A tree has something in common with the weeds and mushrooms growing around its roots, the squirrels scurrying up its trunk, the birds perched on its branches, and the photographer taking pictures of the scene. They all have genomes and cellular machinery neatly packed into membrane-bound compartments, an organizational system that places them in an immensely successful group of life forms called eukaryotes.

The early history of eukaryotes has long fascinated scientists who yearn to understand when modern life started and how it evolved. But tracing the earliest eukaryotes back through Earth’s history has been difficult. Limited fossil data shows that their first ancestor appeared at least 1.6 billion years ago. Yet other telltale proofs of their existence are missing. Eukaryotes should produce and leave behind certain distinctive molecules, but fossilized versions of those molecules don’t show up in the rock record until 800 million years ago. This unexplained 800-million-year gap in early eukaryotic history, a crucial period when the last common ancestor of all of today’s complex life first arose, has shrouded the story of early life in mystery.

“There’s this massive temporal gap between the fossil record of what we think are the earliest eukaryotes and the first … biomarker evidence of eukaryotes,” said Galen Halverson, a professor at McGill University in Montreal.

There are many possible explanations for that paradoxical gap. Maybe eukaryotes were too scarce during that time to leave behind molecular fossil evidence. Or perhaps they were abundant, but their molecular fossils did not survive the harsh conditions of geologic time.

A recent study published in Nature offers an alternative explanation: Scientists may have been searching for the wrong fossilized molecules this entire time. When the study authors looked for more primitive versions of the chemicals others had been searching for, they discovered them in abundance — revealing what they described as “a lost world” of eukaryotes that lived 800 million to at least 1.6 billion years ago.

“These molecules have been there all along,” said Jochen Brocks, a geochemist with the Australian National University in Canberra who co-led the study with his then-graduate student Benjamin Nettersheim. “We couldn’t find [them] because we didn’t know what they looked like.”

The findings bring new clarity to the dynamics of early eukaryotic life. The abundance of these molecular fossils suggests that the primitive organisms thrived in the oceans for hundreds of millions of years before the ancestors of modern eukaryotes took over, seeding life forms that would one day evolve into the animals, plants, fungi and protists that we see today.

“It’s an elegant hypothesis that seems to reconcile these very disparate records,” said Halverson, who was not involved in the study. “It makes everything make sense.”

The findings were welcome news for paleontologists like Phoebe Cohen, chair of geosciences at Williams College in Massachusetts who long thought something was missing in the biomarker record. “There is a rich and dynamic history of life before animals evolved that is harder to understand because we can’t see it,” Cohen said. “But it’s extremely important because it basically sets the stage for the world that we have today.”

The Protosteroid Puzzle

When the fossil record is underwhelming, scientists have other ways to estimate when different species branched off from one another in the evolutionary tree. Primary among those tools are molecular clocks: stretches of DNA that mutate at a constant rate, allowing scientists to estimate the passage of time. According to molecular clocks, the last common ancestor of modern eukaryotes, which belonged to a diverse collection of organisms known as the crown group, first emerged at least 1.2 billion years ago.

But the eukaryotic story doesn’t start there. Other early eukaryotes, known as the stem group, lived for hundreds of millions of years before our first common ancestor evolved. Researchers know little about them beyond the fact that they existed. The small handful of ancient eukaryote fossils that have been discovered are too ambiguous to be identified as stem or crown.

In the absence of compelling body fossils, researchers look for molecular fossils. Molecular fossils, which preserve separately from body fossils, can be challenging for scientists to pin down. First they have to identify which molecules could have been produced only by the organisms they want to study. Then they have to deal with the fact that not all of those molecules fossilize well.

Organic material decays at different rates, and some parts of eukaryotes preserve in rock better than others. Tissues dissolve first. DNA might stick around for longer, but not too long: The oldest DNA ever found is around 2 million years old. Fat molecules, however, can potentially survive for billions of years.

Eukaryotes create vast quantities of fat molecules known as sterols, a type of steroid that’s a critical component of cell membranes. Since the presence of a cell membrane is indicative of eukaryotes, and fat molecules tend to persist in rock, sterols have become the go-to molecular fossil for the group.

Modern eukaryotes run on three major sterol families: cholesterol in animals, phytosterols in plants and ergosterol in fungi and some protists. Their synthesis starts with a linear molecule, which the cell molds into four rings so that the resulting shape fits perfectly into a membrane, Brocks said. That process has many stages: It takes another eight enzymatic steps for animal cells to make cholesterol, while plant cells require another 11 enzymatic steps to make a phytosterol.

On its way to building its advanced sterol, a cell creates a series of simpler molecules at each step in the process. When plugged into an artificial membrane, even those intermediate sterols provide the permeability and rigidity a cell needs to function as it ought. The biochemist Konrad Bloch, who was awarded the Nobel Prize in 1964 in part for discovering the cellular steps to make cholesterol, “was puzzled by that,” Brocks said. Why would a cell put in extra effort to make a more complicated sterol when a simpler molecule will do the job?

In 1994, Bloch wrote a book in which he predicted that each of these intermediate sterols had once been the end product used in the membrane of an ancestral eukaryotic cell. Each additional step may have required more of the cell’s energy, but the resulting molecule was a slight improvement over the previous one — enough of an upgrade to outcompete the precursor and take hold in evolutionary history.

If that were true, it would explain why no one had been able to find molecular fossils of sterols before the rapid expansion of modern eukaryotes some 800 million years ago. Researchers had been searching for cholesterols and other modern structures in the rock record. They didn’t realize that ancient biochemical pathways were shorter and that stem-group organisms didn’t make modern sterols: They made protosterols.

Molecular Coffee Grind

In 2005, about five years after Bloch died, Brocks and colleagues reported in Nature the first hints that such intermediary molecules once existed. In ancient sediments they had found unusually structured steroids they didn’t recognize. But at the time, Brocks didn’t consider that a eukaryote could have created them. “Back then, I was pretty convinced that they were bacterial,” he said. “No one was thinking about the possibility of stem-group eukaryotes at all.”

He continued sampling ancient rocks and looking for these curious molecules. About a decade into the work, he and Nettersheim realized that many of the molecular structures in rock samples looked “primitive” and not like the ones bacteria typically make, Brocks said. Could they be Bloch’s intermediate sterols?

They needed more proof. In the decade that followed, Brocks and Nettersheim contacted petroleum and mining companies to request samples of any ancient sediments they had accidentally discovered during drilling expeditions.

“Most people would have found two examples and published,” said Andrew Knoll, a professor of natural history at Harvard University who was not involved in the study. (He was Brocks’ postdoctoral adviser years ago.) “Jochen spent the better part of the decade looking at rocks throughout the Proterozoic from all over the world.”

Meanwhile, the researchers created a search template to identify molecules in the sediment. They converted modern-day intermediate molecules made during sterol synthesis into plausible geological steroid equivalents. (Cholesterol, for example, fossilizes as cholestane.) “If you do not know what the molecule looks like, you will not see it,” Brocks said.

In the lab, they extracted fossil molecules from the sediment samples using a process that is “a bit like making coffee,” Nettersheim said. After crushing rocks, they added organic solvents to extract the molecules within — just as hot water is used to extract coffee from roasted and ground beans.

To analyze their samples and compare them against their references, they used mass spectrometry, which determines the molecules’ weights, and chromatography, which reveals their atomic makeup.

The process is arduous. “You analyze hundreds of rocks and find nothing,” Brocks said. When you do find something, it’s often contamination from recent times. But the more samples they analyzed, the more fossils they found.

Some samples were filled to the brim with protosteroids. They found the molecules in rocks dating from 800 million to 1.6 billion years ago. It seemed that not only were ancient eukaryotes present for some 800 million years before modern eukaryotes took off, but they were abundant.

The researchers could even recognize the eukaryotes’ evolutionary process as their steroids became more complex. In 1.3-billion-year-old rocks, for example, they found an intermediate molecule that was more advanced than the 1.6-billion-year-old protosteroids, but not as advanced as modern steroids.

“That was a very clever way to deal with the missing record of molecular fossils,” said David Gold, a geobiologist at the University of California, Davis who was not involved in the study. Their discovery immediately filled an 800-million-year gap in the story of how modern life came to be.

A Lost World

The molecular findings, put together with genetic and fossil data, reveal the clearest picture yet of early eukaryotic dynamics from around 1 billion years ago during the mysterious mid-Proterozoic era, experts said. Based on Brocks and Nettersheim’s evidence, stem- and crown-group eukaryotes likely lived together for hundreds of millions of years and probably competed with each other during a period that geologists call the Boring Billion for its slow biological evolution.

The absence of the more modern steroids during this time suggests that the crown group didn’t immediately take hold. Rather, the membrane-bound organisms started small as they found niches in the ancient ecosystem, Gold said. “It takes a long time for [eukaryotes] to become ecologically dominant,” he said.

At first, the stem group may have had an advantage. Oxygen levels in the atmosphere were significantly lower than they are today. Because building protosterols requires less oxygen and energy than modern sterols require, stem-group eukaryotes were likely more successful and abundant.

Their influence declined when the world hit a critical transition known as the Tonian Period. Between 1 billion and 720 million years ago, oxygen, nutrients and other cellular raw materials increased in the oceans. Fossils of modern eukaryotes, like algae and fungi, start to appear in the rock record, and modern steroids start to outnumber protosteroids in fossilized biomarkers — evidence that suggests crown-group eukaryotes had begun to thrive, increase in number and diversify.

Why would sterols become more complicated over time? The authors suggested that the more complex sterols bestowed some evolutionary advantage on their owners — perhaps related to dynamics in the creatures’ cell membranes. Whatever the reason, the sterol shift was evolutionarily significant. The makeup of modern sterols likely gave crown-group eukaryotes a boost over the stem group. Eventually, “this lost world of ancient eukaryotes was replaced by the modern eukaryotes,” Brocks said.

A Bacterial Wrinkle

The researchers’ evolutionary sterol story is compelling, but it’s not rock solid.

“I wouldn’t be surprised” if their interpretation is correct, Gold said. However, there is another possibility. Although scientists tend to associate sterols with eukaryotes, some bacteria can also make them. Could the molecular fossils in the study have been left by bacteria instead?

Gordon Love, a geochemist at the University of California, Riverside, thinks the bacterial scenario makes more sense. “These protosteroids turn up in rocks of all ages,” he said. “They don’t just disappear, which means that something other than stem eukaryotes is capable of making those.” He argued that bacteria, which dominated the sea at the time, could have easily produced protosteroids.

The authors can’t rule out that possibility. In fact, they suspect that some of their fossil molecules were made by bacteria. But the possibility that their vast collection of fossilized protosteroids, stretching for hundreds of millions of years, was made entirely by bacteria seems unlikely, Brocks said.

“If you look at the ecology of these bacteria today, and their abundance, there is just no reason to believe that they could become so abundant that they could have produced all these molecules,” he said. In the modern world, bacteria produce protosterols only in niche environments such as hydrothermal springs or methane seeps.

Cohen, the Williams College paleontologist, agrees with Brocks. The interpretation that these molecules were made by eukaryotes “is consistent with every other line of evidence,” she said — from the fossil record to molecular clock analyses. “I’m not as worried” about that possibility, she said.

Either interpretation presents more questions than answers. “Both stories would be absolutely crazy weird,” Brocks said. They are “different views of our world,” he added, and it would be nice to know which one is true.

Lacking a time machine, the researchers are searching for more evidence to improve their certainty one way or the other. But there are only so many ways to reconstruct or perceive ancient life — and even scientists’ best guesses can never completely fill the gap. “Most life didn’t leave any traces on Earth,” Nettersheim said. “The record that we see is limited. … For most of Earth’s history, life might have looked very different.”

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