Multiple mitochondrial haplotypes within individual specimens affect biodiversity estimation by DNA metabarcoding

In the global action for biodiversity conservation, the most urgent need
is to delimit and identify species. DNA barcoding and metabarcoding based
on molecular markers have been increasingly used in species delimitation
and species diversity assessment, respectively, owing to their advantages
of standardization and high throughput. The molecular markers used in
these methods for animals are mainly derived from mitochondrial DNA. The
principle involved is that inter-specific genetic distances are greater
than intra-specific genetic distances. However, if multiple mitochondrial
haplotypes exist within individual specimens and the divergence between
these haplotypes is greater than the assumed maximum intra-specific
divergence, the species delimitation and species diversity assessment may
be affected. In our recent studies, a widespread phenomenon of multiple
mitochondrial haplotypes within individual specimens was discovered in fig
wasps (Hymenoptera, Insecta), having negative impact on DNA barcoding and
metabarcoding. In this study, we expanded the taxa to include up to 480
specimens from 82 species, 66 genera, 45 families, and 13 orders of
Insecta and Arachnida. By high-throughput sequencing of mitochondrial cox1
fragments within individual specimens, we studied the pattern of multiple
mitochondrial haplotypes within individual specimens. Three DNA
metabarcoding strategies, one of which was simulated, were applied. The
results revealed that multiple mitochondrial haplotypes within individual
specimens could lead to an overestimation of species diversity by
metabarcoding. Simultaneously, we found that the infection with
intracellular endosymbiotic bacteria Wolbachia was significantly
negatively related with this effect. These results suggest that additional
attention should be paid to the interference of multiple mitochondrial
haplotypes within individual specimens on the results of DNA metabarcoding
in species diversity assessment of animals.