Anim. Syst. Evol. Divers. Vol. 35, No. 3: 136-139, July 2019 https://doi.org/10.5635/ASED.2019.35.3.017
Short communication DNA Barcoding of the Endangered Species Ellobium chinense (Mollusca, Gastropoda, Ellobiidae) from Coastal Areas of South Korea
Chang Ho Yi1,2, Tae Won Jung3, Il-Hun Kim1, In-Young Cho1, Min-Seop Kim1, Moongeun Yoon1, Won Kim2,*
1National Marine Biodiversity Institute of Korea, Seocheon 33662, Korea 2School of Biological Science, College of Natural Science, Seoul National University, Seoul 08826, Korea 3Research Center for Endangered Species, National Institute of Ecology, Yeongyang 36531, 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. Keywords: DNA barcode, cytochrome c oxidase subunit I, marine endangered species, pulmonate
INTRODUCTION of South Korea. Additionally, we analyzed genetic distances among E. chinense individuals, and compared with an ellobiid Ellobium chinense (Pfeiffer, 1864) is a conoidal pulmonate species Auriculastra duplicata (Pfeiffer, 1854) (Gastropoda, gastropod mollusk (Fig. 1) that inhabits the intertidal and ter- Ellobiidae), which exhibits similar ecological and morpholog- restrial zones of estuaries and coastal regions. This species’ ical characteristics to those of E. chinense (see Lee and Lee, whole distribution range is known to be confined to the north- 2015; Yi et al., 2017). Unfortunately, there was no compatible western Pacific coasts of Asia, including China, Japan, and sequences of Ellobium species in public databases. South Korea (Yoo, 1976). In South Korea, it mostly inhabits [Permission for sampling, analysis, and storage of E. chin- saltmarshes along the western and southern coasts, where it ense was obtained from the Geum River Basin Environmental has been severely affected by human activities, such as land Office (Permit No. 2015-14), Saemangeum Regional Environ- reclamation and coastal development, during the last few de- mental Office (Permit No. 2015-09), Yeongsan River Basin En- cades (Lim et al., 2015). Therefore, this species is regarded as vironmental Office (Permit No. 2015-21), and Nakdong River a being in need of conservation in South Korea, and has been Basin Environmental Office (Permit No. 2015-18), as this registered as an endangered species by the Korean Govern- species is protected as a type of endangered wildlife by law.] ment (Ministry of Environment, 2016; Ministry of Oceans and Fisheries, 2017). Although genetic resources of endangered species are essential for the success of conservation strategies RESULTS AND DISCUSSION concerned with the preservation of their populations and hab- itat restoration, only limited genetic data have been published The COI sequences of E. chinense were obtained from five in public databases on them until recently. In this study, we coastal areas in South Korea: Seocheon (36°8ʹ23ʺN, 126°34ʹ reported the cytochrome c oxidase subunit 1 (COI) sequenc- 16ʺE), Gochang (35°31ʹ50ʺN, 126°35ʹ47ʺE), Haenam (34°24ʹ es of E. chinense specimens collected from five coastal areas 41ʺN, 126°38ʹ3ʺE), Yeosu (34°41ʹ42ʺN, 127°34ʹ49ʺE), and
This is an Open Access article distributed under the terms of the Creative *To whom correspondence should be addressed Commons Attribution Non-Commercial License (http://creativecommons.org/ Tel: 82-2-880-6695, Fax: 82-2-872-1993 licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, E-mail: [email protected] and reproduction in any medium, provided the original work is properly cited. eISSN 2234-8190 Copyright The Korean Society of Systematic Zoology DNA Barcode of Ellobium chinense
A
B
Fig. 1. Photographs of Ellobium chinense from Sacheon. A, Dorsal; B, Ventral (photo taken in July 2015).
Sacheon (35°2ʹ57ʺN, 128°0ʹ31ʺE). In total, 25 adult (4-6 cm ATY GAW ACT ATG GC-3ʹ), and Mmt00019f (5ʹ-ACA AAY in shell length) specimens were collected between March and CAY AAA GAT ATT GG-3ʹ) and Mmt00826r (5ʹ-ACA ATA August of 2015, with five individuals collected from each of AAM CCY AAA ATY CC-3ʹ). Amplified sequences were the sites. Morphological identification was performed based then aligned using Geneious 9.1.8 (Biomatters Ltd., Auck- on the description of this species given by Yoo (1976), and all land, New Zealand). Newly obtained 631 bp COI sequences specimens were deposited in the National Marine Biodiver- were registered in the GenBank nucleotide database (accession sity Institute of Korea (MABIK) (Seocheon, Korea). Voucher Nos. MK696944-696968). Pairwise genetic distances among numbers are given in Table 1. From the extracted genomic sequences were calculated in the MEGA X program (Kumar DNA, COI sequences were amplified with the following new- et al., 2018) using the Kimura two-parameter model (Kimura, ly designed two-primer sets: Mmt00002f (5ʹ-TGC GTT GGY 1980). The COI sequences of A. duplicata (NC036959) were TAT TYT CMA CAA A-3ʹ) and Mmt00811r (5ʹ-ATC CCA compared as outgroups to those of E. chinense. In our results,
Anim. Syst. Evol. Divers. 35(3), 136-139 137 Chang Ho Yi, Tae Won Jung, Il-Hun Kim, In-Young Cho, Min-Seop Kim, Moongeun Yoon, Won Kim 25 0.225 24 0.225 0.000 23 0.008 0.008 0.228 22 0.010 0.010 0.221 0.008 21 0.011 0.011 0.228 0.011 0.006 20 0.010 0.010 0.221 0.011 0.013 0.005 19 0.011 0.011 0.219 0.011 0.010 0.011 0.006 18 0.008 0.008 0.228 0.006 0.008 0.006 0.008 0.003 17 0.011 0.011 0.219 0.006 0.011 0.010 0.011 0.006 0.000 16 0.225 0.011 0.008 0.011 0.010 0.011 0.010 0.008 0.000 0.000 15 0.013 0.230 0.013 0.013 0.011 0.008 0.011 0.013 0.002 0.013 0.008 14 0.010 0.221 0.010 0.013 0.010 0.011 0.008 0.011 0.011 0.013 0.005 0.000 13 0.008 0.221 0.008 0.008 0.011 0.008 0.010 0.006 0.010 0.008 0.010 0.008 0.006 12 0.006 0.221 0.006 0.005 0.010 0.010 0.006 0.008 0.005 0.008 0.010 0.008 0.006 0.005 11 0.221 0.010 0.010 0.008 0.013 0.010 0.011 0.008 0.011 0.011 0.013 0.005 0.010 0.000 0.000 10 0.230 0.006 0.010 0.006 0.008 0.010 0.010 0.006 0.008 0.002 0.008 0.010 0.008 0.010 0.005 0.006 9 0.230 0.006 0.010 0.006 0.008 0.010 0.010 0.006 0.008 0.002 0.008 0.010 0.008 0.010 0.005 0.006 0.000 8 0.223 0.008 0.005 0.005 0.008 0.005 0.006 0.008 0.008 0.008 0.006 0.003 0.006 0.008 0.006 0.008 0.003 0.008 7 0.230 0.006 0.005 0.010 0.006 0.008 0.010 0.010 0.006 0.008 0.002 0.008 0.010 0.008 0.010 0.005 0.006 0.000 0.000 6 0.223 0.011 0.010 0.011 0.014 0.011 0.011 0.008 0.010 0.014 0.014 0.011 0.013 0.010 0.013 0.014 0.013 0.005 0.010 0.011 5 0.225 0.011 0.008 0.006 0.010 0.006 0.006 0.006 0.008 0.010 0.013 0.011 0.008 0.011 0.010 0.011 0.010 0.008 0.000 0.000 0.000 4 0.230 0.013 0.011 0.013 0.008 0.010 0.013 0.010 0.010 0.010 0.011 0.013 0.003 0.013 0.011 0.008 0.011 0.013 0.002 0.010 0.008 0.013 3 0.221 0.010 0.010 0.005 0.010 0.008 0.010 0.013 0.010 0.010 0.006 0.008 0.013 0.013 0.010 0.011 0.008 0.011 0.013 0.011 0.008 0.010 0.000 2 0.225 0.003 0.013 0.016 0.014 0.003 0.011 0.010 0.013 0.010 0.010 0.010 0.011 0.013 0.016 0.003 0.014 0.011 0.014 0.013 0.014 0.013 0.011 0.003 (MABIK). Bold numbers indicate no difference between specimens.
1 0.223 0.010 0.010 0.013 0.010 0.011 0.010 0.005 0.006 0.010 0.006 0.006 0.003 0.008 0.010 0.010 0.010 0.008 0.005 0.008 0.010 0.008 0.010 0.005 0.010 I I J I L F E E E P A B C K E H C D G N G G - O M M Haplotype Voucher No. Voucher MO00163733 MO00163122 MO00163264 MO00163265 MO00163266 MO00163268 MO00163267 MO00163115 MO00163114 MO00163113 MO00163117 MO00163116 MO00163230 MO00163231 MO00163232 MO00163233 MO00163234 MO00163157 MO00163158 MO00163159 MO00163160 MO00163161 MO00163118 MO00163119 MO00163120 MO00163121 NCBI No. NCBI NC036959 MK696968 MK696944 MK696945 MK696946 MK696948 MK696947 MK696951 MK696950 MK696949 MK696953 MK696952 MK696954 MK696955 MK696956 MK696957 MK696958 MK696959 MK696960 MK696961 MK696962 MK696963 MK696964 MK696965 MK696966 MK696967 2 3 1 5 4 8 7 6 9 26 25 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 No. Yeosu Haenam Location Sacheon Gochang Ganghwa Seocheon Species duplicata Ellobium chinense Auriculastra Auriculastra Specimens were deposited in National Marine Biodiversity Institute of Korea Specimens were deposited in National Marine Biodiversity Pairwise genetic distances among individuals, with their collecting locations, NCBI accession numbers, and specimen voucher numbers genetic distances among individuals, with their collecting locations, NCBI accession numbers, and specimen voucher Table 1. Pairwise
138 Anim. Syst. Evol. Divers. 35(3), 136-139 DNA Barcode of Ellobium chinense a total 16 haplotypes of the mtDNA COI sequences were Lee JS, Lee YS, 2015. A report of three unrecorded Ellobiid identified from the 25 individuals examined. Among the five species (Gastropoda, Eupulmonata) from Korea. Korean sampling sites, only specimens from Gochang did not share Journal of Malacology, 31:323-326. https://doi.org/10.9710/ any haplotypes with specimens from any other sites (Table 1). kjm.2015.31.4.323 The maximum intraspecific genetic variation was 1.6% among Lim SY, Lee CS, Kim MS, Yoo SH, 2015. The conservation val- ue of endangered marine species: the case of the Ellobium all E. chinense specimens, while the interspecific genetic dif- chinense. Journal of the Korean Society of Marine Envi- ference of E. chinense from A. duplicata ranged from 21.9 to ronment & Safety, 21:645-654. https://doi.org/10.7837/ko- 23.0%. In conclusion, the use of the mtDNA COI region of E. somes.2015.21.6.645 chinense was found to be appropriate for identifying this spe- Ministry of Environment, 2016. Wildlife Protection and Manage- cies and its related taxon due to their low intraspecific genetic ment Act [Internet]. Korea Ministry of Government Legisla- variations and high interspecific variations in this gene. In tion, Sejong, Accessed 10 Apr 2019,
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