DNA Barcoding of a Worldwide Colonial Ascidian, Diplosoma listerianum (Ascidiacea: Aplousobranchia: Didemnidae), from East Sea, Korea

2021·1.1 MB·

by  Taekjun Lee

Anim. Syst. Evol. Divers. Vol. 37, No. 2: 160-164, April 2021
https://doi.org/10.5635/ASED.2021.37.2.074
Short communication
DNA Barcoding of a Worldwide Colonial Ascidian,
Diplosoma listerianum (Ascidiacea: Aplousobranchia:
Didemnidae), from East Sea, Korea
Taekjun Lee, Sook Shin*
Department of Animal Biotechnology and Resource, Sahmyook University, Seoul 01795, Korea
ABSTRACT
Diplosoma listerianum (Milne Edwards, 1841) is a globally distributed species that encompasses the existence of
multiple cryptic species. In this study, partial sequences of cytochrome c oxidase subunit I (COI) from D. listerianum
in South Korea were determined and compared with COI sequences of known D. listerianum and other Diplosoma
species retrieved from GenBank. The results showed that Korean D. listerianum matched with clade A of D.
listerianum, which is a group distributed globally. These new DNA barcodes for Korean D. listerianum may be useful
in the identification of colonial ascidians, which is difficult to morphological identify.
Keywords: DNA barcoding, COI, colonial ascidian, Didemnidae, Diplosoma listerianum
INTRODUCTION and population genetic network structure (Pérez-Portela et al.,
2013). The COI gene has been shown to be extremely useful
Ascidians are among the most important introduced species and accurate for the detection of cryptic speciation and spe-
in the sea (Lambert, 2007). Populations of some introduced cies identification in ascidians (Turon et al., 2003; Tarjuelo et
ascidians clearly belong to a single species; in other cases, al., 2004; Rius and Teske, 2013). In this study, we obtained
cryptic speciation has been found: Botryllus schlosseri, Ciona partial sequences of COI from D. listerianum in South Korea
intestinalis, and Diplosoma listerianum (Bock et al., 2012; and compared it with known COI sequences of D. listeria-
Pérez-Portela et al., 2013; Brunetti et al., 2015). Among num and other Diplosoma species.
these, D. listerianum is truly cosmopolitan, recorded in the The colonies were collected from artificial materials in Juk-
tropical to temperate waters of the Pacific, Atlantic, and In- byeon harbor, South Korea (Fig. 1). All examined colonies
dian Oceans and the Mediterranean and North Seas (Kott, were deposited in a 95% ethyl alcohol solution at the Marine
2001). Analysis of D. listerianum revealed the existence of Biological Resource Institute of Sahmyook University. Total
multiple cryptic species worldwide, and identified a complex genomic DNA was extracted from a single zooid in a colony
geographic structure and multiple clades that occurred in in accordance with the DNeasy Blood & Tissue kit protocol
sympatry (clades A-D), based on the dataset of mitochondrial (Qiagen, Hilden, Germany). The partial sequences of COI
cytochrome c oxidase subunit I (COI) (Pérez-Portela et al., were amplified using a universal primer pair: dinF-Nux1R
2013). The results indicated that D. listerianum is a species (Brunetti et al., 2017). Polymerase chain reaction (PCR) was
complex of at least four evolutionarily distinct lineages; how- executed in a reaction volume of 20.0 μL AccuPower PCR
ever, they were unable to substantiate any clear diagnostic PreMix & Master Mix (Bioneer, Seoul, Korea) including 1.0
morphological character owing to the scarcity of candidate μL of each primer (10 mM) and 1.5 μL of DNA template (>50
characters (e.g., the lack of calcareous spicules in the tunic of ng/μL) using the following thermocycling profile: 94℃ for 3
this genus), and the high variability in the shape and color of min; 35 cycles of 94℃ for 30 s, 52℃ for 60 s, and 72℃ for
the colonies (Pérez-Portela et al., 2013). Among these, clade 60 s; and a final extension at 72℃ for 5 min. The pairwise
A of D. listerianum appears to have been introduced in differ- distance (p-distance) was calculated using the Kimura 2-pa-
ent biogeographical areas, as shown by phylogenetic analyses rameter model (Kimura, 1980) in MEGA 7.0 (Kumar et al.,
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: [phone], Fax: [phone]
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 Barcoding of Diplosoma listerianum
Fig. 1. Various color morph of Diplosoma listerianum from South Korea: A, B, Artificial materials (acrylic plate) covered with D. lies-
terianum in Jukbyeon harbor.
Table 1. Pairwise distance values (%) based on 516 bp mitochondrial COI sequences in this study, calculated using the Kimura
2-parameter method
Species Clade na 1 2 3 4 5 6 7 8 9 10 11 12 13 14
1 Diplosoma listerianum Korea 3 0.0
2 Ab 28 4.2 5.8
3 A.1b 8 12.6 12.2 1.3
4 Bb 3 18.9 9.4 19.2 5.2
5 Cb 4 23.3 23.0 22.3 23.0 4.1
6 Db 2 20.5 20.3 19.8 22.4 19.3 0.2
7 D. gelatinosa - 1 18.6 17.8 11.0 24.9 25.5 26.2 0.0
8 D. multifidum - 1 25.7 24.4 24.1 32.7 30.8 30.5 27.2 0.0
9 D. simile - 1 25.6 25.1 23.3 25.4 23.5 23.3 27.0 31.8 0.0
10 D. spongiforme - 1 26.5 26.0 21.5 23.0 26.3 22.9 26.8 32.6 22.7 0.0
11 D. swamiensis - 1 19.1 18.2 7.3 25.3 26.9 24.6 16.8 30.0 27.9 25.8 0.0
12 Diplosoma sp. - 1 17.7 17.7 16.7 18.1 18.4 18.3 21.7 26.4 20.3 20.2 21.4 0.0
13 Diplosoma sp. - 1 17.7 17.7 16.7 18.1 18.4 18.3 21.7 26.4 20.3 20.2 21.4 0.0 0.0
14 Didemnum vexillum - 1 33.1 32.6 29.3 32.3 36.3 35.1 31.8 38.9 29.3 38.7 34.8 29.0 29.0 0.0
aNumber of cytochrome c oxidase subunit I (COI) sequences in a clade.
bThe type of clade is according to Pérez-Portela et al. (2013).
2016). The best-fit model of nucleotide substitution for the Rho and Huh, 1984; Kott, 2001; Shenkar et al., 2021), col-
COI dataset was selected by jModelTest v. 2.1.1 (Darriba et lected colonial ascidians in Jukbyeon harbor were identified
al., 2012) using the Akaike Information Criterion for Maxi- as D. listerianum (Fig. 1). The newly obtained COI sequenc-
mum Likelihood (ML). The ML tree was constructed using es (867 bp) of D. listerianum were registered in GenBank
PhyML 3.0 (Guidon et al., 2010) under HKY I G model (accession No. MT983895-MT983897). The type of clade
for COI dataset, and which included Korean and GenBank of in this study was according to Pérez-Portela et al. (2013).
+ +
D. listerianum, and D. spongiforme as an outgroup. The p-distance values showed that D. listerianum could be
separated into four groups, with a different species in each
group (Table 1). The Korean D. listerianum were closer to
RESULTS AND DISCUSSION
clade A of D. listerianum, than other clades (clade A.1-D)
Based on previous morphological studies (Tokioka, 1970; (Table 1). The variation within the Korean and clade A were
Anim. Syst. Evol. Divers. 37(2), 160-164 161
Taekjun Lee, Sook Shin
Fig. 2. The maximum likelihood tree of Diplosoma listerianum based on the cytochrome c oxidase subunit I (COI) dataset (518 bp).
The Korean D. listerianum indicated with red letters. Bootstrap values (>50) are indicated on each node. The type of clade is
according to Pérez-Portela et al. (2013).
4.2% and this value is lower than the intraspecific varia- standing taxonomic relationship of Diplosoma species.
tion of clade A (5.8%) (Table 1). Among them, the varia-
tion within the Korean and closer group in clade A were
ORCID
the mean of 0.8% and the range of 0.0-3.4% (Appendix 1).
The variation within Korean and clade A.1-D were 12.6%,
Taekjun Lee: https://orcid.org/0000-0003-4407-7862
18.9%, 23.3%, and 20.5%, respectively, and which were
Sook Shin: https://orcid.org/0000-0003-1799-9466
close to interspecific variations within D. listerianum and
other Diplosoma (Table 1). Pérez-Portela et al. (2013) veri-
CONFLICTS OF INTEREST
fied that D. listerianum had high intraspecific variations, but
they did not found any distinct morphological differences
No potential conflict of interest relevant to this article was
between each clade. The ML tree revealed the same as the
p-distances result and Pérez-Portela et al. (2013) (Fig. 2). reported.
Among them, Korean D. listerianum was distinctly clus-
tered with clade A of D. listerianum, and this clade is clear- ACKNOWLEDGMENTS
ly divided into another clade of D. listerianum (Fig. 2). This
study contributes in identifying D. listerianum and under- This study was the project titled ‘Improvement of manage-
162 Anim. Syst. Evol. Divers. 37(2), 160-164
DNA Barcoding of Diplosoma listerianum
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17 1.9 8.7 9.4 7.8 7.6 8.0 7.8 7.8 8.3 8.2 8.2 8.0 8.5 2.1 9.8 0.8 0.3 0.3 0.1 0.1 0.3 0.6 0.3 0.4 6.6 1.2 0.7 1.6 0.9 8.2 8.5 6.6 3.6 3.1 3.5 6.1 6.3 6.3 1.5
1 1 1 1 1 1 1 2 2 1 2 2 2 2 1 1 1 2 2 2 1 1 1 3
16 8.0 8.5 3.7 2.8 1.5 2.1 9.7 9.2 9.5 0.2 9.7 9.9 0.4 9.9 8.3 2.6 3.6 2.5 2.0 2.3 2.8 3.1 2.8 2.9 0.1 4.5 3.6 3.1 4.6 3.3 0.8 0.8 8.3 5.4 5.3 6.2 8.8 7.4 7.4 2.8
1 1 1 1 1 1 1 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
15 0.2 8.3 8.8 3.9 3.0 1.7 2.4 9.9 9.4 9.7 0.4 9.9 0.2 0.6 0.2 8.5 2.8 3.8 2.8 2.3 2.5 3.1 3.3 3.0 3.2 0.4 4.8 3.9 3.3 4.8 3.6 1.1 1.1 8.6 5.7 5.5 6.5 9.1 7.7 7.7 3.1
1 1 1 1 1 1 1 1 1 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
14 0.2 9.9 8.7 8.7 0.4 1.1 0.2 9.5 0.8 0.8 1.1 0.2 0.8 1.1 9.9 1.1 8.9 1.8 1.8 2.3 2.3 2.1 2.1 1.8 2.1 1.2 0.2 8.0 4.5 2.4 3.6 2.7 0.4 0.4 7.0 2.5 4.3 6.3 7.7 8.0 8.0 1.8
1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
13 0.4 0.6 0.4 8.5 8.5 3.7 2.8 1.5 2.1 0.2 9.7 9.9 0.2 0.2 0.4 0.4 0.4 8.3 2.0 3.6 2.0 1.5 1.8 2.3 3.1 2.8 2.9 0.1 4.5 3.0 2.5 3.9 2.7 0.8 0.8 8.3 5.4 5.3 6.2 8.8 7.4 7.4 2.8
1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
12 0.4 9.9 0.6 0.4 8.0 8.5 3.2 2.8 1.5 2.1 9.7 9.2 9.5 0.2 9.7 9.9 0.4 9.9 8.3 2.0 3.6 2.0 1.5 1.8 2.3 3.1 2.8 2.3 9.5 4.0 3.0 2.5 3.9 2.7 0.2 0.2 8.3 5.4 5.3 6.5 8.8 7.4 7.4 2.8
1 1 1 1 1 1 1 1 2 1 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
11 9.5 9.9 0.6 9.7 9.5 8.0 8.0 0.4 1.1 9.7 9.0 0.2 0.2 0.4 9.7 0.2 0.4 9.5 0.4 8.2 1.3 1.3 1.8 1.8 1.6 1.6 1.3 1.6 1.2 0.1 8.0 4.4 2.7 3.9 3.0 0.4 0.4 6.9 2.4 4.3 5.7 7.1 7.7 7.7 1.8
1 1 1 1 1 1 1 1 1 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
10 8.7 1.1 1.1 9.2 0.8 0.6 7.3 7.8 4.3 3.4 2.1 2.8 9.0 8.5 8.7 0.8 9.0 9.2 1.1 9.2 7.6 2.1 3.1 2.0 1.6 1.8 2.3 2.6 2.3 2.6 9.8 4.8 3.6 3.3 4.8 3.6 0.5 0.5 8.0 5.0 5.8 6.5 8.3 7.7 7.7 2.5
1 1 1 1 1 1 1 1 2 1 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
d
etho 9 0.8 9.7 0.2 0.2 10.2 0.4 0.2 8.3 8.8 3.4 2.6 1.3 1.9 9.9 9.5 9.7 0.0 9.9 10.2 0.2 10.2 8.5 12.3 13.8 12.3 11.8 12.0 12.6 13.3 13.1 22.6 19.8 14.3 23.3 22.8 24.2 23.0 20.5 20.5 18.6 25.7 25.6 26.5 19.1 17.7 17.7 33.1
m
eter 8 0.0 0.8 9.7 0.2 0.2 10.2 0.4 0.2 8.3 8.8 3.4 2.6 1.3 1.9 9.9 9.5 9.7 0.0 9.9 10.2 0.2 10.2 8.5 12.3 13.8 12.3 11.8 12.0 12.6 13.3 13.1 22.6 19.8 14.3 23.3 22.8 24.2 23.0 20.5 20.5 18.6 25.7 25.6 26.5 19.1 17.7 17.7 33.1
m
a 2 2 1 9 4 4 4 6 4 5 0 6 8 5 1 2 7 9 2 2 4 4 4 8 5 1 5 0 3 8 6 3 9 1 5 6 0 5 3 8 8 8 4 8 8 3 0 0 5
ar 7 0. 0. 1. 9. 0. 0. 0. 0. 0. 8. 9. 3. 2. 1. 2. 0. 9. 9. 0. 0. 0. 0. 0. 8. 2. 4. 2. 2. 2. 2. 3. 3. 2. 0. 4. 3. 3. 4. 3. 0. 0. 8. 5. 5. 6. 9. 8. 8. 3.
p 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
-
ra 2 6 0.0 0.2 0.2 1.1 9.9 0.4 0.4 0.4 0.6 0.4 8.5 9.0 3.6 2.8 1.5 2.1 0.2 9.7 9.9 0.2 0.2 0.4 0.4 0.4 8.8 2.5 4.1 2.5 2.0 2.3 2.8 3.6 3.3 2.9 0.1 4.5 3.6 3.0 4.5 3.3 0.8 0.8 8.8 5.4 5.8 6.8 9.3 8.0 8.0 3.5
u 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 2 2 2 2 2 2 1 2 2 2 1 1 1 3
m
e Ki 5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
h
g t 0 0 0 2 2 1 9 4 4 4 6 4 5 0 6 8 5 1 2 7 9 2 2 4 4 4 8 5 1 5 0 3 8 6 3 9 1 5 6 0 5 3 8 8 8 4 8 8 3 0 0 5
sin 4 0. 0. 0. 0. 0. 1. 9. 0. 0. 10. 0. 0. 8. 9. 3. 2. 1. 2. 10. 9. 9. 0. 10. 10. 0. 10. 8. 12. 14. 12. 12. 12. 12. 13. 13. 22. 20. 14. 23. 23. 24. 23. 20. 20. 18. 25. 25. 26. 19. 18. 18. 33.
u
d 2 2 2 2 0 0 8 7 2 2 2 4 2 3 8 4 6 3 9 9 5 7 0 9 2 2 2 5 3 8 3 8 0 6 3 1 6 8 3 3 8 2 0 5 5 6 7 6 5 1 7 7 1
ate 3 0. 0. 0. 0. 0. 0. 0. 9. 0. 0. 10. 0. 0. 8. 8. 3. 2. 1. 1. 9. 9. 9. 0. 9. 10. 0. 10. 8. 12. 13. 12. 11. 12. 12. 13. 13. 22. 19. 14. 23. 22. 24. 23. 20. 20. 18. 25. 25. 26. 19. 17. 17. 33.
ul
y, calc 2 0.0 0.2 0.2 0.2 0.2 0.0 0.0 0.8 9.7 0.2 0.2 10.2 0.4 0.2 8.3 8.8 3.4 2.6 1.3 1.9 9.9 9.5 9.7 0.0 9.9 10.2 0.2 10.2 8.5 12.3 13.8 12.3 11.8 12.0 12.6 13.3 13.1 22.6 19.8 14.3 23.3 22.8 24.2 23.0 20.5 20.5 18.6 25.7 25.6 26.5 19.1 17.7 17.7 33.1
stud 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
s
hi s
16bp mitochondrial COI sequences in t Localilty Jukbyeon, Korea Jukbyeon, Korea Jukbyeon, Korea California, USA California, USA California, USA California, USA California, USA Worldwide: California, USA; Plymouth, UK; Cape Town, South Africa; Hout Bay, South Africa; Bastimentos Is., Panama; Santander Bay, Spain; Arenys, Spain; Coquimbo, Chile; Melbourne, Australia California, USA Worldwide: California, USA; Plymouth, UK; Cape Town, South Africa; Hout Bay, South Africa; Santander Bay, Spain; Arenys, Spain; Coquimbo, Chile; Antofagasta, Chile; Melbourne, Australia Worldwide: Washington, USA; Coquimbo, Chile; Antofagasta, Chile Worldwide: Washington, USA; Coquimbo, Chile Plymouth, UK Plymouth, UK Worldwide: Plymouth, UK; Cape Town, South Africa; Hout Bay, South Africa Port Alfred, South Africa Port Alfred, South Africa Port Alfred, South Africa Port Alfred, South Africa Santander Bay, Spain Santander Bay, Spain Coquimbo, Chile Coquimbo, Chile Worldwide: Coquimbo, Chile; BastimentoIs., Panama; Bacas Marina, Panama Coquimbo, Chile Coquimbo, Chile Coquimbo, Chile Coquimbo, Chile Arenys, Spain Arenys, Spain Port Alfred, South Africa Bacas Marina, Panama Bacas Marina, Panama Bacas Marina, Panama Bacas Marina, Panama Bacas Marina, Panama Bacas Marina, Panama Bacas Marina, Panama Kanagawa, Japan Kanagawa, Japan Kanagawa, Japan Hout Bay, South Africa Bastimentos Is., Panama Port Alfred, South Africa Port Alfred, South Africa Bastimentos Is., Panama Bastimentos Is., Panama
5
ed on Clade - - - A A A A A A A A A A A A A A A A A A A A A A A A A A A A A.1 A.1 A.1 A.1 A.1 A.1 A.1 A.1 B B B C C C C D D
s
a
b
e values(%) Accession No. MT983895 MT983896 MT983897 KY235585 KY235588 KY235590 KY235593 KF791867 KF791868 KF791869 KF791870 KF791871 KF791872 KF791873 KF791874 KF791875 KF791880 KF791881 KF791882 KF791883 KF791894 KF791895 KF791900 KF791901 KF791903 KF791904 KF791905 KF791906 KF791907 KF791908 KF791909 KF791884 KF791885 KF791886 KF791887 KF791888 KF791889 KF791890 KF791891 KF791896 KF791897 KF791898 KF791876 KF791877 KF791878 KF791879 KF791892 KF791893 KX650792 KX650796 KU221218 KF309624 KX650797 MH258861 MH258862 KM259616 nit I.
c u
n b
a u
Taekjun Lee, Sook Shin Appendix 1. Pairwise dist Species 1Diplosoma listerianum 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49D. gelatinosa 50D. multifidum 51D. simile 52D. spongiforme 53D. swamiensis 54Diplosoma sp. 55Diplosoma sp. 56Didemnum vexillum COI, cytochrome c oxidase s 164