Multiple mitochondrial haplotypes within individual specimens may interfere with species identification and biodiversity estimation by DNA barcoding and metabarcoding in fig wasps

Accurate assessment of biodiversity is important for scientists, conservationists and policy makers. With the development of DNA barcoding and metabarcoding, many cryptic species have been identified based on the sequence diversity of a ‘barcode’ region in the mitochondrial genome. These molecular identifications have increased the estimated number of species on Earth ten‐fold. Mitochondrial DNA heteroplasmy, in which multiple mitochondrial haplotypes coexist in a single organism, can interfere with the accuracy of these estimates. However, this phenomenon is still infrequently documented. The main reason is that it is difficult to distinguish heteroplasmic sequences from nuclear mitochondrial segments (Numts). We investigated the prevalence of multiple haplotypes in DNA barcoding sequences in fig wasps (Insect: Hymenoptera) by performing high‐throughput sequencing on individual specimens. DNA metabarcoding was artificially simulated by combining all the haplotype sequences. Using conventional DNA barcoding by Sanger sequencing, the 28 morphological species we studied were divided into 38 taxa, suggesting the existence of ten cryptic species. However, the haplotypes originating from a single specimen clustered into different cryptic species clades, which conflicted with the conventional barcoding results. Furthermore, when we artificially simulated DNA metabarcoding by mixing all haplotype sequences, the number of molecular operational taxonomic units (or putative species) was 3.2× the number of morphological species. These results indicate that the prevalence of multiple haplotypes within individual specimens may have an important impact on DNA barcode‐based species identification and biodiversity assessment more broadly. We obtained multiple haplotypes within individual specimens by high‐throughput sequencing. Multiple haplotypes originating from a single specimen could mislead DNA barcoding result. Multiple haplotypes within individual specimens could result in overestimation of biodiversity in DNA metabarcoding.