ITS2 reference sequences improve species discrimination for some Lake Biwa fish

A reference-library study in the Lake Biwa basin found that the nuclear ribosomal ITS2 region separated some freshwater fish species more clearly than the mitochondrial MiFish region, while remaining insufficient for other closely related species. Researchers developed an ITS2 primer pair and assembled reference sequences for 63 of 84 target species, providing a regional resource for environmental DNA metabarcoding studies.

The work was a sequence-collection and sequence-comparison study, not a field validation of environmental DNA monitoring. The target list was based primarily on a comprehensive inventory of fish inhabiting the Lake Biwa basin. Mugil cephalus and Lateolabrax japonicus, distributed in the downstream Yodo River, were also included. Specimens came mainly from the Lake Biwa and Yodo River basins, although some species, including non-native species, were collected in Gifu and Kanagawa prefectures. Other specimens were obtained from the Lake Biwa Museum, freshwater fish researchers, aquarium shops and a fishery cooperative in Shiga Prefecture.

The forward primer, Fish_ITS2_F, was placed in the conserved 5.8S region, and the reverse primer, Fish_ITS2_R, was placed in the 28S region, allowing amplification of the intervening ITS2 sequence. The primers were designed manually from NCBI sequences aligned with MUSCLE, with target melting temperatures of approximately 60 °C. Forward-primer design used sequences from 113 species in 64 genera, 36 families and 12 orders, while reverse-primer design used sequences from 96 species in 58 genera, 34 families and 11 orders.

The researchers tested the primer pair with tissue-derived DNA from 12 fish species inhabiting Lake Biwa, and PCR products of the expected size were obtained for every tested species. Reported amplified lengths among taxa in the study area ranged from 298 base pairs for Channa argus to 481 base pairs for Micropterus nigricans. This laboratory test showed that the primers could amplify tissue DNA, but it did not assess their detection performance in environmental water samples.

For reference-library construction, genomic DNA was extracted from tissue or pelvic-fin samples representing the 84 target species. Amplicons were prepared for Illumina MiSeq sequencing, which produced 12,651,181 reads across two runs. The researchers obtained new ITS2 sequences for 61 species, represented by 291,855 reads. Together with previously available sequences for Cyprinus carpio and Plecoglossus altivelis altivelis, the collection covered 63 species, or 75% of the targets. Sequences were unavailable for 21 species because samples were unavailable for three species or sequencing quality was poor for 18 species.

Species distinguishability was assessed using BLAST percent identities and maximum-likelihood phylogenetic trees. The study used the MiFish identity threshold of 98.5% to define four categories. Identities at or below 98.5% and lower than the MiFish value were considered easily distinguishable; identities at or below the threshold but comparable to MiFish were considered distinguishable. Identities above 98.5% or substantially overlapping within-species values were treated as difficult to distinguish, and identical sequences were classified as indistinguishable.

Results differed among taxonomic groups. Oncorhynchus biwaensis had ITS2 identities 1.9–2.8 percentage points lower than its MiFish identities when compared with O. masou ishikawae and O. mykiss, allowing the study to classify O. biwaensis as easily distinguishable from them. The other two Oncorhynchus species were classified as distinguishable because their interspecific identities were no higher than 98.5% and were comparable to MiFish. In Rhinogobius, R. flumineus had ITS2 identities 1.0–3.0 percentage points lower than MiFish when compared with Rhinogobius sp. OM and R. biwaensis, making it easily distinguishable by the study’s criteria. However, Rhinogobius sp. OM and R. biwaensis exceeded 99.5% identity and overlapped with their within-species values, so they remained difficult to distinguish.

ITS2 also failed to resolve every species pair. Cobitis magnostriata and C. minamorii oumiensis shared identical ITS2 sequences and were classified as indistinguishable. Carassius sp. and C. buergeri grandoculis likewise shared identical sequences. The two BIWAE types of Cobitis exceeded 99.5% identity and overlapped with within-species values, while Carassius cuvieri had identities above 98.5% relative to Carassius sp. and C. buergeri grandoculis. These findings show that the marker’s discriminatory value was taxon-dependent rather than uniformly superior to MiFish.

The study has several bounded limitations. Reference coverage was incomplete, with sequences recovered for 63 of 84 targets, and only a small number of individuals contributed to some species comparisons. The analysis used tissue-derived reference DNA rather than a controlled comparison of environmental DNA detection, so it did not establish field sensitivity, false-detection rates or performance across habitats. The authors concluded that ITS2 can be effective for some species that are difficult to distinguish with MiFish and recommended combining both markers for broader biodiversity monitoring. They also called for field validation in the Lake Biwa basin and expansion of the reference database with additional species and individuals. The generated sequences were deposited in DDBJ, EMBL and GenBank under accession numbers LC876517–LC876631.

Subject of Research: ITS2 reference sequences for freshwater fish in the Lake Biwa basin

Article Title: Collection of ITS2 region reference sequences for freshwater fish in the Japanese archipelago for environmental DNA metabarcoding applications

Article References: Ito, G., Sasaki, D., Sakata, M. K., Kawase, S., Minamoto, T., & Yamanaka, H. (2026). Collection of ITS2 region reference sequences for freshwater fish in the Japanese archipelago for environmental DNA metabarcoding applications. Metabarcoding and Metagenomics, 10, Article e162895. https://doi.org/10.3897/mbmg.10.162895

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.162895

Keywords: environmental DNA, metabarcoding, ITS2, Lake Biwa, freshwater fish, MiFish, reference library, species discrimination, biodiversity monitoring, Collection, region, reference