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The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. The resultant increase in number of samples and polymorphic sites assayed and the number of studies published may give the impression that all technological hurdles associated with aDNA technology have been overcome. However, analysis of aDNA is still plagued by two issues that emerged at the advent of aDNA technology, namely the inability to amplify a significant number of samples and the contamination of samples with modern DNA. Herein, we analyze five well-preserved skeletal specimens from the western United States dating from 800-1600 A.D. These specimens yielded DNA samples with levels of contamination ranging from 0-100%, as determined by the presence or absence of New World-specific mitochondrial markers. All samples were analyzed by a variety of protocols intended to assay genetic variability and detect contamination, including amplification of variously sized DNA targets, direct DNA sequence analysis of amplification products and sequence analysis of cloned amplification products, analysis of restriction fragment length polymorphisms, quantitation of target DNA, amino acid racemization, and amino acid quantitation. Only the determination of DNA sequence from a cloned amplification product clearly revealed the presence of both ancient DNA and contaminating DNA in the same extract. Our results demonstrate that the analysis of aDNA is still an excruciatingly slow and meticulous process. All experiments, including stringent quality and contamination controls, must be performed in an environment as free as possible of potential sources of contaminating DNA, including modern DNA extracts. Careful selection of polymorphic markers capable of discriminating between ancient DNA and probable DNA contaminants is critical. Research strategies must be designed with a goal of identifying all DNA contaminants in order to differentiate convincingly between contamination and endogenous DNA. KEY WORDS: aDNA; mitochondrial DNA; PCR contamination; New World

Mitochondrial and nuclear DNA diversifies were determined for two Choco-speaking Amerind populations, the Embera and Wounan, sampled widely across their geographic range in eastern Panama. These data were compared with mitochondrial and nuclear diversities determined here and previously for neighboring Chibcha-speaking Ngobe and Kuna populations. Chocoan groups exhibited mitochondrial diversity levels typical for Amerind populations while Chibchan groups revealed reduced mitochondrial diversity. A slight reduction in autosomal levels of heterozygosity was determined for the Chibcha while X and Y variation appeared equivalent in all populations. Genetic distinctiveness of the two linguistic groups contradicts the anthropological theory that Paleoindians migrated repeatedly through the isthmian region and, instead, supports the idea of cultural adaptation by endogenous populations. Reduced genetic diversity in Chibchan populations has been proposed to represent a population bottleneck dating to Chibchan ethnogenesis. The relative sensitivities of haplotype pairwise difference distributions and Tajima's D to detect demographic events such as population bottlenecks are examined. Also, the potential impact of substitution rate heterogeneity, population subdivision, and genetic selection on pairwise difference distributions are discussed. Evidence is presented suggesting that a larger effective population size may obscure the historical signal obtained from nuclear genes while the single mitochondrial locus may provide a moderately strong signal.

High levels of mitochondrial DNA (mtDNA) diversity were determined for Mongolian populations, represented by the Mongol-speaking Khalkha and Dariganga. Although 103 samples were collected across Mongolia, low levels of genetic substructuring were detected, reflecting the nomadic lifestyle and relatively recent ethnic differentiation of Mongolian populations. mtDNA control region I sequence and seven additional mtDNA polymorphisms were assayed to allow extensive comparison with previous human population studies. Based on a comparative analysis, we propose that indigenous populations in east Central Asia represent the closest genetic link between Old and New World populations. Utilizing restriction/deletion polymorphisms, Mongolian populations were found to carry all four New World founding haplogroups as defined by WALLACE and coworkers. The ubiquitous presence of the four New World haplogroups in the Americas but narrow distribution across Asia weakens support for GREENBERG and coworkers' theory of New World colonization via three independent migrations. The statistical and geographic scarcity of New World haplogroups in Asia makes it improbable that the same four haplotypes would be drawn from one geographic region three independent times. Instead, it is likely that founder effects manifest throughout Asia and the Americas are responsible for differences in mtDNA haplotype frequencies observed in these regions.