Effective Characterisation of the Complete Orang-Utan Mitochondrial DNA Control Region, in the Face of Persistent Focus in Many Taxa on Shorter Hypervariable Regions

PLoS One. 2016 Dec 29;11(12):e0168715. doi: 10.1371/journal.pone.0168715. eCollection 2016.

Abstract

The hypervariable region I (HVRI) is persistently used to discern haplotypes, to distinguish geographic subpopulations, and to infer taxonomy in a range of organisms. Numerous studies have highlighted greater heterogeneity elsewhere in the mitochondrial DNA control region, however-particularly, in some species, in other understudied hypervariable regions. To assess the abundance and utility of such potential variations in orang-utans, we characterised 36 complete control-region haplotypes, of which 13 were of Sumatran and 23 of Bornean maternal ancestry, and compared polymorphisms within these and within shorter HVRI segments predominantly analysed in prior phylogenetic studies of Sumatran (~385 bp) and Bornean (~323 bp) orang-utans. We amplified the complete control region in a single PCR that proved successful even with highly degraded, non-invasive samples. By using species-specific primers to produce a single large amplicon (~1600 bp) comprising flanking coding regions, our method also serves to better avoid amplification of nuclear mitochondrial insertions (numts). We found the number, length and position of hypervariable regions is inconsistent between orang-utan species, and that prior definitions of the HVRI were haphazard. Polymorphisms occurring outside the predominantly analysed segments were phylogeographically informative in isolation, and could be used to assign haplotypes to comparable clades concordant with geographic subpopulations. The predominantly analysed segments could discern only up to 76% of all haplotypes, highlighting the forensic utility of complete control-region sequences. In the face of declining sequencing costs and our proven application to poor-quality DNA extracts, we see no reason to ever amplify only specific 'hypervariable regions' in any taxa, particularly as their lengths and positions are inconsistent and cannot be reliably defined-yet this strategy predominates widely. Given their greater utility and consistency, we instead advocate analysis of complete control-region sequences in future studies, where any shorter segment might otherwise have proven the region of choice.

MeSH terms

  • Animals
  • DNA, Mitochondrial / genetics*
  • Feces
  • Genetic Variation*
  • Phylogeny
  • Pongo pygmaeus / genetics*
  • Sequence Analysis, DNA

Substances

  • DNA, Mitochondrial

Grants and funding

Collection of samples at Camp Leakey was generously supported by the University of Aberdeen (UoA; http://www.abdn.ac.uk/) Expedition Fund; UoA Small Grants Fund; UoA Alumni Annual Fund; the John Reid Trust; the Royal Geographical Society with Institute of British Geographers (http://www.rgs.org/); the Royal Scottish Geographical Society (http://www.rsgs.org/); the Gilchrist Educational Trust (http://www.gilchristgrants.org.uk/); the Orangutan Foundation UK (http://www.orangutan.org.uk); the North of England Zoological Society at Chester Zoo (http://www.chesterzoo.org/); Darwin College, Cambridge (http://www.darwin.cam.ac.uk/); the Primate Society of Great Britain (http://www.psgb.org/), through a Charles A Lockwood Memorial Grant; the ARCUS Foundation (http://www.arcusfoundation.org/); the Leverhulme Centre for Human Evolutionary Studies, University of Cambridge (http://www.human-evol.cam.ac.uk/); the Ridgeway-Venn Studentship; the Miss Millie Foundation and the American Association of Zoo Keepers Los Angeles Chapter. The long-term research conducted at Camp Leakey, which facilitated sample collection in Tanjung Puting National Park, was primarily funded by the Orangutan Foundation International (http://www.orangutan.org/). Collection of samples in the United States, and subsequent laboratory analyses, was funded by the Sacramento Zoo Conservation Fund (http://www.saczoo.org/); Como Park Zoo and Conservatory (http://www.comozooconservatory.org/); Orangutan Outreach (http://www.redapes.org/); Henry Vilas Zoo (http://www.vilaszoo.org/); The Orang-utan Conservation Genetics Trust; the Max Planck Society (http://www.mpg.de); the Chinese Academy of Sciences (http://www.cas.ac.cn) and the National Natural Science Foundation of China (http://www.nsfc.gov.cn/; grant numbers 31450110070 and 91331203). GLB was supported by the Orang Utan Republik Foundation (http://www.orangutanrepublik.org/) through the 2012 LP Jenkins Memorial Fellowship and by the German Academic Exchange Service (DAAD; https://www.daad.de/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.