MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species

Abstract

We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163-185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.

Keywords: MiSeq; community ecology; environmental DNA; metabarcoding; mitogenome; resource management.

Figure 1.

(a–d) Four tanks used for water sampling in the Okinawa Churaumi Aquarium and (e,f) a sampling site in the coral reefs near the aquarium: (a) Kuroshio (water volume =7500 m³); (b) tropical fish (700 m³); (c) deep-sea (230 m³); and (d) mangrove (35.6 m³) tanks; (e,f) sampling site in Bise (arrow; 26°42′35^′′ N, 127°52′48^′′ E) and the Okinawa Churaumi Aquarium (star; 26°41′39^′′ N, 127°52′41^′′ E).

Figure 2.

Schematic representation of the paired-end library preparation using a two-step tailed PCR. The workflow is derived from a document ‘16S metagenomic sequencing library preparation: preparing 16S ribosomal gene amplicons for the Illumina MiSeq system’ distributed by Illumina (part no. 15044223 Rev. B) and the figure was drawn with reference to a website of the Genomics and Sequencing Center at the University of Rhode Island (http://web.uri.edu/gsc/next-generation-sequencing/).

Figure 3.

Neighbour-joining trees of the seven species of tunas based on the amplified regions with multiplex PCR using MiFish-U (12S rRNA gene) and MiFish-tuna (ND5 gene) primers. Two species contained in the Kuroshio tank (yellowfin and Pacific bluefin) are highlighted in bold. Distances are calculated by using the Kimura's two-parameter model of base substitution with gaps being completely deleted. Numerals beside the internal branches are bootstrap probabilities based on 300 pseudo-replicates, and branch lengths are proportional to substitutions per site. Photos of the two tuna species are courtesy of H. Senou (Kanagawa Prefectural Museum of Natural History).

Figure 4.

Compositions of the non-tank species (with more than or equal to 97% sequence identity to reference sequences in the custom database) for eDNA from the four tanks in the Okinawa Churaumi Aquarium. Percentages in parentheses are based on the total number of reads with sequence identity of more than or equal to 97% (table 6). For classification of the non-tank species, see text.

Figure 5.

Temporal accumulation of the number of whole mitogenome sequences (ca 16 500 bp) curated in MitoFish and the MiFish sequences (ca 170 bp) in the custom database. The former data were taken from a change log recorded in MitoFish (http://mitofish.aori.u-tokyo.ac.jp/about/log.html).