Document Type


Original Publication Date


Journal/Book/Conference Title

BMC Evolutionary Biology





DOI of Original Publication



Originally published at

Date of Submission

August 2014



A widely-used approach for screening nuclear DNA markers is to obtain sequence data and use bioinformatic algorithms to estimate which two alleles are present in heterozygous individuals. It is common practice to omit unresolved genotypes from downstream analyses, but the implications of this have not been investigated. We evaluated the haplotype reconstruction method implemented by PHASE in the context of phylogeographic applications. Empirical sequence datasets from five non-coding nuclear loci with gametic phase ascribed by molecular approaches were coupled with simulated datasets to investigate three key issues: (1) haplotype reconstruction error rates and the nature of inference errors, (2) dataset features and genotypic configurations that drive haplotype reconstruction uncertainty, and (3) impacts of omitting unresolved genotypes on levels of observed phylogenetic diversity and the accuracy of downstream phylogeographic analyses.


We found that PHASE usually had very low false-positives (i.e., a low rate of confidently inferring haplotype pairs that were incorrect). The majority of genotypes that could not be resolved with high confidence included an allele occurring only once in a dataset, and genotypic configurations involving two low-frequency alleles were disproportionately represented in the pool of unresolved genotypes. The standard practice of omitting unresolved genotypes from downstream analyses can lead to considerable reductions in overall phylogenetic diversity that is skewed towards the loss of alleles with larger-than-average pairwise sequence divergences, and in turn, this causes systematic bias in estimates of important population genetic parameters.


A combination of experimental and computational approaches for resolving phase of segregating sites in phylogeographic applications is essential. We outline practical approaches to mitigating potential impacts of computational haplotype reconstruction on phylogeographic inferences. With targeted application of laboratory procedures that enable unambiguous phase determination via physical isolation of alleles from diploid PCR products, relatively little investment of time and effort is needed to overcome the observed biases.


© 2010 Garrick et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Is Part Of

VCU Biology Publications

1471-2148-10-118-s1.pdf (98 kB)
Increase over time in the use of PHASE in empirical studies relating to phylogeography, speciation or hybridization.

1471-2148-10-118-s2.pdf (130 kB)
Frequency distribution of the relationship between number of segregating sites (S) and number of different alleles (AN) in the 500 simulated datasets from which 35 (solid circles) were arbitrarily selected for further analysis using PHASE.

1471-2148-10-118-s3.pdf (65 kB)
Correlation coefficients between the four measures of dataset polymorphism.

1471-2148-10-118-s4.pdf (126 kB)
Supplementary references.

1471-2148-10-118-s5.pdf (195 kB)
Relationship between alternative measures of dataset polymorphism (x-axis) and the number of unresolved genotypes (y-axis).