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Development of novel PCR primer sets for DNA barcoding of aquatic insects, and the discovery of some cryptic species

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DNA barcoding is a powerful tool that provides rapid, accurate, and automatable species identification using standardized genetic region(s), such as for revealing the existence of cryptic species and/or rare species in biodiversity monitoring. DNA barcoding techniques require the development of sets of universal PCR primers for DNA barcoding. We tried to develop universal primer sets, and succeeded in designing not only universal primer sets for DNA barcoding regions of almost all insects, which were designed to include a hypervariable site between highly conserved sites, but also primer sets for longer fragment sequences for registration in a database. We confirmed successful amplification for 14 orders, 43 families, and 68 species with DNA barcoding in the mtDNA 16S rRNA region, and for 13 orders, 42 families, and 66 species with DNA barcoding in the mtDNA 12S rRNA region, including Apterygota and Pterygota (Paleoptera, Polyneoptera, Paraneoptera, and Oligoneoptera). A key feature is that the DNA fragments of the DNA barcoding regions amplified by these primer sets are both short at about 200-bp, and longer fragment sequences will increase the level of data registration in the DNA database. In addition, we evaluated the sensitivity of these newly developed primers using Epeorus aesculus (Heptageniidae), which inhabits a relatively wide range of river systems. The results of this study revealed the existence of a cryptic species or an undescribed species. Such resulting database enhancements will provide opportunities for increasingly accurate assessment of biodiversity and genetic diversity.

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Data availability

All DNA sequences analysed in this study have been deposited in the public repository of GenBank systems. All methods information is included in this manuscript.


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We express our thanks to Dr. Y. Iwasaki (National Institute of Advanced Industrial Science and Technology), Dr. N. Uchida (Tohoku University), Dr. N. Kondo (National Institute of Environmental studies), Dr. Y. Hasebe (Kanagawa Prefectural Government) for valuable advice and encouragement. We are indebted to Tojo Lab. members (Shinshu University) and Niimi Lab. members (NIBB) for their cooperation with the field research and collection of specimens. This study was supported by Grants from the River Environment Fund (22-1215-021 to KT, 2017-5211-025 to KT, 2021-5311-005 to MT) of River and Watershed Environment Management, and was also partly supported by the River Works Technology Research and Development Program from Ministry of Land, Infrastructure, Transport and Tourism, Japan.

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MT designed, managed the study, and performed sample collection; MT, KY mainly performed laboratory work and phylogenetic analyses; MT, KY, TS designed Figures and Tables; MT, KT gathered funds; MT, KY, TS, KT wrote and reviewed the manuscript.

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Correspondence to Masaki Takenaka or Koji Tojo.

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The authors declare that they have no competing interests. The experiments comply with the current laws of the country in which they were performed.

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Handling Editor: Teruhiko Takahara.

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Takenaka, M., Yano, K., Suzuki, T. et al. Development of novel PCR primer sets for DNA barcoding of aquatic insects, and the discovery of some cryptic species. Limnology 24, 121–136 (2023).

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