Reassessment of the mitochondrial 12S-rRNA gene for DNA barcoding of museum specimens of shelled marine gastropods from Japan

Nao Fukunaga,Naoto Tomobuchi,Shinnosuke Teruya,Satoko Nakashima,Moe Shimizu,Takenori Sasaki,Davin H E Setiamarga,Nazifa Naziha Razali

doi:10.1051/e3sconf/202132201028

Abstract

DNA barcoding is an effective and powerful tool for taxonomic identification and thus very useful for biodiversity monitoring. This study investigated the usefulness of the mitochondrial 12S-rRNA gene for the DNA barcoding of shelled marine gastropods. To do so, we determined partial 12S-rRNA sequences of 75 vouchered museum specimens from 69 species of shelled gastropods from Japan. The specimens have been identified morphologically, and natural history data catalog. Sequence analyses through BLAST searches, maximum likelihood phylogenetic analysis, and species delimitation analysis suggested that the 12S-rRNA gene is helpful for barcoding shelled marine gastropods. They thus could be helpful to complement barcoding studies using other markers such as COI. The analyses successfully confirmed all samples’ identity at higher taxonomy (subfamily and above), but much less so at the species level. Our result thus also underlines the lingering problem of DNA barcoding: The lack of comprehensive reference databases of sequences. However, since we provided sequences of properly curated, vouchered museum specimens in this study, our result reported here has thus also helped to give taxonomically reliable reference sequences for biodiversity monitoring and identifications of shelled gastropods which include many important fisheries species.

Highlights

With its ca. 35,000 of recorded species, Gastropoda, a class of shelled mollusks (Conchifera), is one of the most prominent invertebrate groups, in which, during its long evolutionary history, has radiated and occupied a diverse array of ecological niches in marine, freshwater, and biofouling/invasive organisms [2]
Our result presented here, which was based on BLAST searches, phylogenetic analysis, and species delimitation analysis, has confirmed the usefulness of the 12S-rRNA as a genetic marker for DNA barcoding of shelled gastropods, which include many fisheries species
For the 12S-rRNA gene marker, we successfully amplified and obtained ca. 450 bp for 12Sma / 12Smb primer pairs (60 samples) and ca. 430 bp of 12S97L / 12Smb primer pairs (15 samples), making us successfully obtain the 12S-rRNA sequences of all individuals used in this study

Summary

Introduction

With its ca. 35,000 of recorded species, Gastropoda, a class of shelled mollusks (Conchifera), is one of the most prominent invertebrate groups, in which, during its long evolutionary history, has radiated and occupied a diverse array of ecological niches in marine, freshwater, and biofouling/invasive organisms [2]. Recent development in DNA sequencing and the building up of DNA sequence databases have allowed for the usage of DNA sequences for the quick and effective identifications and classifications of samples collected from the field with relatively high accuracy, a method called “DNA barcoding” [5]. Further development of DNA sequencing technology (e.g., Generation Sequencing) has allowed for the development of a non-invasive method of biodiversity monitoring using DNA sequences, called the eDNA (environmental DNA = eDNA) method, which is essential barcoding of DNA fragments shed off into the environment by living organisms [6,7,8]. For DNA barcoding and e-DNA to work, the availability of a robust, reliable, and exhaustive reference database of DNA sequences collected from target taxa, is essential. At present, biases in biodiversity studies might have caused such a reliable database to be not available for some taxa, including gastropods Some taxa might become unidentifiable and become “dark taxa.”This becomes very problematic in monitoring studies, especially those conducted by people with inadequate taxonomical skills and resources, or if target taxa contain many possible undescribed or cryptic species, or when conducting eDNAbased monitoring for which morphological samples are unavailable

Results

Discussion

Conclusion