Genotyping errors
Genotyping errors were quantified by comparing original AFLP profiles to replicates for
the 47 individuals replicated. Mean error rate per phenotype was calculated following
Pompanon et al. (2005). In addition, mean error rate per fragment amplified was defined
as ef = mf / n, where n is the number of loci x individuals for which at least one of the two
replicates had a band, and mf is the fraction of n that presented a mismatch between
replicates. Thus, ef assumes that genotyping errors are essentially due to fragment
dropouts and is less conservative because n does not include recessive phenotypes
without mismatches. This assumption appears plausible since false bands at a truly
recessive phenotype position must be rare given the perfect adaptor/primer match and the
selectivity of the 3’-end of the AFLP primer.
The mean error rate per AFLP phenotype was 0.85%, a value within the range reported in
other studies (Pompanon et al. 2005). However, two samples accounted for 46% of all
errors. Clearly, the replicate for these samples did not amplify well and were thus
excluded from subsequent analyses. Error rate per phenotype without these two samples
was 0.48%. Distribution of errors across loci was close to uniformity (KolmogorovSmirnov goodness-of-fit : d = 7, n = 32 loci with errors, P = 0.05) but not across samples
(d = 9, n = 18 samples with errors, P < 0.001), when considering loci or samples with at
least one genotypic mismatch. Hence, eliminating problematic individuals rather than loci
should better improve the accuracy of our results. In fact, whenever an individual was
suspected to exhibit errors because of an “unusual” genetic dissimilarity with other birds,
we did re-amplify for verification. As predicted, error rate per amplified fragment was
higher (ef = 0.59) than error rate per phenotype (i.e., per locus x sample) but only slightly
so. Overall, error rates are low and likely to have a negligible effect on results. For
example, for a fixed (monomorphic) fragment to become erroneously polymorphic under
the 5%-criterion, it would necessitate a fragment dropout in at least 20 wandering
albatrosses or 1.7 (≈2) Amsterdam albatrosses, while our error rate predicts dropouts for
2 and 0.17 birds, for the two species respectively, assuming uniform distribution of errors
across loci.
Homoplasy
Homoplasy, i.e. the occurrence of non-homologous loci of identical size, may also bias
estimates of genetic diversity. Vekemans et al. (2002) showed that the AFLP technique
generates a non-uniform distribution of fragment sizes, such that the proportion of
detectable fragments greatly decreases with size. This, in turn, leads to a greater level of
homoplasy for small fragments. As a consequence, the proportion of polymorphic
fragments increases as a function of size (Vekemans et al. 2002). In order to assess
whether polymorphism varies with fragment density/size, we calculated the proportion of
polymorphic loci (P5%) separately for each 50 bp interval between 50 and 300 bp.
Fragments above > 300 bp were rare and thus pooled. Larger fragments (150-300 bp)
were rarer than shorter fragments (50-150 bp; χ2 = 15.71, P < 0.01, df = 5; Table 1). They
also showed a higher level of polymorphism, but this result was not significant (d = 3.8, n
= 12 polymorphic loci, P > 0.2). The density of scored markers was very low for CGprimers (respectively 1.0 locus/10 bp and 0.9 loci/10 bp of AFLP profile for the 50-150
bp and 150-300 bp intervals). With such small densities, it appears unlikely that
homoplasy would strongly affect our data. On the other hand, non-CG primers had a
density of selected markers about twice greater for the 50-150 bp class (density = 2.5
loci/10 bp) than for the 150-300 bp class (1.2 loci/10 bp). In addition, a large number of
potential markers were not scored for these primers, because they presented peaks either
too small or unclear. Such discarded markers were rare for CG-primers. Overall, P5%
remained low in albatrosses whatever the range of fragment size considered.
References
Pompanon, F., Bonin, A., Bellemain, E. & Taberlet, P. 2005 Genotyping errors: causes,
consequences and solutions. Nature Rev. Genet. 6, 847-859.
Vekemans, X., Beauwens, T., Lemaire, M. & Roldan-Ruiz, I. 2002 Data from amplified
fragment length polymorphism (AFLP) markers show indication of size
homoplasy and of a relationship between degree of homoplasy and fragment size.
Mol. Ecol. 11, 139-151.
Table 1 Polymorphism in wandering and Amsterdam albatrosses
for different ranges of AFLP fragment size.
Size range (base pairs)
number of loci
% polymorphic loci
050-100
65
3.1
100-150
68
1.5
150-200
44
9.1
200-250
28
3.6
250-300
17
23.5
>300
12
0