Homo neaderthalensis (humans) had two nagging worries during theLate Pleistocene period, which ended circa 11,000 years ago. Onewas to keep from freezing to death during the last Ice Age. Anotherhazard was the European cave bear (Ursus spelaeus), which rivaledin size and ferocity the Alaskan Kodiak grizzly bear, the largest landcarnivore alive today.

Neanderthal humans flourished from about 85,000 to 35,000 yearsago; U. spelaeus outlived that hominoid species by many millennia;the cave bear became extinct circa 20,000 years ago, done in by thelast ice-age glaciation.

Now extinction threatens a much smaller beast of the same ursineilk, the European brown bear (U. arctos) _ironically at the hands ofHomo sapiens. Once spread thickly over nearly all of Europe, U.arctos today is spread so thin that fewer than 10 animals have beencounted alive in the French Pyrenees mountains. Humans hunt themand civilization encroaches fatally on their habitat.

To save this endangered species from going the way of its collateralancestor, the cave bear, France's environment ministry has plansafoot to reinforce the brown bear's Pyrenean presence by importingbreeding populations from elsewhere in Europe, where they are stillrelatively plentiful. To maintain the genetic purity of the localremnant animals, the government has turned to molecular ecologistPierre Taberlet, who directs the laboratory of high-altitudepopulations biology in the mountain city of Grenoble.

Taberlet described his approach and recommendations in the March1994 issue of the Proceedings of the Royal Society of London. Hispaper bears the title, "Mitochondrial DNA polymorphism,phylogeography and conservation genetics of the brown bear Ursusarctos in Europe."

This genetic analysis drew its tissue from hairs of wild, captive orkilled animals, and extracted DNA from the hair roots. Of 60individuals thus analyzed, 272 base pairs of the mitochondrialgenome's control region yielded 16 different haplotypes, or geneticvariants.

Mitochondrial DNA, Taberlet explained to BioWorld Today, lendsitself to polymerase chain reaction (PCR) sequence amplification,and has a specific advantage in that using, as he did, primers specificfor bears "avoided contamination by human DNA."

"We analyzed the organelle's control region," he added, "because itis not possible to amplify genomic DNA. The control region is themost viable; it evolves faster than coding genes, so we were able toget a lot of information with relatively short sequences."

All four bears of the Pyrenees population had identical haplotypesequences; so did seven from Sweden, seven from Lapland and 13from Slovenia. These are all from the western European lineage ofbrown bears, which also embraces Italy, Spain and several othercountries, where the populations are fragmented, scarce anddwindling. Russia and Romania, the eastern lineage, are home tolarge U. arctos concentrations.

"In Europe," Taberlet's article reported, "a remarkable degree ofconcordance was found between the geographic distribution of thebrown bears and their mtDNA haplotypes." This he explainedmainly by "the low dispersal habits of females," and noted thatmtDNA is maternally inherited.

Lineages Diversed 850,000

Taberlet estimated that these two main mtDNA lineages divergedevolutionarily some 850,000 years ago, by a genetic distance of 7.13percent. But he hedged these figures due in part to the "molecularclock calibration, which is based on human" mitochondrial control-region evolution. He assumed that the two groups drifted apart "dueto geographic separation during the early Quaternary cold periods."

Responding to the environment ministry's need to repopulate thePyrenees, Taberlet postulated three possible but improbablestrategies:

* round up and send in breeding bears from all eastern and westernEuropean lineages;

* split the western lineage into two distinct conservation units;

* preferentially, turn to the northern Spanish units, most closelyrelated genetically and geographically to the French.

In practice, he pointed out, none of the above are feasible, so as a lastpragmatic resort he recommended pooling selected western lineagesfor use. Accordingly, the Ministry of the Environment is nowcounting on animals from southern Scandinavia and Bulgaria "toprovide for a ready source of bears for transplantation to thePyrenees."

Contamination of DNA samples in PCR analysis is (literally) thebug-bear of Taberlet's work with Ursus species. To refine thetechnology, he has collaborated with post-doctoral molecularpaleontologist Catherine Hnni at the Pasteur Institute in Lille, whereursine DNA is unknown. HSnni is first author of their paper in thecurrent Proceedings of the National Academy of Sciences (PNAS),dated Dec. 6. Its title: "Tracking the origins of the cave bear (Ursusspelaeus) by mitochondrial DNA sequencing."

Cave-bear bones are no rarity; Europe-wide, remains of more than100,000 skeletons have been discovered. In the mountainous fissuresof the high French Alps, HSnni and her associates chose four _ two40,000-year-old femurs and a child's mandible from three caves, anda femur 25,000-30,000 years old from a fourth.

"To separate the DNA from the pulverized bone," she told BioWorldToday, "we used EDTA [ethylene diamine tetraacetic acid], which isa chelator of calcium. After this extraction, we quantitated the DNAin the bone, then PCR-amplified the control region of themitochondrial genome."

To minimize the risk of contamination, Hnni emphasized, "theextractions were carried out in our laboratory, where no bear DNA ishandled, and the PCR amplifications in a DNA-free room whereDNA is never handled."

Of the three 40,000-year-old samples tested, only one, the metatarsus(foot bone), yielded usable genomic sequence polymorphisms. "Toour knowledge," says the PNAS article, "this represents the oldestDNA fragment yet sequenced from ancient bones."

In finding a common origin for U. spelaeus and U. arctos on thephylogenetic family tree of European cave and brown bears,"Taberlet explained, "we used the American black bear (U.americanus) as an out group to root the tree."

A Third Lineage?

What they discovered, he added, was that "the same genetic distanceexists between the east-west lineages of the brown bear and that ofthe cave bear. So maybe the cave bear could be a third lineage."

But this result is based, he said, "disappointingly" on only a singlesample, "We are now planning, with Hnni, to amplify cave-bearmtDNA from bones that give a lot of proteins, using only goodbones for protein analysis."

In Lille, Hnni has already lined up a source: "I have a lot of bonescoming from a man who has extracted cave-bear proteins, forexample, collagen. I want to know if there is a correlation betweenthe quantity of protein present in a bone, and the quantity of DNA."

Taberle and Hnni allow that the physical evolution of both H.neanderthalensis and U. spelaeus may have been partly driven by theharsh glaciations of the period. "It could have influenced thedevelopment of strong bones in both," Taberlet speculated.

"I think of the cave bear as a model for analyzing ancient DNA,"Hnni added. It has great applications in human population geneticsto know the differentiation between different populations, the historyof migration, and so on." She cited forensic and paternity testing asother examples of the technology's utility.

The fact that cave bears and Neanderthal humans were alive at thesame time," Hnni suggested, "may show that if we can have someDNA from U. spelaeus, perhaps we can get some from theNeanderthal people as well."

She knows that "a lot of researchers are working on Neanderthalbones, "but with no results yet published. "

Asked if these are likely in future, she said, "Oh, I'm sure. Yes."

Bone biochemist Noreen Tucross, a researcher at the SmithsonianInstitution's Conservation Analytical Laboratory, told BioWorld,"I'd certainly be interested in seeing that Neanderthal research, but Idon't have such bones."

She pointed out that "there is a major problem of contamination withhuman DNA, and that is why I think a lot of people are cutting theirteeth on these extinct megafauna _ a very good approach." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

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