DNA Barcoding of the Endangered Species Ellobium chinense (Mollusca, Gastropoda, Ellobiidae) from Coastal Areas of South Korea

Abstract

The pulmonate gastropod Ellobium chinense (Pfeiffer, 1864) is an endangered marine species along the South Korean coasts due to habitat destruction and population declines. We sequenced the cytochrome c oxidase subunit 1 (COI) of 25 E. chinense specimens collected from five coastal sites in South Korea, and identified 16 unique haplotypes. The maximum intraspecific variation among individuals was 1.6%, while interspecific differences from another ellobiid species, Auriculastra duplicata (Pfeiffer, 1854), ranged from 21.9 to 23.0%. Our barcoding data will be useful to elucidate the phylogenetic relationships among pulmonate gastropods and infer the population genetic structure of E. chinense.

INTRODUCTION

Ellobium chinense (Pfeiffer, 1864) is a conoidal pulmonate gastropod mollusk (Fig. 1) that inhabits the intertidal and terrestrial zones of estuaries and coastal regions. This species’ whole distribution range is known to be confined to the northwestern Pacific coasts of Asia, including China, Japan, and South Korea (Yoo, 1976). In South Korea, it mostly inhabits saltmarshes along the western and southern coasts, where it has been severely affected by human activities, such as land reclamation and coastal development, during the last few decades(Lim et al., 2015). Therefore, this species is regarded as a being in need of conservation in South Korea, and has been registered as an endangered species by the Korean Government(Ministry of Environment, 2016; Ministry of Oceans and Fisheries, 2017). Although genetic resources of endangered species are essential for the success of conservation strategies concerned with the preservation of their populations and habitat restoration, only limited genetic data have been published in public databases on them until recently. In this study, we reported the cytochrome c oxidase subunit 1 (COI) sequences of E. chinense specimens collected from five coastal areas of South Korea. Additionally, we analyzed genetic distances among E. chinense individuals, and compared with an ellobiid species Auriculastra duplicata (Pfeiffer, 1854) (Gastropoda, Ellobiidae), which exhibits similar ecological and morphological characteristics to those of E. chinense (see Lee and Lee, 2015; Yi et al., 2017). Unfortunately, there was no compatible sequences of Ellobium species in public databases.

Fig. 1. Photographs of Ellobium chinense from Sacheon. A, Dorsal; B, Ventral (photo taken in July 2015).

[Permission for sampling, analysis, and storage of E. chinense was obtained from the Geum River Basin Environmental Office (Permit No. 2015-14), Saemangeum Regional Environmental Office (Permit No. 2015-09), Yeongsan River Basin Environmental Office (Permit No. 2015-21), and Nakdong River Basin Environmental Office (Permit No. 2015-18), as this species is protected as a type of endangered wildlife by law.]

RESULTS AND DISCUSSION

The COI sequences of E. chinense were obtained from five coastal areas in South Korea: Seocheon (36°8ʹ23ʺN, 126°34ʹ 16ʺE), Gochang (35°31ʹ50ʺN, 126°35ʹ47ʺE), Haenam (34°24ʹ 41ʺN, 126°38ʹ3ʺE), Yeosu (34°41ʹ42ʺN, 127°34ʹ49ʺE), and Sacheon (35°2ʹ57ʺN, 128°0ʹ31ʺE). In total, 25 adult (4-6 cm in shell length) specimens were collected between March and August of 2015, with five individuals collected from each of the sites. Morphological identification was performed based on the description of this species given by Yoo (1976), and all specimens were deposited in the National Marine Biodiversity Institute of Korea (MABIK) (Seocheon, Korea). Voucher numbers are given in Table 1. From the extracted genomic DNA, COI sequences were amplified with the following newly designed two-primer sets: Mmt00002f(5ʹ-TGC GTT GGY TAT TYT CMA CAA A-3ʹ) and Mmt00811r (5ʹ-ATC CCA ATY GAW ACT ATG GC-3ʹ), and Mmt00019f(5ʹ-ACA AAY CAY AAA GAT ATT GG-3ʹ) and Mmt00826r (5ʹ-ACA ATA AAM CCY AAA ATY CC-3ʹ). Amplified sequences were then aligned using Geneious 9.1.8 (Biomatters Ltd., Auckland, New Zealand). Newly obtained 631 bp COI sequences were registered in the GenBank nucleotide database (accession Nos. MK696944-696968). Pairwise genetic distances among sequences were calculated in the MEGA X program (Kumar et al., 2018) using the Kimura two-parameter model(Kimura, 1980). The COI sequences of A. duplicata (NC036959) were compared as outgroups to those of E. chinense. In our results, a total 16 haplotypes of the mtDNA COI sequences were identified from the 25 individuals examined. Among the five sampling sites, only specimens from Gochang did not share any haplotypes with specimens from any other sites(Table 1). The maximum intraspecific genetic variation was 1.6% among all E. chinense specimens, while the interspecific genetic difference of E. chinense from A. duplicata ranged from 21.9 to 23.0%. In conclusion, the use of the mtDNA COI region of E. chinense was found to be appropriate for identifying this species and its related taxon due to their low intraspecific genetic variations and high interspecific variations in this gene. In addition, the relatively high haplotype diversity of this region will make it possible to analyze population genetic diversity and structure to help in establishing conservation strategies for E. chinense.

Table 1. Pairwise genetic distances among individuals, with their collecting locations, NCBI accession numbers, and specimen voucher numbers​​​​​​​