Molecular Paleontology and Biochemical Evolution

Carl Woese’s drive for a unified system of biological classification didn’t just open the microbial world to exploration: it reshuffled the entire taxonomic system and revolutionized the way that biologists study evolution, reigniting interest in preanimal evolution. Studies of evolution from the mid-nineteenth through most of the twentieth century relied on the comparison of forms in living and fossil organisms and were limited to the complex multicellular organisms that developed over the past 550 million years. In other words, much was known about the evolution of animals and land plants that left distinctive hard fossils, and very little was known about the unicellular algae and microorganisms that occupied the seas for most of the earth’s history. Woese’s Tree of Life, derived from nucleic acid sequences in ribosomal RNA, has revealed ancestral relationships that form and function don’t even hint at, allowing biologists to look beyond the rise of multicellular life and link it with less differentiated, more primal forms—which was precisely Woese’s intention. But evolution is a history, not just a family tree of relationships. If the information stored in the genes of extant organisms is to provide true insight into that history, it needs to be anchored in time, linked to extinct organisms and to past environments. Ultimately, we must look to the record in the rocks and sediments, just as paleontologists and biologists have been doing for the past two centuries. In Darwin’s time, that record comprised rocks from the past 550 million years, a span of time that geologists now call the Phanerozoic eon, based on Greek words meaning visible or evident life. The eon began with the rocks of the Cambrian period, in which nineteenth- and early-twentieth-century paleontologists discovered a fabulous assortment of fossils—traces of trilobites, anemones, shrimp, and other multicellular animals that were completely missing from any of the earlier strata. Thousands of new animals and plants, including representatives of almost all contemporary groups, as well as hundreds of now-extinct ones, appeared so suddenly between 542 and 530 million years ago that paleontologists refer to the phenomenon as the Cambrian “explosion.”

Black Gold: An Alchemist’s Guide to Petroleum

For many of us who studied and came of age in the last two decades of the twentieth century, there was nothing more prosaic, lacking in romance, and less worthy of our scientific curiosity than petroleum. The basic questions about its composition and origin had been answered, and it was no longer one of Nature’s secrets luring us to discovery, but rather the dull stuff of industry and business, money and technology. Some of us even imagined, naively, that we would witness the end of the age of fossil fuels: they were the bane of modern man, the source of pollution, environmental disaster, and climate change that threatened to disrupt ecosystems and civilizations around the entire globe. Finding new reserves, we reasoned, would only forestall the inevitable, or exacerbate the havoc. But when Jürgen joined Germany’s government-funded Institute of Petroleum and Organic Geochemistry in 1975, there was still a sense of mission in finding new reserves. The energy crisis of the early 1970s had created a heightened awareness of the value of fossil fuels and the need for conservation, but the accepted wisdom remained that oil was the key to the future and well-being of civilization. And the chemistry, it seems, was anything but banal—it was, in fact, leading not just to a better success rate in finding new reserves of oil, but also to a new understanding of life that no one had foreseen. Certainly for Geoff and the generations of organic chemists that came before him, the oils that occasionally seeped out of a crack in a rock, or came spouting out of the earth if one drilled a hole in the right place, were as intriguing as the life some said they came from. Liquid from a solid, organic from mineral, black or brown or dark red, it was as if blood were oozing from stone, an enigma that inspired inquiry from scientists long before it found its place among man’s most coveted commodities.

Deep Sea Mud: Biomarker Clues to Ancient Climates

Though the concept of the biomarker emerged from attempts to infer the provenance of petroleum and the incidence of life on the young earth—for all the successes and disappointments of the early studies on Precambrian rocks, lunar dust, and oil shales—it was in the sediments of the deep sea that biomarkers really came into their own. The Deep Sea Drilling Project (DSDP) was initiated in the 1960s by a consortium of American oceanographic research institutions, but institutions in Russia, the United Kingdom, France, and Germany were quick to sign on. In what began as an effort to understand the makeup and dynamics of the earth’s crust and mantle, the DSDP’s special research ship traveled the world’s oceans, drilling thousands of meters into the seafloor to retrieve sediment cores that soon became coveted objects of study for geologists, oceanographers, biologists, paleontologists, and geochemists around the world. When Geoff’s group started analyzing the DSDP sediments in the early 1970s, most of the organic chemists involved with the program were from the oil industry and formed part of the drill ship’s safety program, monitoring the cores as they were brought on deck to ensure that dangerous accumulations of gas or liquid hydrocarbons weren’t being penetrated. But Geoff saw the DSDP as the perfect opportunity to wean his Bristol lab of its dependence on NASA’s Apollo program—a chance to bring his full attention back to Earth and its still largely unexplored realm of fossil molecules. The British Natural Environment Research Council had earmarked a large pot of funding for work on the cores, which would be unencumbered by the narrow commercial goals and secrecy that surrounded the limited offerings from oil-company bore holes. Geoff’s budding Organic Geochemistry Unit would be aligned with a multidisciplinary community of scientists who were all studying the same cores, working cooperatively, and publishing freely. And, unlike the lunar samples, ocean sediments were rife with interesting organic compounds, including many entirely unforeseen structures. Most of the cores consisted of sediments that had been laid down and buried sequentially without ever being subjected to the tectonic turmoil of stretching and subsidence, and the overlying kilometers of cold water had kept their temperatures relatively low.

Thinking Molecularly, Anything Goes: From Mummies to Oil Spills, Doubts to New Directions

Though the biomarker saga began with attempts to understand the ancient provenance of petroleum and with the concept of “fossil molecules” and search for early forms of life, the explorations of the past 50 years have led organic geochemists far afield of these first endeavors. As Geoff and Max Blumer recognized back in the 1960s, and as microbiologists began to realize in the early 1980s, the usefulness of the biomarker concept is not restricted to geologic time. Most organic geochemists have, at one time or another, applied their techniques and expertise to the resolution of environmental problems, or found a way to address some archaeological mystery. One of the Bristol group’s most vibrant research programs now has its chemists brushing shoulders not with geologists and oceanographers, but with archaeologists and anthropologists concerned with the evolution of human civilizations and societies. Much of the impetus for the application of biomarker concepts to archaeologists’ questions in the 1970s and 1980s came from petroleum geochemists, not least from Arie Nissenbaum, a geochemist at Israel’s Weizmann Institute of Science who developed a keen interest in the role that geological events and circumstance might have played in the history of civilizations in the Fertile Crescent region. Nissenbaum was fascinated by the bizarre geology and chemistry of the Dead Sea Basin area, where oil seeps and impressive raftlike chunks of asphalt floating on the surface of the lake had long tempted oil prospectors to no avail. Renewed interest in the area’s oil potential in the early 1980s attracted a wave of geochemical studies, and Israeli geochemists scrambled for laboratory resources and funding from abroad. Jürgen, still with Dietrich Welte’s group, did a detailed biomarker study at the behest of an Israeli colleague, and when Nissenbaum saw the results he suggested to Jürgen that they apply Jülich’s considerable GC-MS capability to solving an entirely different sort of mystery. Excavations of archaeological sites in the vicinity of the Dead Sea had turned up solid chunks of black, sticky material that was used as early as 3000 B.C., either in materials used for construction or as a glue to attach tool heads to wooden handles.

From the Moon to Mars: The Search for Extraterrestrial Life

“But did anyone really expect to find anything?” I ask Geoff, as he shows me the canister that had contained his sample of moon dust from the 1969 Apollo 11 mission. “Well, no,” he replied, “we didn’t think there’d ever been life on the moon. But we didn’t know. We thought there might be organic compounds.” And why not? People had been finding organic compounds in meteorites for more than a century, and no one was quite sure where they’d come from or how they’d formed. In 1834, the Swedish chemist Jöns Jakob Berzelius noted the high carbon content of a meteorite that had fallen in southern France a couple of decades earlier. Meteor showers in Europe were described as early as 1492, and their extraterrestrial provenance had been documented in 1803, when the distinguished French physicist Jean-Baptiste Biot featured among the scores of citizens who witnessed the stones falling from the sky above the village of l’Alsace. But the source of the carbon compounds Berzelius and others found in meteorites would remain controversial far into the next century. Another carbonaceous meteorite fell in Hungary in 1857, and the eminent chemist Frederick Wöhler—Berzelius’s student, and the first to show that one could create carbon compounds like those made by organisms from inorganic substances in the lab—found organic compounds that he was convinced were of extraterrestrial biological origin. A decade later, Marcellin Berthelot found what he called “petroleum-like hydrocarbons” in a meteorite that had fallen near Orgueil, France, in 1864. He postulated that the hydrocarbons had formed abiotically from reaction of metal carbides with water, but in the next few years there was a spate of meteorite treatises in which the fossils of an astounding assortment of exotic extraterrestrial creatures were described in minute detail. Louis Pasteur had just presented his famous experiment showing that a protected, sterile medium remained devoid of life ad infinitum and debunked the popular theory that life could burst spontaneously into being from nonliving matter, but now the debate shifted to the possibility that life on Earth had originated with live cells or spores delivered by meteorites from space.

Molecular Informants: A Changing Perspective of Organic Chemistry

Lodged in the earth’s outermost layer, ephemeral scratch on a mineral skin, life plays cards with a handful of elements—builds molecular extravaganzas of carbon and hydrogen, oxygen, nitrogen, sulfur, or precious phosphorus, and forms the pieces to the parts that, assembled, define it. When the game is over, the cards reshuffled, the parts dismantled—membranes ruptured, shells dissolved, bones ground to dust—a few of those organic molecules remain in the sediments and rocks, bearing witness to the distant moments of their creation. Imagine the most humble bit of life, a microscopic alga basking in the sun-graced surface of the sea. Think of the tiny animal that grazes on the alga, dismantling its parts, using the pieces and discarding the difficult-to-digest fats and sturdy membrane lipids in tiny pellet-like feces that sink slowly into the dark waters of the deep sea—a thousand meters, two, three, maybe more. Imagine the bacteria that cling to the pellets as they settle onto the seafloor, zealous recyclers of organic molecules, using some and transforming others, leaving them stripped down or broken but still recognizable among the generic mineral bits of shell and clay that accumulate, particle by particle, year by year, layer by layer. Dig down, dig back, through meters and kilometers of sediments, through millennia and epochs, and you’ll find them yet, those molecular relics, testaments to that tiny, light-loving bit of bygone life. What do those molecules know, what do they have to say? Might they remember their maker’s name and environment, how that tiny alga lived and died? Was it rich or poor, food plentiful or scarce, the water warm or cold? Perhaps there was a current from the south, or cold nutrient-rich waters upwelling from the deep. Maybe there was a drought in Africa and dry winds blew nutrient-laden dust over the Atlantic, the continent’s misfortune a literal windfall for marine algae. Perhaps a meteor fell that year and the light went out of the sky, the temperature dropped suddenly, and the world died in a blink.

Early Life Revisited

That the evolution of organisms depends in large part on the evolution of their environment is something paleontologists have been noting since the early nineteenth century, and indeed, it is so inherent in Darwinian theory as to seem almost banal. That this dependency might have been two-way—that the earth’s minerals, atmosphere, oceans, and climate have been in large measure determined by the evolution of different life-forms—was somewhat harder to document and accept, partly because the most dramatic evidence was hidden, at the molecular level, in the elusive Precambrian rocks. The concept of the coevolution of Earth and life saw its first cohesive and most provocative expression when James Lovelock presented his Gaia hypothesis in the early 1970s, but not until the end of the twentieth century were the basic tenets of the hypothesis accepted as a valid theory. Lovelock began conceiving the Gaia hypothesis when he was designing instruments for NASA’s first extraterrestrial explorations and it occurred to him that, unlike the moon and Mars, the earth had an atmosphere composed of gases that couldn’t and wouldn’t coexist without life’s intervention. At the same time, a handful of paleontologists and geochemists had been conceiving similar if less provocatively formulated hypotheses based on their studies of the earth’s most ancient rocks and sediments. In 1979, a decade after Geoff, Thomas Hoering, and Keith Kvenvolden had more or less given up on the prospect of garnering clues about early life-forms from the fossil molecules in Archean and early Proterozoic rocks, one of those paleontologists inadvertently inspired a certain Australian chemist to give it another go. Roger Summons met the paleontologist Preston Cloud when Cloud was on sabbatical at the Australian Institute of Marine Science. Summons was working in the biology department at Australian National University and had been assigned to play guide and chauffeur for Andrew Benson, a visiting American plant physiologist who was staying out at the marine institute. “There was a couple living in the guesthouse next to us,” Summons tells me. “And this guy was a jogger. He’d leave every morning at 5:00 A.M. and run past the house, clump clump clump clump, and I’d wake up.”

Weird Molecules, Inconceivable Microbes, and Unlikely Environmental Proxies: Marine Ecology Revised

Anaerobic methanotrophs are not the only ecologically important archaea to surprise microbiologists in the last decade. And their isoprenoid ethers are not the only useful lipids—and certainly not the strangest—to have joined the lexicon of microbial biomarkers. Though much of that lexicon is still too generic to be of much use in understanding geologic history, some of these structures have allowed geochemists to transcend biological complexity and garner clues to past climates and environments. In the 1990s, when Stefan Schouten first started finding ring-containing biphytanyl ethers in his sediment samples, he was still working on his doctorate at NIOZ. Like everyone else at the time, he assumed that they derived from the lipids of methanogenic archaea and that it was only a matter of time before ring-containing biphytanyl tetraethers would be identified among the lipids of some newly isolated culture of methanogens, as Guy Ourisson had predicted. Schouten was studying oxygen- and sulfur-bound biomarkers, which meant he treated his sediment extracts chemically to cleave the ether and sulfur bonds, and the treatments often turned up biphytanes. But then, he says, he and another student started finding the ring-containing compounds in some really unlikely places, such as the oxic surface layer of marine sediments where neither methanogens nor extreme thermophilic and halophilic archaea were likely to make a home. The only thing they could think of at the time was that the tetraethers had come from methanogens that lived in the oxygen minimum zone, the layer of water beneath the photic zone where heterotrophic bacteria are active, sometimes to the point of using up all of the oxygen. When Schouten presented these ideas at the 1995 organic geochemistry meeting, Stuart Wakeham immediately piped up with the suggestion that they look for the lipids in the water column—and offered the perfect samples for the enterprise. He had collected particulate matter at different depths in the Black Sea and Cariaco Basin, just the sort of anoxic environments where one might expect to find methanogens in the water column. . . .

Microbiologists (Finally) Climb on Board

In the half century since paleontologists began finding putative microfossils in Precambrian sedimentary rocks, it has become apparent that not only is most of life’s history absent from the visible fossil record, but huge sectors of extant life remain to be discovered. Until the early 1980s, the only way to identify and study species of microbes—many if not most of which are morphologically indistinct from each other—was by growing them in the laboratory, isolating the separate colonies of organisms that developed as they reproduced, and then noting differences and similarities in what they consumed and produced. But the capacity to read the information in microbial genes that was developed in the 1980s and 1990s opened an entirely new world for study—much as the invention of the microscope had in the eighteenth century—and laid bare the unnerving fact that the vast majority of microbes on the planet had been boycotting the microbiologists’ carefully prepared cultures. Microbiologists had spent almost a century painstakingly cultivating, isolating, and classifying microorganisms, and yet they had failed to identify the most abundant microbes in natural waters, sediments, and soils. Indeed, it now appears that the hundreds of thousands of microbes named and maintained in the world’s bacteria zoos, or “culture collections,” invaluable as they are, comprise but a tiny and somewhat random sampling of the microbial world. These microbial cultures provide the only means by which biologists can directly manipulate and study the biochemistry and physiology of this huge sector of life in the laboratory—but they are distinguished more by their ability to prosper under laboratory conditions than by their importance in natural ecosystems. In the past few years, application of new techniques from molecular biology has resulted in the discovery of thousands of strange new types of microorganisms, and there is the implication of countless more: in the twenty-first century we find ourselves unexpectedly gathered at the threshold of a new world, looking not to Mars or Jupiter or to some distant galaxy, but gazing awestruck at the mud beneath our feet and the water in our seas.

More Molecules, More Mud, and the Isotopic Dimension: Ancient Environments Revealed

Throughout the 1980s, while the molecule collectors were busy exploring the ocean sediments, tracking their finds into the past, and learning to read the messages hidden in the carbon skeletons, one analytical chemist cum geochemist at Indiana University was finding that important elements of the lexicon lay not only in the molecular structures, stereochemistry, and distributions of the carbon skeletons, but in the carbon atoms themselves. John Hayes had done his graduate work at MIT in the mid-1960s under Klaus Biemann, one of the doyens of mass spectrometry who, like Carl Djerassi, was interested in natural products with biomedical applications. When Hayes told Biemann he wanted to do his doctoral thesis on the organic constituents in meteorites, Biemann was uninterested, to say the least. Forty years later, Hayes can still quote the eminent scientist’s response to his proposal, replete with thick Austrian accent: “Don’t talk to me about zat junk.” Biemann walked away from the discussion without another word, and Hayes was so mortified by his own foolishness that he couldn’t bring himself to tell his wife about the incident. When he went into the lab the next day, he was convinced that his graduate career was over—but Biemann had done some homework and had a change of heart. “It seems we can get lots of money for zat junk!” he exclaimed as soon as he saw Hayes. NASA was, at the time, offering generous funding for such projects. For all his skepticism, Biemann was eventually seduced by the extraterrestrial “junk” and even ended up designing the mass spectrometer for the Viking Mars mission. Hayes remembers him commenting, a couple of years into the meteorite project, that it was actually “much more interesting than the thousandth alkaloid in the thousandth tree,” though Hayes himself says his doctoral thesis was unexceptional, completed before the Murchison meteorite fell and things really got interesting.