Complete mitochondrial genome and phylogeny of Pleistocene mammoth Mammuthus primigenius

Abstract

Phylogenetic relationships between the extinct woolly mammoth (Mammuthus primigenius), and the Asian (Elephas maximus) and African savanna (Loxodonta africana) elephants remain unresolved. Here, we report the sequence of the complete mitochondrial genome (16,842 base pairs) of a woolly mammoth extracted from permafrost-preserved remains from the Pleistocene epoch--the oldest mitochondrial genome sequence determined to date. We demonstrate that well-preserved mitochondrial genome fragments, as long as approximately 1,600-1700 base pairs, can be retrieved from pre-Holocene remains of an extinct species. Phylogenetic reconstruction of the Elephantinae clade suggests that M. primigenius and E. maximus are sister species that diverged soon after their common ancestor split from the L. africana lineage. Low nucleotide diversity found between independently determined mitochondrial genomic sequences of woolly mammoths separated geographically and in time suggests that north-eastern Siberia was occupied by a relatively homogeneous population of M. primigenius throughout the late Pleistocene.

Figure 1. The Right Back Leg of the Woolly Mammoth(M. primigenius) Found in Siberia

The well-preserved mammoth body fragment with foot (33 × 36 cm), shin, and ankle-joint (the total length is ~88 cm) was found in the Enmynveem River valley (north-eastern Siberia, Chukotka). The tissue material (bones, muscles, and skin) had no visible marks of tissue damage by insects or other animals. Radiocarbon dating of the skin and muscle tissue determined that the mammoth lived 32,850 ± 900 y ago [12].

Figure 2. The Unusually Well-Preserved Mammoth DNA

(A) Nuclei with DNA clearly detectable by DAPI staining in muscle cells of ~33,000-y-oldM. primigenius. (B) Total genomic DNA isolated from the mammoth muscle tissue (lane 1 is 1/10 dilution of the DNA on lane 2); control DNA from fresh human blood samples in lanes 3 and 4. (C) Examples of PCR products (~300–600 bp) for mammoth mitochondrial genome. (D) PCR amplification recovers long sequences for complete mitochondrial genes (1,317 bpCytB and 1,613 bpATP6 genes), but PCR of larger fragments (3,054 bpND5) is failed.

Figure 3. Mitochondrial Genome of the Woolly MammothM. primigenius

The complete mitochondrial genome was determined independently in two different laboratories using designs with multiple primers for overlapping PCR fragments ranged from ~325 to ~650 bp; the longer PCR fragments were also produced and sequenced. The overlapped PCR products used for sequencing and cloning are shown by the inner circle. Only PCR fragments produced from different pairs of primers are shown. Two genes,ATP6 andND4L, overlap with neighboring genes.

Figure 4. Paenungulata Tree and Phylogenic Relationship of the Woolly Mammoth

The analysis of complete mtDNA sequences placesM. primigenius withE. maximus on the tree. The Sirenia(D. dugon) and Hyracoidea(P. capensis), most closely related species among extant taxa to Elephantinae, were taken as outgroups. Bootstrap values and posterior probabilities were calculated using a Bayesian approach [29,31] assuming a gamma distribution of the rates of evolution across sites with a General Time Reversible model (normal font), HKY model (bold), with a parsimony approach (italic), and by neighbor joining (italic and bold) [28]. The scale is 0.1 substitutions per site. The mitochondrial genomes ofM. primigenius, E. maximus A, E. maximus B, L. africana A, L. africana B, D. dugon, andP. capensis were used in the analysis.