We extracted DNA from a dried tissue sample of the more recently describedS. bucculentus specimen¹ (kindly supplied by C. P. Groves). The D-loop, 12S and cytochromeb regions of mitochondrial DNA were amplified, sequenced and compared with a wide range of sequences from GenBank and from our laboratory database. The comparison included three samples ofS. scrofa from archaeological sites on Pacific islands; these were extracted in a dedicated ancient-DNA laboratory using standard precautions to avoid contamination², and were independently replicated by J. Hay (Massey University, New Zealand) in an ancient-DNA laboratory in which porcine DNA had not previously been processed.

Phylogenetic analysis of the 12S-rRNA gene would be uninformative because of poor species representation in GenBank, so we used a barcode approach³ to compare intraspecific and interspecific sequence distances⁴. For cytochromeb and D-loop, phylogenetic trees were estimated using both bayesian⁵ and maximum-likelihood⁶ methods.

For the 12S-rRNA gene, the mean pairwise distance betweenS. bucculentus andS. scrofa (0.006) is identical to that withinS. scrofa (0.006), and only a tenth of the pairwise distance (0.055) betweenS. scrofa and the African warthog (Phacochoerus africanus). Unlike the 7% sequence difference reported earlier¹, this 0.6% difference is consistent with the conserved nature of the 12S-rRNA gene. For cytochromeb and D-loop, all four phylogenetic trees have similar topology, placingS. bucculentus within the Asian subclade ofS. scrofa. Furthermore, in the D-loop analyses (Fig. 1),S. bucculentus sits in a clade that includesS. scrofa from Thailand and Myanmar, countries that border Laos. Coincidentally, an archaeological specimen from Tikopia in the Solomon Islands, albeit from an uncertain chronological context⁷, had a D-loop sequence identical to that ofS. bucculentus.

Triangles represent monophyletic clades and circles represent paraphyletic groups and single sequences. Clades are coloured and named according to ref.9. Nodes marked by asterisks have posteriorP < 80%. All clades representS. scrofa except when labelled: b,S. barbatus; ce,S. celebensis; cr,S. cristatus; s,S. scrofa; v,S. verrucosus. GenBank accession numbers for the new sequences used in this study are DQ444703–DQ444711. Sample details, methods of DNA extraction, amplification and sequencing, computational methods, the full phylogenetic trees based on the D-loop and cytochromeb regions of the mitochondrial DNA, and the barcoding analysis of the 12S gene are available from the authors (http://www.cebl.auckland.ac.nz/~hros001/Susbucc/).

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These results undermine the species status ofS. bucculentus. The partial skull from 1997 was morphologically identical to the 1892 skull¹. But Groves and colleagues have recently judged the key taxonomic characters of the mandibular canine and malar tuberosity to be less distinctive than once thought, even though multivariate analyses of cranial and dental measurements tend to separate the two skulls from samples of IndochineseS. scrofa⁸. Our phylogenetic results are consistent with a recently shared ancestry between the 1997S. bucculentus sample from the Annamite Range and Asian domestic and wildS. scrofa. Also, ourS. bucculentus 12S-rRNA gene sequence was within the range of intraspecific variation shown byS. scrofa.

There are three possible explanations for these findings, a view shared by C. P. Groves (personal communication). First, hybridization may have introduced a locally occurring mitochondrial haplotype fromS. scrofa intoS. bucculentus. Second, speciation and morphological differentiation ofS. bucculentus may have occurred relatively recently and so subsequent mitochondrial lineage sorting remains incomplete. A third possibility, however, is that the morphology previously^1,8 designated as that of a separate species,S. bucculentus, is no more than one extreme of the natural range of variation shown by the wild IndochineseS. scrofa.

Authors and Affiliations

1. Department of Anthropology and Allan Wilson Centre for Molecular Ecology and Evolution, The University of Auckland, Private Bag, Auckland, 92019, New Zealand

   Judith H. Robins & Elizabeth Matisoo-Smith

2. Bioinformatics Institute and School of Biological Sciences, The University of Auckland, Private Bag, Auckland, 92019, New Zealand

   Howard A. Ross

3. Department of Anthropology, The University of Auckland, Private Bag, Auckland, 92019, New Zealand

   Melinda S. Allen

Corresponding author

Correspondence toJudith H. Robins.

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