DURHAM, N.C. -- Classifying kangaroos and platypuses together on the evolutionary family tree is as absurd as adding your neighbors to your own family ancestral line simply because they share your love of the opera, according to scientists at Duke University.
But the current molecular method of using mitochondrial DNA to classify how mammals evolved is so flawed that it might have erroneously linked very different mammals, the scientists said. The mitochondrial DNA method of analyzing mammals has turned on its head the common-sense approach of linking mammals by similar anatomical traits or "morphology," they said.
Using a more comprehensive method to analyze the genetic material of 15 types of mammals, Duke researchers have shown that the mitochondrial DNA method that links disparate animals (hippo and whale, kangaroo and platypus) is statistically unreliable when it comes to evolutionary genetics, said Randy Jirtle, professor of radiation oncology at Duke University Medical Center. Their own research using nuclear genes (genes from the nucleus or core of cells) has shown a nearly 100 percent statistical likelihood that the Duke results are correct.
Mitochondria are the cell's power plants and possess their own genes that are inherited through the maternal line. Scientists use this method because mitochondrial DNA is more accessible, easier to sequence, and all multi-cellular animals have mitochondria, whereas all animals do not share the same nuclear genes.
Results of the study are published in the July 1, 2001 issue of the journal Mammalian Genome.
Such conclusive results led the researchers to strongly support the Theria hypothesis of classifying the three major groups of mammals. The Theria hypothesis holds that eutherians (humans, rats, pigs, whales, etc.) and marsupials (kangaroos, wallabies, koalas, etc.) have evolved from a common ancestor, and monotremes (platypus, echidna) have evolved from a different ancestor and on a separate land mass. The mitochondrial method of studying evolution, however, supports the Marsupionta hypothesis, which places the platypus and kangaroo together. This controversy has lasted for more than two centuries since the discovery that the platypus lays eggs.
"Our study is the first to provide statistically unambiguous results in favor of classifying mammals using the Theria hypothesis, as paleontologists have long done through studying fossils," Jirtle said. "Now we need to retest the results generated by scientists who have used mitochondrial DNA sequences to link mammals such as hippos to whales."
The Duke scientists generated their results by isolating a whole nuclear gene from the genetic material of 15 different mammals, and then determining the unique genetic code or sequence that distinguishes each gene from the others in the respective mammals.
Then, by plugging molecular traits of the gene into a computer software program -- similar to entering eye color and earlobe structure into a family tree software program -- the scientists identified which animals shared common DNA traits and which did not. The data they derived from studying nuclear genes clearly identified marsupials (kangaroos, opossums, etc.) as having a common evolutionary background with eutherians (humans, pigs, etc.), and monotremes (platypus, echidna) as having evolved separately. Jirtle said these conclusions contradict the highly publicized assertions Ulfur Arnason and colleagues reported in a 1997 article in the journal Proceedings of the National Academy of Science.
Aside from its purely academic value, the scientists said that classifying mammals correctly is critical because it helps biologists apply the information learned from one mammal to others in the same class, without having to conduct identical molecular studies on each mammal.
"The family tree is a crucial evolutionary roadmap," said Keith Killian, a Duke researcher in molecular development and evolution. "If you are trying to trace, for example, the evolutionary steps of fetal heart development to better understand how fetal defects occur, it helps to know which mammals are related so that you can make accurate inferences about one mammal from another mammal's development."
In recent years, scientists have increasingly relied on using mitochondrial DNA to make comparisons among mammals and thereby link those that are related on the evolutionary tree.
But Killian said that mitochondrial DNA provides misleading results for a variety of reasons. Most importantly, it requires more human input to decide which information is fed to the computer, thereby raising the risk of human bias. In fact, when the data were given to three different laboratories for analysis, they generated three different family trees, Killian said.
Secondly, the "bootstrap value" - a measure of the relatedness of genes -- which the computer assigns to the accuracy of its predictions has been quite low in mitochondrial studies on mammalian associations, hovering between 40 percent and 60 percent.
In contrast, the Duke study generated 97 percent to 100 percent bootstrap support in its nuclear gene comparisons of animals representing all three mammalian groups. Mammals they studied included the platypus, echidna, opossum, wallaby, hedgehog, mouse, rat, rabbit, cow, pig, bat, tree shrew, colugo, ringtail lemur and humans.
"This is the first molecular evolutionary study that seriously and powerfully says the paleontologists have been right all along in grouping mammals the way they did," said Killian. "It turns out that common sense is correct."
The Duke scientists realized the power of using large nuclear genes for molecular evolutionary studies when they were sequencing a gene called the insulin-like growth factor II receptor (IGF2R) to determine its evolutionary history in relation to cellular growth and mammalian overgrowth in cloned animals. Killian said this gene is especially amenable to evolutionary research because it is very large; it is shared by all mammals and is even present in fish and mollusks; and it provides critical information on the evolution of a genetic phenomenon called imprinting. Thus, it provides considerable amounts of statistically meaningful data for determining the evolutionary relationship between mammals.
"Using prominent genes like IGF2R to classify mammals has allowed us to close the Theria versus Marsupionta debate once and for all," Killian said.
Other researchers on the paper include former Duke Research Fellow Thomas R. Buckley and Niall Stewart and Barry L. Munday of the School of Aquaculture at the University of Tasmania, Australia.
The study was funded by grants from the National Institutes of Health, Department of Defense, Sumitomo Chemical Company Ltd. and AstraZeneca Pharmaceuticals Ltd.