No loci were in linkage disequilibrium. One locus (Ame-22) significantly deviated from HWE after sequential Bonferroni correction (Rice 1989) due to an excess of homozygotes. Ame-22 and Pnov02 showed signs of potential null alleles as identified by MICROCHECKER. This may have resulted from weighted sampling (30 of 50 samples) from an area of potentially related groups of birds on breeding
territories possessing alleles identical by descent from a common ancestor. This
can be resolved by further study of site philopatry in the tūī (Hedrick 2009). Three loci had indels in one or both allele copies in some individuals. These were reproducible, and were found to be inherited when mother-offspring pairs were compared. When sequenced, the Ame-20 indel was found to result from the occasional presence of a 1 bp insertion in the flanking sequence of the repeat. This indel could be found in all three alleles and was visualised as a 1 bp shift on the microsatellite profile. This could be used to provide further resolution for parentage analyses. Pnov23 has a deletion of two T’s in some copies of the repeat causing alleles not to be consistently 5 bp apart, as evidenced by clean peaks without stuttering. Similarly, Ame-14 has an allele in-between two repeat lengths caused by a TC deletion in one of the repeat units.
The mean error rate across all loci based on differing allele scoring between repeated genotypes was 1.6%. Mother-offspring mismatches produced the same error rate to that identified by repeat genotyping (1.6%). These error rates were predominantly a result of allelic drop-out of some of the longer Pnov03, Pnov25,
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and Ame-9 alleles, and therefore in all further analyses, all apparent homozygotes at any loci were re-genotyped separately to minimise scoring errors. An error rate of 1.6% was subsequently input as an error rate for paternity analyses to allow for potential low frequency null alleles.
As is common among longer tandem repeat microsatellites (Bacher and Schumm 1998), no loci exhibited stuttering, resulting in profiles that were easily read with minimal chance for scoring errors. This study shows that longer tandem repeats can be highly polymorphic, and with increasingly commonly reported genotyping errors found in microsatellite studies (Dewoody et al. 2006), researchers should consider these repeats more frequently for genetic studies.
In summary, this study reports the first microsatellites developed for the tūī, as well as the cross-amplification of loci designed for another honeyeater, the bellbird. These markers will be useful in studies of population genetics, and behavioural aspects such as mating systems, of the New Zealand honeyeaters and potentially honeyeaters of the Austro-Papuan region.
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2.5
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Male size predicts extra-pair
paternity
in
a
socially
monogamous passerine with
extreme sexual size dimorphism
Male tūī engaged in territorial song display
3
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