Research Stories

by Margaret Coulombe

James Elser has built a career asking questions about evolutionary biology and energy and material flows in ecosystems. To find answers, he travels from icy Antarctica to the alpine lakes of Norway and Colorado to the Mongolian grasslands of China. Elser's world view is known as "stoichiometric theory." Understanding the balance of carbon, nitrogen, and phosphorus in systems forms the backbone of that view.

Elser has taught more than 12,000 students as a professor in the School of Life Sciences at Arizona State University. His pioneering studies have shaped young minds and jump-started new research approaches. His work provides insights into nutrient limitation, trophic dynamics, and biogeochemical cycling.

However, it was while working in a unique part of Mexico's Chihuahuan desert that Elser found something elemental. At Cuatro Ciénegas, the ASU scientist thinks he found a key to unlock secrets about microbial evolution, the Cambrian Explosion, speciation, extraterrestrial life, and just about everything else. That key is phosphorus.

A microbial Galapagos
Cuatro Ciénegas is a living laboratory that some call "a microbial Galapagos." Like the Galapagos Islands made famous by Charles Darwin, Cuatro Ciénegas is unlike most places on Earth. It is a hotbed, literally, of biodiversity. The area includes more than 200 warm springs that harbor more than 70 endemic species (species found nowhere else). Scientists say that it is home to "the largest collection of vertebrates, invertebrates and floral endemic biodiversity in North America."

The pools (pozas in Spanish) function like aquatic islands in the vast Chihuahuan desert. They are fed by volcanic activity and waters shed from the Sierra Madre mountains.

Cuatro Ciénegas is also a hot spot for microbial diversity. The abundance is perhaps due to the unusual range of water temperatures, mineral compositions, salinities, pH, and substrates—and the region's ancient history. Even the limestone used to build the local Catholic church harbors the layered remains of cyanobacteria. The microbes were once the dominant form of early life on Earth. Now they are fossilized, scoured the color of bleached bones by an unrelenting Coahuila sun.

Stromatolites are the living descendants of these bacteria. They look like laminated microbial mats. Isolated, protected by their harsh environment, stromatolites have built up in the pozas much like modern coral reefs. Cuatro Ciénegas is one of few places left in the world that shelters these communities.

One radiocarbon estimate places them in the Coahuila basin for more than 31,000 years, according to Elser's colleague Valeria Souza, a microbiologist from Universidad Nacional Autonoma de Mexico. She conducts genomic studies of microbial DNA, teasing out the origins of the ponds' bacterial inhabitants.

Each poza can be environmentally radically different. Each poza isolates its bacterial occupants from nearest neighbors and the outside world. The result is the creation of conditions ripe for adaptive radiation and the emergence of new species.

Cuatro Ciénegas

Together, Souza and Elser discovered an intriguing genetic treasure trove. They found 38 unique phylotypes (genetically distinct microbes) from 10 major lineages of bacteria. That discovery was enhanced by the finding that at least half of the microbial freshwater inhabitants of Cuatro Ciénegas were not even related to freshwater microbes from other similar systems. They are related to marine species more than 500 miles distant.

As Souza artfully explains, "Their genes bore the genetic memory of the sea." They remember the ocean that once covered this area during the opening of Pangaea, the ancient supercontinent.

The discoveries are exciting. However, Souza thinks that what has helped most to define understanding about Cuatro Ciénegas and its diversity is Elser's discovery of the area's severe phosphorus limitation.

"This lack of phosphorus was probably one of the main characteristics of early Earth for billions of years," she says. "And so, what Cuatro Ciénegas might tell us, that nowhere else on Earth can, is why there are so many species on this planet, how they diversify, and what the rules were that imposed constraints on speciation."

Phosphorus a key evolutionary force
Cuatro Ciénegas is a specialized place. It is home to unique microbial species found only in its warm pools. How can a place like this take the world closer to understanding larger evolutionary questions?

Let's take the example of Charles Darwin, Elser says. Darwin's observations on the Galapagos archipelago ultimately fueled his theory of evolution. He examined species that were isolated on distinct islands. He noted the development of endemism and local specializations (for example in the beaks of finches). Darwin proposed that these changes arose through the combination of mutation/selection driving local adaptation and, in turn, reproductive isolation. The changes were a result of the geographic barriers that allowed each island's population to diverge—the essence of speciation.

But most scientists (and people on the street) have long viewed bacteria as being everywhere all the time. So how then could so many endemic microbes evolve at Cuatro Ciénegas, to be found nowhere else in the world?

Cuatro Ciénegas provides a springboard, much as the Galapagos did for Darwin, to understanding about species evolution. Elser conducted lots of studies at Cuatro Ciénegas with Souza and Janet Siefert of Rice University. In 2008, they published a speculative paper in the journal Nature Reviews Microbiology. The scientists proposed that the long history, isolation among various pools, and, intriguingly, lack of phosphorus could have limited genetic exchange between microbial occupants in much the same way as Darwin perceived in finches on the Galapagos.

But how could lack of phosphorus regulate exchange of genetic material in species that don't have sex? The answer lies in a process called horizontal gene transfer.

Transfer of DNA between microbes can and does commonly occur in several different ways. When bacteria come into close cell-to-cell contact, they can swap DNA directly. In addition, when a cell dies or is killed, free DNA is released and is available to be taken up by another living cell. The cell incorporates that piece of free DNA into its own DNA sequence.

Viruses can also can carry gene sequences from past to future hosts. All of these processes tend to even out genetic variations among populations. It works just as if there had been a free ferry service on the Galapagos to bring one island's finches to visit another island's during mating season.

Microbes in Cuatro Ciénegas live in a severely isolated and phosphorus-limited environment. As a result Elser and Souza think that have experienced less horizontal gene transfer. That is one reason for the high levels of endemism.

Phosphorus is valuable as an essential building block of DNA. The scientists reason that low phosphorus in the environment means that microbes are more likely to break down any free DNA to salvage the phosphorus to use in metabolism. They don't incorporate the intact DNA into their genomes. So there is less free DNA available for other microbes to assimilate.

In addition, Elser and colleagues think that gene transfer is further diminished because phosphorus-limited host cells have greatly reduced viral production per cell. This in turn limits the potential of viral transfers of DNA from one cell to another.

Phosphorus limitation can also cause organisms to develop thrifty genomes—to limit the amount of phosphorus invested in DNA. For example, Souza discovered a bacterium at Cuatro Ciénegas called Bacillus coahuilensis. The microbe has the smallest genome ever found in this genus.

Lack of phosphorus has altered the metabolism of B. coahuilensis. This species can only produce sulfur-based lipids. It has completely lost the capacity to build phospholipids. These are basic structural units used to make the cell membranes of most living things.

"What we are arguing is that microbial evolution and advent of new species is not as different as people imagine from that characterized by Darwin," Elser explains. "This is true especially if you focus on horizontal gene transfer in microbes as playing the same role as sexual reproduction in macroorganisms."

A little phosphorus can go a long way
The scientists think that lack of phosphorus might have acted in very large, less localized ways to shape evolutionary processes. Elser points to the Cambrian Explosion as an example. The Cambrian Explosion was a period of rapid evolution, believed to be roughly 5 to 10 million years in length, that started 545 million years ago. This period led to the development of large-bodied animals (Metazoans) and most of the basic body plans of life we see around us today.

Many paleontologists have long wondered what might have held back the appearance and diversification of Metazoans during the billions of years of life. That was when stromatolites dominated the Earth and was millions of years after multicellular life forms first appeared.

Lots of potential causes have been hotly debated in scientific circles. But Elser looks through his stoichiometric lens. He poses this hypothesis: Junk food.

We can't go back to 545 million years ago. Instead, Elser has taken advantage of the fact that Cuatro Ciénegas' stromatolites are analogs of those ancient systems on Earth.

"We wanted to test the hypothesis that the quality of algal or stromatolite production was so poor throughout much of the history of early Earth that the Metazoan lifestyle could not succeed or evolve," Elser says. "If the Precambrian era was severely phosphorus limited, it could have been a major constraint on animal proliferation."

To test this idea, Elser selected a poza. Using experimental tanks maintained on the shoreline, he added phosphorus to the water. The stromatolite cyanobacteria, algae, and microbes quickly removed the element from the water. They raised the phosphorus content of their own tissues. It then stimulated growth, reproduction, and survivorship of the snails consuming them—compared to snails feeding on unenriched stromatolites.

The snails at Cuatro Ciénegas appeared to be suffering, Elser says, "from junk food; food low in phosphorus." Indirect evidence to be sure, but a suggestion that lack of phosphorus might have indeed delayed Metazoan's first footsteps for millions of years.

Preservation of a living legacy
"If we don't think of Cuatro Ciénegas as a place of very little phosphorus, and phosphorus as a keystone element in the evolution of life, then Cuatro Ciénegas has less meaning—besides simply being beautiful. It wouldn't have been discovered to be so important for science," Souza says. "The point of view of Jim Elser as an expert in stoichiometry has been really enlightening with respect to understanding evolution and, ultimately, important to the preservation of this unique site."

Innovative study, educational and public outreach, and the dedication of Elser, Souza and others, such as the late Wendell Minkley (professor in ASU's former Department of Zoology), who devoted much of his life to the area's preservation, has led to the designation of Cuatro Ciénegas as a federal protected area. The designation has the support from the governor of Coahuila, the Secretary of the Environment, and the Mexican president, Phillipe Calderon. In addition, the World Wildlife Federation, Mexico's National Commission on Biodiversity (CONABIO), and government organizations such as PRONATURA and the Nature Conservancy have classified the Cuatro Ciénegas valley as "globally outstanding due to its endemism and representation of evolutionary radiations."

Changes in policy and protections in Mexico have preserved not just Cuatro Ciénegas, but the surrounding valleys and mountain ranges key to the hydrology of the region. Most importantly, Souza believes, is how their work in the area has captured the imagination of the region's children.

"I think what is most important is environmental education. We had 2,000 kids from kindergarten to high school attend last year," she says. "I think putting the ecosystems of Cuatro Ciénegas in front of their eyes is what matters. They now can carry away with them how important and special it is."

Cuatro Ciénegas is special. But Elser says that there are areas elsewhere in the world too, for example on the Kamchatka Peninsula in Russia, that are microbial hotspots and ripe for discovery.

"At present, we don't know where these places are or what amazing creatures or weird enzymes are out there. But they need to be protected, just as we place high value on the habitats of giant pandas or orchids."

Listen to SOLS Science Studio podcast about Souza and Elser's work at Cuatro Cienegas.

Research at Cuatro Ciénegas is supported by the NASA Astrobiology Institute and National Science Foundation. For more information, contact James J. Elser, Ph.D., School of Life Sciences, College of Liberal Arts and Sciences, 480.965.9747.