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. 2010 Dec 21;8(12):e1000564.
doi: 10.1371/journal.pbio.1000564.

Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants

Nadin Rohland  1 , David Reich, Swapan Mallick, Matthias Meyer, Richard E Green, Nicholas J Georgiadis, Alfred L Roca, Michael Hofreiter
Affiliations

Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants

Nadin Rohland et al. PLoS Biol. .

Abstract

To elucidate the history of living and extinct elephantids, we generated 39,763 bp of aligned nuclear DNA sequence across 375 loci for African savanna elephant, African forest elephant, Asian elephant, the extinct American mastodon, and the woolly mammoth. Our data establish that the Asian elephant is the closest living relative of the extinct mammoth in the nuclear genome, extending previous findings from mitochondrial DNA analyses. We also find that savanna and forest elephants, which some have argued are the same species, are as or more divergent in the nuclear genome as mammoths and Asian elephants, which are considered to be distinct genera, thus resolving a long-standing debate about the appropriate taxonomic classification of the African elephants. Finally, we document a much larger effective population size in forest elephants compared with the other elephantid taxa, likely reflecting species differences in ancient geographic structure and range and differences in life history traits such as variance in male reproductive success.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Strategy for obtaining overlapping DNA from four elephantids and a mastodon.
(a) Mastodon shotgun 454 sequencing. We ligated 454-adaptors (green and blue) to the ends of the DNA molecules and sequenced the libraries on a Roche 454 GS. (b) Bioinformatic analysis of shotgun 454 sequences. To identify proboscidean sequence, we compared the sequences to databases consisting of the savanna elephant draft genome (loxAfr1), the human genome (hg18), the mouse genome (mm8), NCBI's nucleotide database of environmental samples (env), and NCBI's non-redundant nucleotide database (nr). The 454 sequences with a best match to loxAfr1 (in red) were aligned to loxAfr1. Alignments of at least 90 bp in length and with a similarity higher than 87% were used for primer design after filtering out known repeat elements (using the UCSC RepeatMasker database). Primers were based on loxAfr1 sequence flanking the mastodon sequence. (c) Multiplex PCR and sequencing of the targeted loci in modern elephants and mammoth. We show the protocol for the first of four rounds of the project (Table S3 provides details of the further rounds). A total of 213 primer pairs were randomly divided into 5 multiplex primer mixes with 41–44 primer pairs per mix. These mixes were used for the first step of the two-step multiplex PCR approach, for each of the 5 samples (La, Loxodonta africana; Lc, L. cyclotis; Em 1, Elephas maximus 1; Em 2, E. maximus 2; Mp, Mammuthus primigenius). Dilutions of these products were used as templates to amplify the loci individually in the second step (shown for L. africana), resulting in 213 distinct products per sample. These products were quantified, normalized, and merged into one pool per sample. A 454 library was prepared and sequenced on 1/16th of a picotiter plate of a Roche 454 GS.
Figure 2
Figure 2. Demographic model for the history of the Elephantidae.
Demographic model that is fit by MCMCcoal, in which all population splits are instantaneous (without subsequent gene flow), and all population sizes are assumed to be constant over intervals. Here, T FS refers to forest-savanna elephant population divergence time, T AM refers to Asian elephant-mammoth population divergence time, T Lox-Eur refers to African-Eurasian population divergence time, and T Elephantid-Mastodon refers to elephantid-mastodon population divergence time, presented here in millions of years. The Ν quantities refer to constant diploid effective population sizes ancestral to each of these splits (in thousands). For obtaining estimates of years and population sizes, we assume that the elephantids have an average of 31 years per generation, based on estimates of 17–20 years for females , and 40–49 years for males ,. A lower or higher number of years per generation would produce a proportionate effect on the population size estimates. For each parameter, two sets of numbers are shown. The upper set shows the range consistent with the fossil record, calibrating to an assumed African-Eurasian population split of T Lox-Eur = 4.2–9 Mya (justified in Text S5). For example for forest-savanna population divergence, this leads to T FS = 2.6–5.6 Mya given that MCMCcoal estimates T FS /T Lox-Eur = 62%. The lower set of numbers (in parentheses) provides MCMCcoal's 90% credible interval for the parameters as a fraction of the best estimate (e.g. 76%–126% for T FS). In the main text, we conservatively quote a range that combines the uncertainty from the fossil record and from MCMCcoal (e.g. T FS = 1.9–7.1 Mya).
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