”. Their evolutionary story went something like, "once upon a time, there was a bacterium that ate (or was infected) by another bacterium. Instead of digesting the newcomer, our host conserved the foreigner and made use of its biology". This is why in eukaryotic cells, mitochondria are not coded for in the genome and divide like bacteria. This antique integration is referred to as the?endosymbiosis theory.

Lynn Margulis was not the first to ideate this theory — Schimper, in the mid-1880s, coined the term chloroplast and wrote that the resulting plant cells “would be somewhat reminiscent of a symbiosis”. The Russian Merezhkowsky claimed the idea as his in 1905, although he had read Schimper. In 1927, Ivan Wallin added mitochondria to the endosymbiosis theory. Margulis was aware of that past and she never claimed the paternity (maternity?) of the theory, but she unearthed it from its grave and made it her life’s mission to demonstrate its truth. It was not well received - the 70s were not ready to challenge the core of our identity and share it with mere bacteria.

“I greatly admire Lynn Margulis’s sheer courage and stamina in sticking by the endosymbiosis theory, and carrying it through from being an unorthodoxy to an orthodoxy.” — Dawking. (me too)

Other researchers including Ford Doolittle and Linda Bonen helped confirm that ribosomal RNA from chloroplasts and mitochondria resemble bacterial ribosomal RNA, and not the rRNA of their host.

Beyond its challenge to our sense of self, this finding presented a tremendous impact on the way we had looked at the organization of species and evolution until then. Variation is not only explained by a cumulation of tiny random mutations.

“Rather, the important transmitted variation that leads to evolutionary novelty comes from the acquisition of genomes” — Lynn Margulis.

In other words, evolutionary novelty comes from symbiosis.?

“The evolutionary biologists believe the evolutionary pattern is a tree. It’s not. The evolutionary pattern is a web — the branches fuse.” — Lynn Margulis.

From there, key questions remained to answer. How much did the integration of external organisms and gene transfer play a role in evolution? Was it a one-event-in-a-geological lifetime type of thing, or much more common in the daily swings of life’s dances? Lynn thought it was a major dynamic overall evolution, while Woese thought it was confined to one or two events at the beginning of a more complex life.

Griffith: gene transfer of antibiotic resistance

From the “pabulum” description of virulence transfer in?pneumococcus?by Griffith to transduction and conjugation, to realizing that antibiotic resistance spreads horizontally and quickly, was a relatively quick process. Indeed, penicillin was discovered in 1928 by Fleming, started being used in humans in 1942, and resistant strains turned up in 1959 — to become a concern in multiple countries by 1972.?

Chickens treated with tetracycline led to acquired resistance in less than a week, and jumped to the bowels of farm workers within a few months. The observation of horizontal gene transfer (HGT) had important public health implications.

Jones and Sneath: barriers between species begin to?fall

Jones and Sneath observed in 1970 that genes could cross boundaries between species and even between genuses to “almost every group in the enteric bacteria”.

“Barriers between species might begin to fall, they wrote. This in turn could favor extremely reticulate modes of evolution, with numerous partial fusions of phyletic lines” — Jones and Sneath, 1970.

In the 90s, there was an explosion of awareness of HGT.

Sonea and Panisset: the superorganism

In 1983, Sonea and Panisset made a case that HGT happened beyond bacteria, including in Eukaryotes (8% of rotifer’s genes appeared acquired from bacteria and other dissimilar creatures), and thus that all life could be considered as a single superorganism. They found that bacterial DNA was also common in human cells, and 210 times more common in tumor cells than in healthy ones.

Venter: backward trees don’t?match

Venter et al. published in 1996 the first whole genome sequence of the Archae?Methanococcus jannaschii, and scientists analyzed it through selected proteins essential to all forms of life, to trace back its evolutionary history. The result was telling: the trees behind the different proteins’ history didn’t match. They seemed to have evolved separately from the Archaea, and joined later on.

Evidence built up that “HGT was not a rarity but a rampant phenomenon”. For example, Gogarten et al published that?18% of?E. coli’s genome was acquired through HGT.

HGT impacts can be understood in 4 ways, according to Quammen and Doolittle:

• new sideways genes may allow a microbe population to colonize an entirely new niche;
• it may allow organisms a new sort of abrupt adaptation without passing through the dangerous half-adapted stage;
• it’s a fast transformation;
• it’s a source of innovation, bringing new supplies of variation upon which selection can act.

The Tree of Life started to look more like Doolittle’s famous drawing:

Doolittle had great communications skills and he helped make these scientific discoveries accessible beyond the sphere of evolutionary research.?

Doolittle, Martin, Gogarten, and Lawrence: the 4 horsemen of?HGT

The four friends proposed a synthesis of life’s evolution through both vertical and horizontal gene transmission. They showed these findings were not only applicable among bacteria: Martin found a plant of which 18% of the genome was bacterial in origin. A yeast also was found to host 850 genes from bacteria and Archaea.?

“It’s all endosymbiotic gene transfer” said Martin. “Only 1% of the genes in an average bacterial or archaea genome — and maybe far less in a eukaryote — are so deeply and complexly essential to the organism that they couldn’t be swapped by HGT.”

In a study of over half a million genes from 181 bacteria and archaea, Martin et al found that roughly?80% of genes in each organism had arrived horizontally at some point in their ancestry.

Like Doolittle’s, Martin’s coral-shaped reticulated tree took into account the complexity brought by both divergences and convergences throughout life’s history.

“If there is a tree of life, it’s a small anomalous structure growing out of the web of life” — Dupré.

Of course, it made the classification of microorganisms a headache.?

“If (an organism’s) genes are half bacterial and half archaeal, does that organism belong to Bacteria or Archaea? or is the question impossible or meaningless to answer?” — Doolittle.

“Because of that genetic fluidity, the concept of “species” is useless among Bacteria and Archaea” — Goldenfeld and Woese.

Pace and Alm: HGT in the gut microbiome

Norman Pace and Eric Alm found HGT to be also very common in the microbiome, especially between microorganisms inhabiting the same body sites.?

“These analyses indicate that recent HGT frequently crosses continents and the Tree of Life to connect the human microbiome globally in an ecologically structured network” — Alm.

What he means is that everything is connected, a gene transfer in someone’s belly in Asia can soon bring to the world a better capacity to digest algae for example.

Heidmann: what about viral?DNA?

The whole sequencing of the human genome led to some conundrums and unanswered questions. Why do we comprise so much “junk” DNA? What are these similar repetitive sequences found in humans, opossums, monkeys, and frogs?

Scientists finally understood. Big Aha moment. Viruses.

Sapp says 8% of the human genome consists of remnants of retroviruses that have invaded our lineage. For better or for worse?

Thierry Heidmann works in Gustave Roussy, 8 minutes from my home, and he found such retroviruses to code for syncytin, a protein so important that mice knocked out for syncytin genes (animals to which the genes were removed by bio-engineering) present structural defects in the boundary between the placenta and the fetus, and can’t give birth. Heidmann understood that the “domesticated” viral genes resulted in being key for?two mammalian functions: forming a syncytium between the mother and the fetus to let nutrients and gasses seep in and waste seep out, and suppressing the immune reaction that would otherwise expulse the fetus as recognized as non-self. The capability to carry the next generation internally seems a trait acquired by viral infection!

I don’t know about you, but this blew my mind.?How much our identity is intertwined with microorganisms is endless, at every scale, is… wow.

Microbes: Science is a never-ending process

Trying to make sense of the Biological world brought us humans to define three major concepts: tree, species, and individual. Now we know that each of these categories is wrong.?

“Everyone agrees that puppies are individuals, owls are individuals, humans are individuals, until you consider the disquieting molecular facts. We are mosaics, as Paabo noted, as Bill Martin said, not individuals.” — David Quammen

Microbes are the best teachers for the next phase of Life Sciences: they challenge our need for boundaries and illustrate how deeply and thoroughly everything is connected.