Image based on original art by Charis Tsevis

Blurring Life’s Boundaries

Darwinian theory is based on the idea that heredity flows vertically, parent to offspring, and that life’s history has branched like a tree. Now we know otherwise: that the ‘tree’ of life isn’t that simple.

By David Quammen

Since the late 1970s, there have come three big surprises about what we humans are and about how life on our planet has evolved.

The first of those three surprises involves a whole category of life, previously unsuspected and now known as the archaea. (They look like bacteria through a microscope, but their DNA reveals they are shockingly different.) Another is a mode of hereditary change that was also unsuspected, now called horizontal gene transfer. (Heredity was supposed to move only vertically, from parents to offspring.) The third is a revelation, or anyway a strong likelihood, about our own deepest ancestry. (It seems now that our lineage traces to the archaea.) So we ourselves probably come from creatures that, as recently as forty years ago, were unknown to exist.

One of the most disorienting results of these developments is a new challenge to the concept of “species.” Biologists have long recognized that the boundaries of one species may blur into another—by the process of hybridism, for instance. And the notion of species is especially insecure in the realm of bacteria and archaea.  But the discovery that horizontal gene transfer (HGT) has occurred naturally, many times, even in the lineages of animals and plants, has brought the categorical reality of a species into greater question than ever. That’s even true for us humans—we are composite individuals, mosaics.

It’s not just that—as you may have read in magazine articles—your human body contains at least as many bacterial cells as it does human cells. (This doesn’t even count all the nonbacterial microbes—the virus particles, fungal cells, archaea, and other teeny passengers inhabiting our guts, mouths, nostrils, and other bodily surfaces.) That’s the microbiome. Each of us is an ecosystem.

I’m talking about something else, a bigger and more shocking discovery that has come from the revolution in a field called molecular phylogenetics. (That phrase sounds fancy and technical, but it means merely the use of molecular information, such as DNA or RNA sequences, in discerning how one creature is related to another.) The discovery was that sizeable chunks of the genomes of all kinds of animals, including us, have been acquired by horizontal transfer from bacteria or other alien species.

This is an aspect of evolution that was unimagined by Charles Darwin. Evolution is trickier, far more intricate, than we had realized. The tree of life is more tangled.

1837
On the bottom of page 21 of notebook B, Darwin wrote: “organized beings represent a tree.”

1859
Fifty years later, Darwin published On the Origin of Species.  His book included just one illustration, a diagram of 11 hypothetical lineages proceeding upward through thousands of generations of inheritance—deep evolutionary time.

1990
This diagram asserted what Woese’s RNA fingerprint data showed: that we humans, and all other animals, all plants, all fungi, all eukaryotes, have arisen from an ancestral lineage that was unknown to science before 1977.

1999
Ford Doolittle published an overview of horizontal gene transfer in Science that put it at the center of a new discussion: whether it’s even possible to classify organisms into some “natural order” by placing them on a schematic tree of life.

How could that be possible? How could genes move sideways, between species, not just vertically along ancestral lineages? The mechanisms are complex, but one label that fits most of them is “infective heredity.”  DNA can be carried across boundaries, from one genome to another, by infective agents such as bacteria and viruses. Such horizontal gene transfer, like sex, has been a source of freshening innovation in otherwise discrete lineages, including ours—and it is still occurring.

This is an aspect of evolution that was unimagined by Charles Darwin. Evolution is trickier, far more intricate, than we had realized. The tree of life is more tangled.

Beginning in July 1837, Charles Darwin kept a small notebook, which he labeled “B,” devoted to the wildest idea he ever had. It wasn’t just a private thing but a secret thing, a record of his most outrageous thoughts. The notebook was bound in brown leather, with a tab and a clasp; 280 pages of cream-colored paper, compact enough to fit in his jacket pocket. The B notebook was first of a series on what, to himself only, he called “transmutation.”

At that time, the stability of species represented the bedrock of natural history. It was taken for granted, and was important, not just among clergy and pious lay people but scientists too. Now home from his wildcat voyage on HMS Beagle, the 27-year-old Darwin intended to investigate a radical alternative to scientific orthodoxy: that the forms of living creatures weren’t eternally stable, as God had reputedly created them, but instead had changed over time, one into another—by some mechanism that Darwin didn’t yet understand.

Jotting down clipped phrases, often in bad grammar and punctuation, he laid out a series of questions. “Why is life short,” he asked. Why is reproduction so important? “Each species changes. does it progress.” Put a pair of cats on an island, let them breed there for generations. Do the cats become better cats, or at least better cats for catting on that particular island? If so, how long would it take? How far would it go? What are the logical limits, Darwin wondered, if “every successive animal is branching upwards” and with “different types of organization improving,” new forms arising, old forms dying out?

That one word, branching, was freighted with interesting implications: of directional growth, of divergence, of an arboreal form. Big ideas were coming at him like diving owls. He needed some order as much as he needed the jumble of tantalizing clues. Maybe he needed a metaphor. Then, on the bottom of page 21, Darwin wrote: “organized beings represent a tree.”

He scribbled on. The tree is “irregularly branched,” he told the B notebook. Each branch diverges into smaller branches, he wrote, and then twigs, “Hence Genera,” the next higher category above species, which would be the twiglets or terminal buds. Some buds die away without yielding further growth, while new buds appear, somehow.

Fifteen pages along, after more ruminations, he drew a little sketch, in bold strokes, of a trunk rising into four major limbs and several minor ones. Each major limb diverged into clusters of branches, with certain branches labeled A, B, C, D. The letters were placeholders, meant to represent living species, or maybe genera. Felis, Canis, Vulpes, Gorilla.

This was a thunderous assertion, abstract but eloquent. You can look at the little sketch today, with its four labeled branches amid the limbs and the crown, and imagine the evolutionary divergence of all life from a common ancestor. Just above the sketch, as though gesturing toward it bashfully, Darwin wrote: “I think.”

It was an evolutionary tree of life. Darwin didn’t invent that phrase, “the tree of life,” nor originate its iconic use, though he put it to new purpose in his theory. Like so many other metaphors embedded deep in our thinking, it came down murkily, modified and reechoed, from early versions in Aristotle and the Bible. None represented the notion of change over time—of evolution—until Jean-Baptiste Lamarck’s 1809 book Philosophie Zoologique, which contained a vague evolutionary theory and depicted animal diversity in a branched diagram, descending down the page, with major animal groups connected by dotted lines.

Fifty years later, Darwin published On the Origin of Species.  His book included just one illustration, a diagram of 11 hypothetical lineages proceeding upward through thousands of generations of inheritance—deep evolutionary time. Eight of those 11 lineages came to dead ends—meaning, they went extinct, like trilobites and ichthyosaurs. One rose through the eons without splitting—meaning, it persisted unchanged, much the way horseshoe crabs have survived relatively unchanged over 450 million years. The other two lineages, dominating the diagram, branched often, spread horizontally, and climbed vertically, representing the exploration of different niches by newly evolved forms. So there it all was: evolution and the origins of diversity.

Darwin had seen evolution as arboreal. And the tree image would remain the best graphic representation of life’s history, evolution through time, the origins of diversity and adaptation, until the late twentieth century. Then rather suddenly a small group of scientists would discover: oops, no, it’s wrong.

Darwin had seen evolution as arboreal. And the tree image would remain the best graphic representation of life’s history, evolution through time . . . until the late 20th century. And then rather suddenly a small group of scientists would discover: oops, no, it’s wrong.

Carl Woese
(2004) ©Don Hamerman/CC BY 3.0

Lynn Margulis
(2005) CC BY-SA 2.5

Ford Doolittle
(2009) ©Riley Smith

Lynn Margulis was a forceful 29-year-old adjunct assistant professor from Chicago, divorced and raising two kids, when she brought new attention and credibility to a very strange old idea about the shape of the tree of life. She made her case, in March 1967, with a long paper published in the Journal of Theoretical Biology and titled “On the Origin of Mitosing Cells.” This radical, startling, and ambitious article—previously rejected by more than a dozen journals—proposed to rewrite two billion years of evolutionary history. It laid out an array of evidence supporting the odd conjecture that living ghosts of other life-forms exist and perform functions inside our very own cells. Adopting an earlier term, Margulis called that idea endosymbiosis.

It was the first recognized version of horizontal gene transfer. In these cases, rare but consequential, whole genomes of living organisms—not just individual genes or small clusters—had gone sideways and been captured within other organisms.

The phrase “mitosing cells” in the paper’s title is another way of saying eukaryotic cells, the ones with nuclei and other complex internal structures, the ones that compose all animals and plants and fungi. But the key word in Margulis’s title was “origin.”

“This paper presents a theory,” she wrote—a theory proposing that “the eukaryotic cell is the result of the evolution of ancient symbioses.” Single-celled creatures had entered into other single-celled creatures, like food within stomachs, or like infections within hosts, and by happenstance and overlapping interests, at least a few such pairings had achieved lasting compatibility.

Eventually they became more than partners. The internalized microbes, she argued, had evolved into organelles—working components of a new, composite whole, like the liver or spleen inside a human—with fancy names and distinct functions: mitochondria, chloroplasts, centrioles. They were functional elements of a single new being. A new kind of cell.

The scientific consensus at first, and for some years afterward, was that this smart, knowledgeable, insistent, and charming young woman was in thrall of a loony idea. But eventually the emerging science of molecular phylogenetics confirmed most of her theory of endosymbiosis (mitochondria and chloroplasts as captured bacteria, yes). Margulis became eminent, though never conventional.

As new evidence of horizontal gene transfer continued to accumulate during the 1990s, she and other biologists started questioning the belief that the evolutionary pattern is a tree. “It’s not,” Margulis told a reporter in 2011. “The evolutionary pattern is a web—the branches fuse.”

She was right: the tree of life is not perfectly tree-shaped. There’s something spooky and unnatural about any tree whose limbs grow together, sometimes, rather than always branching apart.

Two years after Margulis published her first provocative paper, challenging the total separation of the prokaryote kingdom (bacteria, lacking nuclei) and the eukaryote kingdom (all other cellular life, including animals and plants), another contrarian scientist embarked on an equally bold quest—to “unravel the course of events” leading to the origin of the simplest cells. His name was Carl Woese. He was an obscure microbiologist at the University of Illinois. In 1969, he confided in a letter to Francis Crick that he hoped to extend the understanding of evolution “backward in time by a billion years or so,” by “using the cell’s ‘internal fossil record’ ” as contained in DNA and RNA. Some years later, Woese’s work would trigger the revolution in molecular phylogenetics and lead to a drastic redrawing of the tree of life, from its roots to its crown.

By 1976, Woese and his team were doing RNA “fingerprinting” of various life forms, including methanogens—microbes that generate swamp gas in muddy wetlands and similar gas in the bellies of cows. Certain bits of structural RNA are built into all life, and the biologists sequenced those pieces, fragment by fragment, and then compared the collections of fragments between one creature and another.

Methanogens were hard to grow in a laboratory, since oxygen poisoned them, but Woese’s collaborators managed it. Under a microscope, these methanogens looked like bacteria. For centuries, they had been considered bacteria. But as Woese examined the fingerprints, he found anomalies. A certain pair of small fragments, common to all bacteria, were missing. Other sequences looked eukaryotic, suggesting a completely distinct form of life: a yeast, a protozoan, what? And still others were just weird.

What was this RNA? Woese wondered, and what manner of organism did it represent? It couldn’t be from a prokaryote. It wasn’t eukaryotic. It wasn’t from Mars, because it contained too many familiar stretches of RNA code.

“Then it dawned on me,” he later wrote. There was “something out there”—out there in the teeming ecosystems of planet Earth, he meant—other than prokaryotes and eukaryotes. A third form of life, separate. A third kingdom. In their seminal 1977 paper on the discovery, published in the Proceedings of the National Academy of Sciences (PNAS), Woese and his postdoc and coauthor, George Fox, gave their kingdom a tentative name: archaebacteria.

The name was misleading, a wrong choice, and would later be changed. It suggested ancient bacteria. By 1990, Woese and other scientists recognized that these creatures weren’t bacterial precursors nor ancient bacterial forms. Bacteria weren’t even their nearest kin. Some evidence had emerged, in fact, that archaea were more closely related to eukaryotes—more closely related to us—than to bacteria. So Woese and two other scientists wrote another paper for PNAS, proposing that there should be three major divisions of life, higher in rank than kingdoms, and those divisions should be domains, which would henceforth be known as the Bacteria, the Eukarya, and the Archaea. The word archaebacteria should now disappear, the authors argued. So should the word prokaryote. Prokaryotes didn’t exist as a phylogenetic category—it was a false unit—because Archaea and Bacteria stood so utterly distinct from each other.

And, of course, there was a tree. It was drawn in straight, simple lines, but it was rich and provocative nonetheless. This diagram asserted what Woese’s RNA fingerprint data showed: that we humans, and all other animals, all plants, all fungi, all eukaryotes, have arisen from an ancestral lineage that was unknown to science before 1977. It was the last of the great classical trees: authoritative, profound, completely new to science, and correct to some degree. But it entirely missed what was coming next.

What came next was an exploding awareness of the role played by horizontal gene transfer in this whole story. That explosion occurred during the 1990s but had deep precedents. The first recognition by science that any such thing might be possible dates to work published in 1928 by an Englishman named Fred Griffith. No one at the time, not even Griffith himself, saw the implications of what he had found.

As a medical officer at the London Pathological Laboratory of Britain’s Ministry of Health, Griffith studied what’s now known as Streptococcus pneumoniae, a dangerous bug that could cause severe, often fatal, pneumonia. During the 1918–19 influenza pandemic, this kind of pneumonia took hold as a secondary infection in many patients and probably killed more millions of people than the flu virus itself.

Griffith’s work, which was pragmatically medical, involved identifying the different types of the streptococcus—there were four—in different patients and parts of the country. He got his data by examining sputum coughed from the lungs of the ill. In 1923, he discovered something important: that each of the four types of the bacterium existed in two different forms—one that was ferociously virulent, one that was mild. Sometimes the virulent form might transmogrify into the mild form, he noticed. He didn’t know why.

His second discovery was far more puzzling: Under certain experimental circumstances, the mild form of, say, Type II bacteria could change into the virulent form of, say, Type I. What? It seemed as though the streptococcus had morphed into a different species.

Recent research has found evidence of bacterial DNA transferred horizontally into the genomes of human tumors. What that dizzying revelation means is still unclear, but there’s at least some chance that such insertions might play a role in causing cancer. 

The transformation Griffith witnessed was later shown to be one of three cardinal mechanisms of horizontal gene transfer, the most counterintuitive phenomenon discovered by biologists in the past century. Griffith’s experiments, and others like them, demonstrated that in its naked form—floating loose in the environment after having been liberated from a busted bacterial cell—DNA is capable of getting into another bacterium and causing heritable change. This sort of sideways passage can carry DNA not just across minor boundaries, type to type among Streptococcus pneumoniae, but also across huge gaps—from one bacterial species to another, from one genus to another, even from one domain of life to another. And the transformations that result from such horizontal transfer can be far more consequential than merely changing a pneumonia bug from mild to virulent.

Today we live with one of those consequences: bacterial resistance to multiple antibiotics, which spreads sideways among different kinds of bacteria. It can happen gradually or in a sudden leap, conveying multidrug resistance from harmless bacteria such as the common form of Escherichia coli into dangerous bacteria such as Shigella dysenteriae. Because of that sideways spread, fast and easy, bacterial resistance has become a dire problem. More than 23,000 deaths annually in the United States and seven hundred thousand deaths globally occur from infection by unstoppable strains of bacteria. This grim, costly trend has been driven not just by overuse of antibiotics, and by incremental adaptation—one strain of bacteria adapting to one antibiotic—but also by horizontal gene transfer, which spreads adaptations instantly.

The implications of horizontal gene transfer go far beyond the problem of antibiotic resistance. Those implications include the whole matter of how evolution works—by classical Darwinian mechanisms, or otherwise?—and how it has worked for much of the past four billion years.

HGT contradicts the conviction that bacterial species are fixed and discrete. If genes routinely cross the boundary between one species of bacteria and another, then in what sense is it really a boundary? New investigations, as time passed, showed that genes have even been transferred sideways between complex eukaryotic organisms. For instance: there’s a peculiar group of tiny animals known as rotifers, notable throughout molecular biology for their massive uploads of alien genes. A big rotifer might be a millimeter long, barely big enough to see, but small as they are, these are not single-celled creatures. They’re multicellular animals.

When Harvard researchers sequenced sections of genome in one rotifer species, they found at least 22 genes that must have arrived by horizontal transfer. Some of those were bacterial genes, some were fungal. One gene had come from a plant. Later work suggested that 8 percent of its genes had been acquired by horizontal transfer from bacteria or other dissimilar creatures.

Genes going sideways among animals? That was definitely supposed to be impossible. It wasn’t.

HGT started showing up among insects as well. The most dramatic case was one species of fruit fly, which had accepted almost the entire genome of a bacterium known as Wolbachia—more than a million letters of genetic code—into its own nuclear genome. Again, this was supposed to be impossible.

By 1999, discoveries had progressed to a point such that Ford Doolittle, a highly respected researcher and theorist based in Halifax, Nova Scotia, published an overview paper in Science that put HGT at the center of a new discussion: whether it’s even possible to classify organisms into some “natural order” by placing them on a schematic tree of life. Doolittle illustrated—literally—the difficulties with his own hand-drawn figure of what he called “a reticulated tree.” To his surprise, the editors of Science published it along with his paper.

And more recent research has found evidence of bacterial DNA transferred horizontally into the genomes of human tumors. What that dizzying revelation means is still unclear, but there’s at least some chance that such insertions might play a role in causing cancer. Putting horizontal gene transfer on the list of suspected human carcinogens brings it out of the realm of microbial arcana.

The cumulative effect of these discoveries has been to challenge three concepts that we have long considered categorically solid: the concepts of species, of individual, and of the tree of life. Now we can understand better. The boundaries between one species and another are not nearly so clear and impervious as we thought. The living individual, including the human individual, is a singular thing, yes, but at the same time a mosaic of life forms and genes of varied origin. And the tree of life, as I’ve said earlier, is not a tree. That is, life’s history doesn’t conform to the pattern of any arboreal plant you’ll ever find in a forest. Again, it’s more tangled.

These discoveries should not merely complicate our magisterial human self-image, but also help lead us toward a wiser and humbler understanding of our place—collectively and as “individuals” within the “species” Homo sapiens—in the story of life on Earth.

It’s a story in which we humans are important protagonists but not the ultimate and predestined heroes. It’s a story in which heredity has moved sideways as well as vertically and all the conventional hierarchies and boundaries have proven more imperfect, transgressible, and leaky than we had supposed. But these revelations don’t diminish our responsibility, as humans, to respect and preserve the diversity of living creatures, with all their own mosaic genomes and tangled lineages, who cohabit the planet with us. On the contrary, I think. All this should make us only more amazed, respectful, and careful. Life on Earth is wondrous precisely because it’s so complicated.

________________

David Quammen is a contributing writer for National Geographic and author of 15 books. This article was adapted from from THE TANGLED TREE: A Radical New History of Life by David Quammen. Copyright ©2018 by David Quammen. Reprinted by permission of Simon & Schuster, Inc.

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