16S_reference16S reference generated as consensus sequences from the Leiopelma hochstetteri 16S sequences available in GenBank.CytB_referenceCyt b reference generated as consensus sequences from the Leiopelma hochstetteri Cyt b sequences available in GenBankTable1Sample details of Leiopelma hochstetteri individuals used in the pooled samples next-generation sequencing (NGS) approach. For each sample species, sampling location, population number, sample number, Cyt b Genbank accession number, 16S Genbank accession number and geographical coordinates of the sampling location are provided. This table corresponds to a modified supplementary material that is available with the online version of the article Fouquet et al. 2010, Conservation Genetics, 11, 907-919 (doi:10.1007/s10592-009-9935-8). Accession numbers in bold indicate unsuccessful PCR amplifications for the given locus and individual. Note that the sequences deposited in the Genbank under these accession numbers are sequences generated by Sanger sequencing in the study of Fouquet et al. 2010.Pop1Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 1. Individuals that belong to population-pool 1 are listed in Table 1.Pop2Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 2. Individuals that belong to population-pool 2 are listed in Table 1.Pop3Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 3. Individuals that belong to population-pool 3 are listed in Table 1.Pop4Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 4. Individuals that belong to population-pool 4 are listed in Table 1.Pop5Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 5. Individuals that belong to population-pool 5 are listed in Table 1.Pop6Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 6. Individuals that belong to population-pool 6 are listed in Table 1.Pop7Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 7. Individuals that belong to population-pool 7 are listed in Table 1.Pop8Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 8. Individuals that belong to population-pool 8 are listed in Table 1.Pop9Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 9. Individuals that belong to population-pool 9 are listed in Table 1.Pop10Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 10. Individuals that belong to population-pool 10 are listed in Table 1.Pop11Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 11. Individuals that belong to population-pool 11 are listed in Table 1.Pop12Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 12. Individuals that belong to population-pool 12 are listed in Table 1.Pop13Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 13. Individuals that belong to population-pool 13 are listed in Table 1.Pop14Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 14. Individuals that belong to population-pool 14 are listed in Table 1.Pop15Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 15. Individuals that belong to population-pool 15 are listed in Table 1.Pop16Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 16. Individuals that belong to population-pool 16 are listed in Table 1.Pop17Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 17. Individuals that belong to population-pool 17 are listed in Table 1.Pop18Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 18. Individuals that belong to population-pool 18 are listed in Table 1.Pop19Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 19. Individuals that belong to population-pool 19 are listed in Table 1.Pop20Standard Flowgram Format (sff) file that contains NGS reads for 16S and Cyt b amplicons from population-pool 20. Individuals that belong to population-pool 20 are listed in Table 1.

Next-generation sequencing (NGS) on pooled samples has already been broadly applied in human medical diagnostics and plant and animal breeding. However, thus far it has been only sparingly employed in ecology and conservation, where it may serve as a useful diagnostic tool for rapid assessment of species genetic diversity and structure at the population level. Here we undertake a comprehensive evaluation of the accuracy, practicality and limitations of parallel tagged amplicon NGS on pooled population samples for estimating species population diversity and structure. We obtained 16S and Cyt b data from 20 populations of Leiopelma hochstetteri, a frog species of conservation concern in New Zealand, using two approaches – parallel tagged NGS on pooled population samples and individual Sanger sequenced samples. Data from each approach were then used to estimate two standard population genetic parameters, nucleotide diversity (π) and population differentiation (FST), that enable population genetic inference in a species conservation context. We found a positive correlation between our two approaches for population genetic estimates, showing that the pooled population NGS approach is a reliable, rapid and appropriate method for population genetic inference in an ecological and conservation context. Our experimental design also allowed us to identify both the strengths and weaknesses of the pooled population NGS approach and outline some guidelines and suggestions that might be considered when planning future projects.