A multi-samples, multi-extracts approach for microsatellite analysis of faecal samples in an arboreal ape

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We investigated the effect of the number of faecal samples, ofextracts per sample and of PCRs per extract on the reliability ofgenotypes for a microsatellite locus in free-living orang-utans.For each individual 36 PCRs were performed using DNA
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  Conservation Genetics  1:  157–162, 2000.© 2000  Kluwer Academic Publishers. Printed in the Netherlands.  157 A multi-samples, multi-extracts approach for microsatellite analysis of faecal samples in an arboreal ape Benoît Goossens 1 , ∗ , Loun`es Chikhi 2 , Sri S. Utami 3 , 4 , Jan de Ruiter 5 & Michael W. Bruford 1 1  Biodiversity and Ecological Processes Group, School of Biosciences, Cardiff University, PO Box 915 CathaysPark, Cardiff CF10 3TL, UK;  2 The Zoological Society of London, Institute of Zoology, Regent’s Park, London NW1 4RY, UK;  3  Ethologie and Socio-Oecologie, University of Utrecht, P.O. Box 80.086, 3508 TB Utrecht, The Netherlands;  4 Fakultas Biologi, Universitas Nasional, Jl Sawo manila, Jakarta 12510, Indonesia;  5  Department of Anthropology, University of Durham, 43 Old Elvet, Durham DH1 3HN, UK ( ∗ Corresponding author: E-mail:goossensbr@cardiff.ac.uk) Received 10 January 2000; accepted 10 April 2000 Key words:  conservation genetics, faeces, microsatellites, non-invasivemethods,  Pongo pygmaeus abelii Abstract We investigated the effect of the number of faecal samples, of extracts per sample and of PCRs per extract on thereliability of genotypes for a microsatellite locus in free-living orang-utans. For each individual 36 PCRs wereperformed using DNA extractions from up to four faecal samples. We found a very large inter-individual variationin positivePCRs (P+)(36/36foroneindividualand0/36foranother).As manyas 30%ofthe cases ledto erroneousgenotypes when only one P+ was obtained. It is preferable to use at least 4 P+ per extract to reduce this proportionto less than 1%. With 3 P+ results, erroneous genotypes were still observed in 26% of the cases together. Theseresults indicate that it is necessary to do a minimum of 4 PCRs per extract. In order to have a chance to observe 4P+, three extracts should be ideally analysed for each sample. We also recommendthat when possible two or moresamples should be collected in the field to increase the chance of having extracts containing DNA and to provideindependent replicates. While we recognise the difficulty of working with faecal samples, we advocate the use of faecal material forgeneticstudies of certainwild animal populationswherethe advantagesof avoidingdisturbance,stress and injury are deemed of critical importance. Introduction Conservation genetics studies of endangered animalsnow often require non-invasive sampling techniques.Inrecentyearsthishasbeenachievedthroughmethodsdeveloped to extract DNA for PCR from minuteamounts of tissue (e.g. using chelex (Walsh et al.1991); silica beads (Höss et al. 1992; Höss and Pääbo1993) and guanidium thiocyanate (Gerloff et al. 1995;Kohn et al. 1995)). In particular, these techniqueshave been applied to a large number of species wherehair or faeces has been collected in the field (Morinand Woodruff 1996; Kohn and Wayne 1997; Taberletet al. 1999). For some primates and other arborealspecies, specific problems arise such as the necessityof climbing trees to collect hairs (Morin et al. 1994)and the risk of mixing samples from different indi-viduals in the nest. Faecal samples collected on thefloorundernestsmaythereforehaveanadvantageoverhair since they are often abundant and the individualcan also sometimes be identified. Furthermore, faecalsamples are not subject to CITES constraints.Despite these advantages, DNA extracted fromfaeces can be degraded and in low quantities, whichdepend on a number of factors (see below) and areknown to generate unreliable data (false alleles and/orallelic dropout, e.g. Taberlet et al. 1996; Gagneux etal. 1997; Goossens et al. 1998; see however, Flag-stad et al. 1999). Based on the very limited amount of DNA extracted and the high variation across samples,Gerloff et al. (1995), Kohn et al. (1995) and Frantzenet al. (1998) have suggested multiple collection of   158faeces for each individual. However, no previousstudies have attempted to deal with this issue in aquantitative manner.Here we demonstrate the potential advantagesof collecting several faecal samples per individualand of carrying out multiple extracts per sampleby measuring amplification success in faecal DNAfrom orang-utans ( Pongo pygmaeus abelii ), collectedfor paternity investigations in a wild population inSumatra (Utami et al. submitted). Materials and methods Multiple faecal samples were collected from 16orang-utans from the Gunung Leuser National Park (Sumatra, Indonesia) between 1993 and 1998. Asmanysamples as possiblewerecollectedforeachindi-vidual, resulting in the following: four samples wereobtained for three individuals, three samples for oneindividual, two samples for 11 individuals, and onesample for one individual.All samples were immersed and stored in 90%ethanol just after defecation. DNA extraction wascarried out using the  Protocol for isolation of  DNA from stool for pathogen detection  from theQIAamp  DNA Stool Mini Kit (pp. 12–14) providedby QIAGEN GMBH (Hilden, Germany, cataloguenumber: 51504). All extractions were performed ina designated room devoid of PCR products and freshtissue, using a Class I microbiological safety cabinet.DNA samples were dissolved in 100  µ l of TE bufferand stored at  − 20  ◦ C. Multiple extractions wereconducted, with 12 DNA extracts produced per indi-vidual (see Table 1 for details). This was done in orderto have at least 3 extracts per faecal sample and thesame numberof extracts for all samples from the sameindividual. Since we had individuals with 4 faecalsamples we had to do 12 extracts for some individuals.We decidedto treatall individualsinthe sameway andtherefore also did 12 extracts for individuals having asmaller number of samples.A multiple-tubes PCR procedure (Navidi et al.1992; Taberlet et al. 1996), three PCR repeats perDNA extract, was conducted using primers for thehuman-derived microsatellite locus D5S1457 (tetra-nucleotide: (GATA)n, http://lpg.nci.nih.gov/ CHLC/).This locus was chosen because it is one of the humanloci that has proved most reliable when tested ondifferent primate species in our laboratory (includingred colobus, savannah baboons, hanuman langurs,gorillas, and chimpanzees). Amplification was carriedout in 12.5  µ l (10 mM Tris-HCl (pH 9.0), 200 mM(NH 4 ) 2 SO 4 , 50  µ M each dNTP, 1.5 mM MgCl 2 , 5ng of BSA, 0.1 U Amplitaq  Gold DNA polymerase(PerkinElmer),0.5 µ MnonfluorescentreverseprimerD5S1457, 0.5  µ M fluorescent (TET) forward primerD5S1457, 2.5  µ l of DNA extract. A PCR amplifica-tion of 50 cycles was carried out (initial denaturation94  ◦ C for 10 min, 94  ◦ C for 15 s, 45  ◦ C for 15 s, 72  ◦ Cfor 30 s) using Perkin Elmer Gene Amp PCR System9600. Standardisation was obtained by using the samePCR machine and validated Taq polymerase batchthroughout the study. The PCR products were visual-ised on a polyacrylamide gel using an ABI PRISM TM 377 DNA sequencer with marker GS350 Tamra. Allgels were analysed using GeneScan TM Analysis 2.0and Genotyper  2.0 software.The data were scored using specific terminology.We defined a P+ or positive PCR when a PCR productwas obtained and alleles were identified; a CCG orcorrectconsensusgenotypeasthemostlikelybasedon36 PCRs (see below). Different genotyping errors (E)were identified: FH or ‘false homozygote’ when oneof the two alleles of the real genotype was not ampli-fied due to allelic ‘dropout’ (Gagneux et al. 1997); FAor ‘false alleles’ when amplification artefacts can bemisinterpreted as true alleles while they are faecal-related because of low template concentration; C or‘contamination’ which is an allele known in human(but also present in orang-utans) but which is not analleleofthetruegenotypeforthe“contaminated”indi-viduals. The CCG was defined following Taberlet etal.’s (1996) recommendations. Based on the analysisof three positive PCRs, the CCG was definedas a ‘truehomozygote’ when a same single allele was obtainedfor three positive PCRs. All homozygotes found inthis study were confirmed by performing a two-stepprocedure defined in Taberlet et al. (1996): fouradditional amplifications for one successful extractshowing the same allele. The CCG was defined as a‘true heterozygote’ when the same two alleles wereobtained three times, or twice, where the third PCRamplified one of the two alleles present in the twoheterozygote PCRs. Results and discussion Table 1 summarises the results of the typing for the 16individuals. For each individual there were 12 DNAextractions from one to four faecal samples, and three  159      T   a     b     l   e     1 .      R   e   s   u     l    t   s   o     f    t     h   e   m   u     l    t     i   p     l   e  -   s   a   m   p     l   e   s   a   n     d   m   u     l    t     i   p     l   e  -   e   x    t   r   a   c    t   s   s    t   r   a    t   e   g   y .     F   o   r   e   a   c     h     i   n     d     i   v     i     d   u   a     l ,     1     2   e   x    t   r   a   c    t   s   w   e   r   e   c   a   r   r     i   e     d   o   u    t .     D   e   p   e   n     d     i   n   g   o   n    t     h   e   n   u   m     b   e   r   o     f   s   a   m   p     l   e   s   o     b    t   a     i   n   e     d     f   o   r   a   g     i   v   e   n     i   n     d     i   v     i     d   u   a     l     (     b   e    t   w   e   e   n     1   a   n     d     4     ) ,    t     h   e   n   u   m     b   e   r   o     f   e   x    t   r   a   c    t   s   p   e   r   s   a   m   p     l   e   v   a   r     i   e     d   c   o   n   s   e   q   u   e   n    t     l   y     b   e    t   w   e   e   n     3   a   n     d     1     2 .     T     h   r   e   e     P     C     R   s   w   e   r   e   p   e   r     f   o   r   m   e     d     f   o   r   e   a   c     h   e   x    t   r   a   c    t     (     f   o   r   a    t   o    t   a     l   o     f     3     6   a   m   p     l     i     fi   c   a    t     i   o   n   s   p   e   r     i   n     d     i   v     i     d   u   a     l     ) .     E   x    n    :   e   x    t   r   a   c    t   n   u   m     b   e   r ,     E   :    t   y   p   e   o     f   e   r   r   o   r ,     F     H   :     f   a     l   s   e     h   o   m   o   z   y   g   o    t   e ,     F     A   :     f   a     l   s   e   a     l     l   e     l   e ,     C   :   c   o   n    t   a   m     i   n   a    t     i   o   n .     P    +   :   p   o   s     i    t     i   v   e     P     C     R ,     C     C     G   :   c   o   r   r   e   c    t   c   o   n   s   e   n   s   u   s   g   e   n   o    t   y   p   e      1   s   a   m   p     l   e  –     1     2   e   x    t   r   a   c    t   s      E   x     1     E   x     2     E   x     3     E   x     4     E   x     5     E   x     6     E   x     7     E   x     8     E   x     9     E   x     1     0     E   x     1     1     E   x     1     2     I   n     d     i   v     i     d   u   a     l     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     O   u .     2     5     3     /     3     3     /     3        0       /       3       0       /       3       0       /       3       0       /       3      3     /     3     3     /     3     2     /     3     2     /     3     3     /     3     3     /     3     1     /     3     1     /     3     1     /     3      0     /     3      C     2     /     3     1     /     3     C     2     /     3     2     /     3      2   s   a   m   p     l   e   s  –     6   e   x    t   r   a   c    t   s     S   a   m   p     l   e     1     S   a   m   p     l   e     2      E   x     1     E   x     2     E   x     3     E   x     4     E   x     5     E   x     6     E   x     1     E   x     2     E   x     3     E   x     4     E   x     5     E   x     6     I   n     d     i   v     i     d   u   a     l     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     O   u .     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     2     /     3     2     /     3        0       /       3       0       /       3      1     /     3     1     /     3        0       /       3       0       /       3      O   u .     7     2     /     3     2     /     3        0       /       3      2     /     3      0     /     3      F     A     2     /     3     1     /     3     F     A     2     /     3     1     /     3     C     1     /     3     1     /     3     2     /     3     2     /     3     2     /     3     1     /     3     F     A     2     /     3     1     /     3     C     2     /     3     2     /     3     1     /     3     1     /     3     3     /     3     2     /     3     C     O   u .     1     2        0       /       3       0       /       3      1     /     3     1     /     3     2     /     3     1     /     3     C     2     /     3     1     /     3     C     1     /     3      0     /     3      C        0       /       3      2     /     3     1     /     3     C     1     /     3     1     /     3     3     /     3     3     /     3     3     /     3     3     /     3        0       /       3      O   u .     1     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     2     /     3     1     /     3     F     H     3     /     3     1     /     3     C     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     1     /     3     F     H     3     /     3     2     /     3     F     H     O   u .     1     4     3     /     3     1     /     3     F     H     3     /     3     3     /     3        0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3      3     /     3     2     /     3     F     H        0       /       3       0       /       3       0       /       3      O   u .     1     6     3     /     3     2     /     3     C     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     1     /     3     F     A     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     2     /     3     C     3     /     3     3     /     3     3     /     3     2     /     3     F     A     O   u .     1     8     3     /     3     2     /     3     F     A     3     /     3     2     /     3     F     A     3     /     3     3     /     3     3     /     3     2     /     3     C     2     /     3     2     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     2     /     3     C     3     /     3     3     /     3     3     /     3     3     /     3     O   u .     2     0     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     O   u .     2     1     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     2     /     3     F     A     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     2     /     3     C     3     /     3     1     /     3     F     A     3     /     3     3     /     3     3     /     3     2     /     3     F     A     O   u .     2     4     2     /     3     2     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     2     /     3     2     /     3        0       /       3       0       /       3      3     /     3     3     /     3        0       /       3      3     /     3     3     /     3        0       /       3      O   u .     2     8     2     /     3      0     /     3      F     H     3     /     3     1     /     3     F     H     3     /     3     3     /     3     3     /     3      0     /     3      F     H     1     /     3     1     /     3     3     /     3     2     /     3     F     H     3     /     3     2     /     3     F     H     3     /     3     1     /     3     F     H     3     /     3     3     /     3     3     /     3     2     /     3     F     H     3     /     3     1     /     3     F     H     3     /     3     2     /     3     F     H      3   s   a   m   p     l   e   s  –     4   e   x    t   r   a   c    t   s     S   a   m   p     l   e     1     S   a   m   p     l   e     2     S   a   m   p     l   e     3      E   x     1     E   x     2     E   x     3     E   x     4     E   x     1     E   x     2     E   x     3     E   x     4     E   x     1     E   x     2     E   x     3     E   x     4     I   n     d     i   v     i     d   u   a     l     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     O   u .     2     7     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3        0       /       3      3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3      4   s   a   m   p     l   e   s  –     3   e   x    t   r   a   c    t   s     S   a   m   p     l   e     1     S   a   m   p     l   e     2     S   a   m   p     l   e     3     S   a   m   p     l   e     4      E   x     1     E   x     2     E   x     3     E   x     1     E   x     2     E   x     3     E   x     1     E   x     2     E   x     3     E   x     1     E   x     2     E   x     3     I   n     d     i   v     i     d   u   a     l     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     P    +     C     C     G     E     O   u .     2     2     /     3     2     /     3     2     /     3     2     /     3     2     /     3     1     /     3     F     A        0       /       3      1     /     3      0     /     3      F     A        0       /       3      1     /     3     1     /     3     1     /     3     1     /     3        0       /       3      3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     3     /     3     O   u .     1     9     2     /     3      0     /     3      F     H     1     /     3      0     /     3      C        0       /       3       0       /       3       0       /       3       0       /       3       0       /       3      3     /     3     1     /     3     C        0       /       3       0       /       3      1     /     3     1     /     3        0       /       3      O   u .     2     6        0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3       0       /       3  160 Table 2.  Number of correct consensus genotypes (CCG = ‘truegenotype’ inferred from 36 total PCRs)observed as afunction of the number ofpositive PCRs(P+). Foreach ofthe 192 extracts, 3PCRs were performed. These three PCRs either did not amplify(i.e. 0 positive PCR out of3 coded as 0/3) orgave positive resultswith varying amounts of success (i.e. from 1/3 to 3/3). Then foreach of the groups of 3 PCRs some led to erroneous genotypes(coded 0/3) while others led to the CCG, again, with varyingamounts of success (from 1/3 to 3/3)CCG3/3 2/3 1/3 0/3P+ 3/3 104 77 17 9 12/3 24 – 11 10 31/3 14 – – 10 40/3 50 – – – 50Total 192 PCRs were carried out for each extract. 374 (65%)of the 576 PCR reactions performed were positive(P+) with a total of 316 (55% of the total, or 85%of the P+) leading to a CCG. However, we found averylargevariabilityacrossindividuals.Ou.26yieldedno positive result out of the 36 PCRs while Ou.20yielded 100% P+ and 100% CCG. The 576 PCR reac-tions were carried out on 192 extracts. In 104 (54%)extractsthe3PCRs gaveaP+, butin50(26%)extractsnone of the reactions were positive (Table 2). Amongthe 142 extracts where there was at least one P+, 8extracts (6%) did not produce the CCG. The propor-tion of cases where the CCG was not observed (i.e.only incorrect genotypes were observed), given thatthere were 3 P+, 2 P+ or 1 P+ was 1/104, 3/24 and4/14, respectively (Table 2). Thus 29% of the 1 P+did not produce the CCG. This proportion reducedto 12.5% when 2 out of 3 PCRs were positive andto less than 1% when all 3 were positive, stronglyindicating the importance of working with extracts forwhich there are three positive PCRs. However, evenwith the 3 P+ extracts there were still 27 (26%) caseswhere at least one genotype was incorrect (Table 2).This proportion was significantly higher for extractswith 2 P+ (13/24 = 54%, binomial test,  p  <  0.01)but is not significantly different from the 1 P+ extracts(4/14). We also found that 10/104 (9.6%) of the 3 P+extracts produced 2 or more (i.e. a majority of) erro-neous genotypes. This strongly indicates that 4 PCRsshould be a minimum (unless many extracts are used,see below). Note that for “hard-to-amplify” samples 4P+ may not be enough.Of the 77 extracts where the 3 P+ gave the samegenotype we found one extract where this was anerroneous genotype when compared to other extracts.Since the two other extracts showed that this could notbe the CCG, the actual risk of misidentifying the CCGwas less than 1/77 (1.3%).We estimated for each sample the probability P n that n extracts would produceno positive PCR. P 1  wasestimated as the proportion of extracts for which allthreePCRs were negative.P n  was thereforesimply P n1 .This allowed us to determine the minimum number of extracts that should be carried out for a given sampleto observe at least one positive PCR. Our resultsshow that when only one extract is taken P 1  variesenormously from individual to individual (from 0 to0.83, excluding individual Ou.26 for which we had nopositive results: P n  is 1 for any n). P 2  varies less, asexpected but can be as high as 0.70, with most valuesbetween 0 and 0.4. In fact unless 3 to 4 extracts for agivensampleare analysedthe probabilityof observingno P+ can still be high for some individuals. We mustnotethatthesevaluesarenotindependentsincewehadmore than one sample for most individuals.To summarise, an absolute minimum of 3 P+should be obtained to minimise the probability of having a majority of P+ giving an erroneous genotypeand to observe the CCG. Also, at least 3 extracts persample should be done to observe 4 P+ for at least onesample. This leads therefore to a recommendation of 12 PCRs per faecal sample, for this study.However, the results obtained here are based on asingle species sampled in a single location. Further,a number of factors could not be controlled. Forinstance, it is now widely accepted that the storagetechniques (Frantzen et al. 1998), the feeding habits(orang-utans are primarily fruit-eaters, MacKinnon1974), the size or part of the faeces sampled, theenvironmental conditions, and the time between drop-ping and collection have an effect on DNA quality. Asa consequencethe estimates providedherewill changefrom one species to another.Variation across samples or extracts indicate thatsloughed orang-utan tissue may not be homogene-ously distributed in their faeces. This confirms theresults of Kohn et al. (1995), who refer to the possi-bility of an uneven distribution of cells shed fromthe intestinal lining in the faeces in brown bears.Frantzen et al. (1998) reviewed the recovery-successof mtDNA and scnDNA in bonobos, European brownbears, Pyrenean brown bears, seals, and baboons (seereferences therein) and showed that on average 31%  161of faecal samples did not yield DNA (varying from0% in baboons to 80% in Pyrenean brown bears).In our study we found that 13% of samples yieldedno DNA, which is clearly at the lower end of thisspectrum. These data are somewhat confounded bydifferent protocols for the number of repeat extrac-tions and PCRs carried out. The large variation acrossindividuals suggests that multiple samples should betaken whereverpossible. This is particularlytruewhenfaeces aresmall anddonot allowmorethanoneortwoDNA extractions.Practical factors such as the humidity (rainfall),exposure to sun or shade, during sampling or the easeof following and identifyingindividuals, could also becrucial but have not yet been investigated. The lattercan be done when long-term behavioural studies arecarried out but may not always be possible for somespecies, particularly when they live in large groups(Gerloff et al. 1999).In the present study we limited laboratory-relatedproblems by working in a designated room and usingonly one batch of Taq polymerase (see above fordetails). In order to avoid slippage artefacts we used atetra-nucleotide locus (Edwards et al. 1991). Howeverthe presence of slippage products may be advan-tageous in identifying the locus when non-specificbands are also detected. A way to improve PCRquality could be to increase the DNA quantity. Thismay not always be possible and will depend on thetotal quantity of DNA available and the number of loci to be typed. Taberlet et al. (1996) suggest thatit is better to work with diluted DNA in order tohave enough material to increase the number of PCRs.In addition to the CCG we observed a number of erroneous genotypes during this study. FH have beenfound to be more common than FA in previous studiesusing hair (Gagneux et al. 1997; Goossens et al.1998) or faeces (Gerloff et al. 1995; Taberlet et al.1996; Launhardt et al. 1998) but the difference herewas slight (observed frequencies: FA = 2.95%, FH= 4.17%). It was not always clear whether we coulddiscriminate FA and C and we conservatively countedas C one allele which is known in human (but alsopresent in orang-utans) even though they could stillbe FA. We thus overestimate contamination in thepresent study. Note that contaminationshould not leadto errors because of the multiple extracts, and multiplePCR strategy. In the absence of other sources of DNA(blood or tissue) we cannot prove that the CCG wascorrect. However, given the large number of PCRs foreach individual we can be confident that the CCG wasthe most likely, since there was only one case out of 77 (less than 1.3%) where we observed 3 P+ givingthe same erroneousgenotype.Eventhoughthis cannotbe an independent test, it strongly indicates that thereis little chance that we misidentified the CCG. Eventhough this study is limited to one locus the results arelikely to be similar for other loci. In particular, Ou.26andOu.20werebothdifficultandveryeasytoamplify,respectively.In conclusion, working with faecal samples canbe very demanding but has a number of advan-tages, which include avoiding stress and contact withendangered populations. Acknowledgements We are grateful to M. Beaumont for helpful discus-sions and to the Indonesian Institute of Science (LIPI)for granting permission to do research in Indonesia,and Universitas Nasional and Leuser ManagementUnit for sponsoring the project. The directorategeneral of Nature Conservation and Forest Protection(PHPA) gave permission to do the work in GunungLeuser National Park. This study was supported byLeverhulme Trust (F/390/U), by the Royal Society(20106) and by a grant from the University of Utrechtand the Lucie Burgers Foundation. References Edwards A, Civitemmo A, Hammond HA, Caskey CT (1991)DNA typing and genetic mapping with trimeric tandem repeats.  American Journal of Human Genetics ,  49 , 746–756.Flagstad Ø, Røed K, Stacy JE, Jakobsen KS (1999) Reliable nonin-vasive genotyping based on excremental PCR of nuclear DNApurified with a magnetic bead protocol.  Mol. Ecol .,  8 , 879–883.Frantzen MAJ, Silk JB, Ferguson JWH, Wayne RK, Kohn MH(1998) Empirical evaluation of preservation methods for faecalDNA.  Mol. Ecol .,  7 , 1423–1428.Gagneux P, Boesch C, Woodruff DS (1997) Microsatellite scoringerrors associated with noninvasive genotyping based on nuclearDNA amplified from shed hair.  Mol. Ecol .,  6 , 861–868.Gerloff U, Hartung B, Fruth B, Hohmann G, Tautz D (1999)Intracommunity relationships, dispersal pattern and paternitysuccess in a wild living community of bonobos ( Pan paniscus )determined from DNA analysis of faecal samples.  Proceedingsof the Royal Society of London B ,  266 , 1189–1195.Gerloff U, Schlötterer C, Rassmann K, Rambold I, Hohmann G,Fruth B, Tautz D (1995) Amplification of hypervariable simplesequence repeats (microsatellites) from excremental DNA of wild living bonobos ( Pan paniscus ).  Mol. Ecol .,  4 , 515–518.Goossens B, Waits LP, Taberlet P (1998) Plucked hair samplesas a source of DNA: reliability of dinucleotide microsatellitegenotyping.  Mol. Ecol .,  7 , 1237–1241.
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