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

M Miya,W Iwasaki,T Sado,H Yamanaka,K Sato,M Kondoh,T Fukunaga,T Minamoto,Y Sato,J Y Poulsen,S Yamamoto,H Araki

doi:10.1098/rsos.150088

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.

Highlights

Environmental DNA in aquatic environments refers to genetic material found in the water column
Such ubiquitous presence of Environmental DNA (eDNA) from fishes in the water column has led to the increasing use of this technique as a tool for detections of invasive [3,7,8,9], rare or threatened species [5,6], investigations of local fauna [10,13], or in a larger mesocosm [12] with known species composition
In order to select a suitable region in the mitogenome for species identification based on eDNA, 1044 whole mitogenome sequences were batch downloaded from the database MITOFISH v. 2.80 [17] in a FASTA format as of 20 April 2013

Summary

Introduction

Environmental DNA (eDNA) in aquatic environments refers to genetic material found in the water column. EDNA from fishes has been detected from various aquatic environments, including ponds [3,4,5], streams [6], rivers [7,8,9,10] and seawater [11,12] Such ubiquitous presence of eDNA from fishes in the water column has led to the increasing use of this technique as a tool for detections of invasive [3,7,8,9], rare or threatened species [5,6], investigations of local fauna [10,13], or in a larger mesocosm [12] with known species composition. These pioneering studies have shown the use of eDNA to be appropriate as a non-invasive genetic monitoring tool in various fields of fish biology

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