Conservation Genetics

, Volume 8, Issue 4, pp 879–884 | Cite as

Single base errors in PCR products from avian museum specimens and their effect on estimates of historical genetic diversity

  • Kristina M. SefcEmail author
  • Robert B. Payne
  • Michael D. Sorenson
Original Paper


Conservation genetic studies often employ DNA extracts from museum specimens for comparisons with extant populations to monitor temporal changes in genetic diversity. Here, we report on artifact base changes in mitochondrial DNA sequences amplified from relatively recent (≤ 35 years) museum specimens of indigobirds (Vidua spp.). Single base errors were confirmed by replicate sequencing and included both double peaks and artifact substitutions at rates of ∼3 × 10−4 and ∼1 × 10−4 per base-pair, respectively, resulting in one or more errors or ambiguities in an 1100 base pair sequence in 21% of 219 samples. Most errors involved C→T changes on the L-strand, presumably due to deamination of cytosine in the template. The error rates encountered here bias comparisons of haplotype number between historical and extant populations, such that the ‘loss’ of artifact haplotypes present in a historical sample could be incorrectly attributed to a population decline or bottleneck. Sequencing errors due to miscoding lesions in template DNA have so far been reported only from ancient and formalin-fixed tissue, but they may also affect relatively recent museum samples, as shown here, and perhaps also non-invasive samples that typically yield low-quality DNA.


PCR artifact Sequence error Museum samples Indigobirds Population decline 



Field work from 1966 to the present has been supported and facilitated by numerous agencies and individuals. Jeff Tetrault assisted with the replicate sequencing. The work was supported by the Austrian Science Fund (P17380-B06; J-2044), the National Science Foundation (IOB 9412399; DEB 0089757), and the Dr. Heinrich-Jörg-Stiftung (Karl-Franzens-Universität Graz, Austria).


  1. Akbari M, Hansen MD, Halgunset J, Skorpen F, Krokan HE (2005) Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. J Mol Diagn 7:36–39PubMedGoogle Scholar
  2. Beaumont MA (2003) Estimation of population growth or decline in genetically monitored populations. Genetics 164:1139–1160PubMedGoogle Scholar
  3. Bonin A, Bellemain E, Bronken Eidesen P, Pompanon F, Brochmann C, Taberlet P (2004) How to track and assess genotyping errors in population genetics studies. Mol Ecol 13:3261–3273PubMedCrossRefGoogle Scholar
  4. Buchan JC, Archie EA, van Horn RC, Moss CJ, Alberts SC (2005) Locus effects and sources of error in noninvasive genotyping. Mol Ecol Notes 5:680–683CrossRefGoogle Scholar
  5. Clark AG, Whittam TS (1992) Sequencing errors and molecular evolutionary analysis. Mol Biol Evol 9:744–752PubMedGoogle Scholar
  6. Cooper A (1994) DNA from museum specimens. In: Herrmann B, Herrmann S (eds) Ancient DNA: recovery and analysis of genetic material from paleontological, archeological, museum, medical and forensic specimens. Springer, New YorkGoogle Scholar
  7. Cooper A, Poinar HN (2000) Ancient DNA: do it right or not at all. Science 289:1139PubMedCrossRefGoogle Scholar
  8. Culver M, Johnson WE, Pecon-Slattery J, O’Brien SJ (2000) Genomic ancestry of the American puma (Puma concolor). J Hered 91:186–197PubMedCrossRefGoogle Scholar
  9. Gagneux P, Boesch C, Woodruff DS (1997) Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair. Mol Ecol 6:861–868PubMedGoogle Scholar
  10. Gilbert MTP, Hansen AJ, Willerslev E, Rudbeck L, Barnes I, Lynnerup N, Cooper A (2003). Characterization of genetic miscoding lesions caused by postmortem damage. Am J Hum Genet 72:48–61PubMedCrossRefGoogle Scholar
  11. Glenn TC, Stephan W, Braun MJ (1999) Effects of a population bottleneck on Whooping Crane mitochondrial DNA variation. Conserv Biol 13:1097–1107CrossRefGoogle Scholar
  12. Godoy JA, Negro JJ, Hiraldo F, Donázar JA (2004) Phylogeography, genetic structure and diversity in the endangered bearded vulture (Gypaetus barbatus, L.) as revealed by mitochondrial DNA. Mol Ecol 13:371–390PubMedCrossRefGoogle Scholar
  13. Hansen AJ, Willerslev E, Wiuf C, Mourier T, Arctander P (2001) Statistical evidence for miscoding lesions in ancient DNA templates. Mol Biol Evol 18:262–265PubMedGoogle Scholar
  14. Hofreiter M, Jaenicke V, Serre D, von Haeseler A, Pääbo S (2001). DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res 29:4793–4799PubMedCrossRefGoogle Scholar
  15. Johnson JA, Dunn PO (2006) Low genetic variation in the Heath Hen prior to extinction and implications for the conservation of prairie-chicken populations. Conserv Genet 7:37–48CrossRefGoogle Scholar
  16. Johnson JA, Bellinger MR, Toepfer JE, Dunn P (2004) Temporal changes in allele frequencies and low effective population size in greater prairie-chickens. Mol Ecol 13:2617–2630PubMedCrossRefGoogle Scholar
  17. Kalinowski ST, Taper ML, Creel S (2006) Using DNA from non-invasive samples to identify individuals and census populations: an evidential approach tolerant of genotyping errors. Conserv Genet 7:319–329CrossRefGoogle Scholar
  18. Lage C, Kornfield I (2006) Reduced genetic diversity and effective population size in an endangered Atlantic salmon (Salmo salar) population from Maine, USA. Conserv Genet 7:91–104CrossRefGoogle Scholar
  19. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–715PubMedCrossRefGoogle Scholar
  20. Luikart G, Sherwin WB, Steele BM, Allendorf FW (1998) Usefulness of molecular markers for detecting population bottlenecks via monitoring genetic change. Mol Ecol 7:963–974PubMedCrossRefGoogle Scholar
  21. MacHugh DE, Troy CS, McCormick F, Olsaker I, Eythórsdóttir E, Bradley DG (1999) Early medieval cattle remains from a Scandinavian settlement in Dublin: genetic analysis and comparison with extant breeds. Proc R Soc Lond B 354:99–109Google Scholar
  22. Miller CR, Joyce P, Waits LP (2002) Assessing allelic dropout and genotype reliability using maximum likelihood. Genetics 160:357–366PubMedGoogle Scholar
  23. Mills LS, Citta JJ, Lair KP, Schwartz MK, Tallmon DA (2000) Estimating animal abundance using noninvasive DNA sampling: promise and pitfalls. Ecol Appl 10:283–294CrossRefGoogle Scholar
  24. Muñoz-Fuentes V, Green AJ, Negro JJ, Sorenson MD (2005) Population structure and loss of genetic diversity in the endangered white-headed duck, Oxyura leucocephala. Conserv Genet 6:999–1015CrossRefGoogle Scholar
  25. Pääbo S, Poinar H, Serre D, Jaenicke-Després V, Hebler J, Rohland N, Kuch M, Krause J, Vigilant L, Hofreiter M (2004) Genetic analyses from ancient DNA. Annu Rev Genet 38:645–679PubMedCrossRefGoogle Scholar
  26. Payne RB, Sorenson MD (2002). Museum collections as sources of genetic data. In: R. van den Elzen (ed) Bird Collections in Europe: promotion of science in ornithology. Alexander Koenig Research Institute and Museum of Zoology Proceedings, Vol. 2, Bonn, GermanyGoogle Scholar
  27. Paxinos EE, James HF, Olson SL, Ballou JD, Leonard JA, Fleischer RC (2002) Prehistoric decline of genetic diversity in the nene. Science 296:1827–1827PubMedCrossRefGoogle Scholar
  28. Quach N, Goodman MF, Shibata D (2004) In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol 4:1–5 PubMedCrossRefGoogle Scholar
  29. Rozas J, Sánchez-Delbarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497PubMedCrossRefGoogle Scholar
  30. Sefc KM, Payne RB, Sorenson MD (2003) Microsatellite amplification from museum feather samples: the effects of fragment size and template concentration on genotyping errors. Auk 120:982–989CrossRefGoogle Scholar
  31. Sefc KM, Payne RB, Sorenson MD (2005). Genetic continuity of brood-parasitic indigobird species. Mol Ecol 14:1407–1419PubMedCrossRefGoogle Scholar
  32. Sorenson MD, Payne RB (2001) A single, ancient origin of obligate brood parasitism in African finches: implications for host-parasite coevolution. Evolution 55:2550–2567PubMedGoogle Scholar
  33. Sorenson MD, Sefc KM, Payne RB (2003) Speciation by host switch in brood parasitic indigobirds. Nature 424:928–931PubMedCrossRefGoogle Scholar
  34. Swofford DL (2002) PAUP* 4.0b10: Phylogenetic analysis using parsimony. Sinauer, Sunderland MassachusettsGoogle Scholar
  35. Taberlet P, Griffin S, Goossens B, Questiau S, Manceau V, Escaravage N, Waits LP, Bouvet J (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res 24:3189–3194PubMedCrossRefGoogle Scholar
  36. Vallianatos M, Lougheed SC, Boag PT (2002) Conservation genetics of the loggerhead shrike (Lanius ludovicianus) in central and eastern North America. Conserv Genet 3:1–13CrossRefGoogle Scholar
  37. Williams C, Pontén F, Moberg C, Söderkvist P, Uhlén M, Pontén J, Sitbon G, Lundeberg J (1999) A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol 155:1467–1471PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Kristina M. Sefc
    • 1
    Email author
  • Robert B. Payne
    • 2
  • Michael D. Sorenson
    • 3
  1. 1.Department of ZoologyKarl Franzens University of GrazGrazAustria
  2. 2.Museum of Zoology & Department of Ecology and Evolutionary BiologyUniversity of MichiganAnn ArborUSA
  3. 3.Department of BiologyBoston UniversityBostonUSA

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