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: 82-2-3399-1717, Fax: 82-2-3399-1762 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
RESULTS AND DISCUSSION group (Table 1). The Korean D. listerianum were closer to 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- fied that D. listerianum had high intraspecific variations, but CONFLICTS OF INTEREST they did not found any distinct morphological differences between each clade. The ML tree revealed the same as the No potential conflict of interest relevant to this article was 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 ment strategies on marine disturbing and harmful organisms lem. Journal of Experimental Marine Biology and Ecology, (No. 20190518)’ funded by the Ministry of Oceans and 342:3-4. https://doi.org/10.1016/j.jembe.2006.10.009 Fisheries, Korea. López-Legentil S, Legentil ML, Erwin PM, Turon X, 2015. Harbor networks as introduction gateways: contrasting dis- tribution patterns of native and introduced ascidians. Bio- REFERENCES logical Invasions, 17:1623-1638. https://doi.org/10.1007/ s10530-014-0821-z Pérez-Portela R, Arranz V, Rius M, Turon X, 2013. Cryptic spe- Bock DG, MacIsaac HJ, Cristescu ME, 2012. Multilocus genet- ciation or global spread? The case of a cosmopolitan marine ic analyses differentiate between widespread and spatially invertebrate with limited dispersal capabilities. Scientific Re- restricted cryptic species in a model ascidian. Proceedings ports, 3:3197. https://doi.org/10.1038/srep03197 of the Royal Society of London, Series B, 279:2377-2385. Rho BJ, Huh MK, 1984. A systematic study on the Ascidians in https://doi.org/10.1098/rspb.2011.2610 Korea. Journal of Korean Research Institute for Better Liv- Brunetti R, Gissi C, Pennati R, Caicci F, Gasparini F, Manni L, ing, Ewha Womans University, 33:99-136. 2015. Morphological evidence that the molecularly deter- Rius M, Teske PR, 2013. Cryptic diversity in coastal Austral- mined Ciona intestinalis type A and type B are different spe- asia: a morphological and mitonuclear genetic analysis of cies: Ciona robusta and Ciona intestinalis. Journal of Zoo- habitat-forming sibling species. Zoological Journal of the logical Systematics and Evolutionary Research, 53:186-193. Linnean Society, 168:597-611. https://doi.org/10.1111/zoj. https://doi.org/10.1111/jzs.12101 12036 Brunetti R, Manni L, Mastrototaro F, Gissi C, Gasparini F, 2017. Shenkar N, Gittenberger A, Lambert G, Rius M, Moreira da Fixation, description and DNA barcode of a neotype for Bot- Rocha R, Swalla BJ, Turon X, 2021. Ascidiacea World Da- ryllus schlosseri (Pallas, 1766) (Tunicata, Ascidiacea). Zoot- tabase. Botrylloides Milne Edwards, 1841 [Internet]. World axa, 4353:29-50. https://doi.org/10.11646/zootaxa.4353.1.2 Register of Marine Species, Accessed 29 Mar 2021,
Anim. Syst. Evol. Divers. 37(2), 160-164 163 Taekjun Lee, Sook Shin
Appendix 1. Pairwise distance values (%) based on 516 bp mitochondrial COI sequences in this study, calculated using the Kimura 2-parameter method
Species Accession No. Clade Localilty 1 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 49 50 51 52 53 54 55 56 References
1 Diplosoma listerianum MT983895 - Jukbyeon, Korea This study 2 MT983896 - Jukbyeon, Korea 0.0 This study 3 MT983897 - Jukbyeon, Korea 0.0 0.0 This study 4 KY235585 A California, USA 0.0 0.2 0.2 Unpublished 5 KY235588 A California, USA 0.0 0.2 0.2 0.0 Unpublished 6 KY235590 A California, USA 0.0 0.2 0.2 0.0 0.0 Unpublished 7 KY235593 A California, USA 0.0 0.2 0.2 0.0 0.0 0.0 Unpublished 8 KF791867 A California, USA 0.0 0.0 0.0 0.2 0.0 0.2 0.2 Perez-Portela et al. (2013) 9 KF791868 A Worldwide: California, USA; Plymouth, 0.0 0.0 0.0 0.2 0.0 0.2 0.2 0.0 Perez-Portela et al. (2013) UK; Cape Town, South Africa; Hout Bay, South Africa; Bastimentos Is., Panama; Santander Bay, Spain; Arenys, Spain; Coquimbo, Chile; Melbourne, Australia 10 KF791869 A California, USA 0.0 0.8 0.8 1.1 0.0 1.1 1.1 0.8 0.8 Perez-Portela et al. (2013) 11 KF791870 A Worldwide: California, USA; Plymouth, 0.0 9.7 9.7 9.9 0.0 9.9 9.9 9.7 9.7 8.7 Perez-Portela et al. (2013) UK; Cape Town, South Africa; Hout Bay, South Africa; Santander Bay, Spain; Arenys, Spain; Coquimbo, Chile; Antofagasta, Chile; Melbourne, Australia 12 KF791871 A Worldwide: Washington, USA; 0.0 0.2 0.2 0.4 0.0 0.4 0.4 0.2 0.2 1.1 9.5 Perez-Portela et al. (2013) Coquimbo, Chile; Antofagasta, Chile 13 KF791872 A Worldwide: Washington, USA; 0.0 0.2 0.2 0.4 0.0 0.4 0.4 0.2 0.2 1.1 9.9 0.4 Perez-Portela et al. (2013) Coquimbo, Chile 14 KF791873 A Plymouth, UK 0.0 10.2 10.2 10.4 0.0 10.4 10.4 10.2 10.2 9.2 0.6 9.9 10.4 Perez-Portela et al. (2013) 15 KF791874 A Plymouth, UK 0.0 0.4 0.4 0.6 0.0 0.6 0.6 0.4 0.4 0.8 9.7 0.6 0.6 10.2 Perez-Portela et al. (2013) 16 KF791875 A Worldwide: Plymouth, UK; Cape Town, 0.0 0.2 0.2 0.4 0.0 0.4 0.4 0.2 0.2 0.6 9.5 0.4 0.4 9.9 0.2 Perez-Portela et al. (2013) South Africa; Hout Bay, South Africa 17 KF791880 A Port Alfred, South Africa 0.0 8.3 8.3 8.5 0.0 8.5 8.5 8.3 8.3 7.3 8.0 8.0 8.5 8.7 8.3 8.0 Perez-Portela et al. (2013) 18 KF791881 A Port Alfred, South Africa 0.0 8.8 8.8 9.0 0.0 9.0 9.0 8.8 8.8 7.8 8.0 8.5 8.5 8.7 8.8 8.5 1.9 Perez-Portela et al. (2013) 19 KF791882 A Port Alfred, South Africa 0.0 3.4 3.4 3.6 0.0 3.6 3.6 3.4 3.4 4.3 10.4 3.2 3.7 10.4 3.9 3.7 8.7 9.2 Perez-Portela et al. (2013) 20 KF791883 A Port Alfred, South Africa 0.0 2.6 2.6 2.8 0.0 2.8 2.8 2.6 2.6 3.4 11.1 2.8 2.8 11.1 3.0 2.8 9.4 9.9 3.9 Perez-Portela et al. (2013) 21 KF791894 A Santander Bay, Spain 0.0 1.3 1.3 1.5 0.0 1.5 1.5 1.3 1.3 2.1 9.7 1.5 1.5 10.2 1.7 1.5 7.8 8.3 3.9 3.0 Perez-Portela et al. (2013) 22 KF791895 A Santander Bay, Spain 0.0 1.9 1.9 2.1 0.0 2.1 2.1 1.9 1.9 2.8 9.0 2.1 2.1 9.5 2.4 2.1 7.6 8.0 4.1 3.2 0.6 Perez-Portela et al. (2013) 23 KF791900 A Coquimbo, Chile 0.0 9.9 9.9 10.2 0.0 10.2 10.2 9.9 9.9 9.0 0.2 9.7 10.2 0.8 9.9 9.7 8.0 8.0 10.6 10.9 9.9 9.2 Perez-Portela et al. (2013) 24 KF791901 A Coquimbo, Chile 0.0 9.5 9.5 9.7 0.0 9.7 9.7 9.5 9.5 8.5 0.2 9.2 9.7 0.8 9.4 9.2 7.8 7.8 10.1 10.9 9.5 8.7 0.4 Perez-Portela et al. (2013) 25 KF791903 A Worldwide: Coquimbo, Chile; Bastimentos 0.0 9.7 9.7 9.9 0.0 9.9 9.9 9.7 9.7 8.7 0.4 9.5 9.9 1.1 9.7 9.5 7.8 7.8 10.4 10.6 9.7 9.0 0.2 0.2 Perez-Portela et al. (2013) Is., Panama; Bacas Marina, Panama 26 KF791904 A Coquimbo, Chile 0.0 0.0 0.0 0.2 0.0 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 Perez-Portela et al. (2013) 27 KF791905 A Coquimbo, Chile 0.0 9.9 9.9 10.2 0.0 10.2 10.2 9.9 9.9 9.0 0.2 9.7 10.2 0.8 9.9 9.7 8.2 8.2 10.6 11.4 9.9 9.2 0.4 0.4 0.6 9.9 Perez-Portela et al. (2013) 28 KF791906 A Coquimbo, Chile 0.0 10.2 10.2 10.4 0.0 10.4 10.4 10.2 10.2 9.2 0.4 9.9 10.4 1.1 10.2 9.9 8.2 8.2 10.9 11.1 10.2 9.5 0.2 0.6 0.4 10.2 0.6 Perez-Portela et al. (2013) 29 KF791907 A Coquimbo, Chile 0.0 0.2 0.2 0.4 0.0 0.4 0.4 0.2 0.2 1.1 9.5 0.4 0.4 9.9 0.6 0.4 8.0 8.5 3.7 2.8 1.5 2.1 9.7 9.2 9.5 0.2 9.7 9.9 Perez-Portela et al. (2013) 30 KF791908 A Arenys, Spain 0.0 10.2 10.2 10.4 0.0 10.4 10.4 10.2 10.2 9.2 0.4 9.9 10.4 1.1 10.2 9.9 8.5 8.5 10.8 11.6 10.2 9.4 0.6 0.6 0.8 10.2 0.6 0.6 9.9 Perez-Portela et al. (2013) 31 KF791909 A Arenys, Spain 0.0 8.5 8.5 8.8 0.0 8.8 8.8 8.5 8.5 7.6 8.2 8.3 8.3 8.9 8.5 8.3 2.1 0.6 8.9 9.7 8.0 7.8 8.2 8.0 8.0 8.5 8.5 8.5 8.3 8.7 Perez-Portela et al. (2013) 32 KF791884 A.1 Port Alfred, South Africa 0.0 12.3 12.3 12.5 0.0 12.5 12.5 12.3 12.3 12.1 11.3 12.0 12.0 11.8 12.8 12.6 9.8 9.6 13.8 13.8 12.0 11.8 11.3 11.1 11.1 12.3 11.6 11.6 12.0 11.8 9.4 Perez-Portela et al. (2013) 33 KF791885 A.1 Bacas Marina, Panama 0.0 13.8 13.8 14.1 0.0 14.1 14.1 13.8 13.8 13.1 11.3 13.6 13.6 11.8 13.8 13.6 10.8 10.1 15.3 15.3 13.6 13.3 11.3 11.1 11.1 13.8 11.6 11.6 13.6 11.8 10.3 1.9 Perez-Portela et al. (2013) 34 KF791886 A.1 Bacas Marina, Panama 0.0 12.3 12.3 12.5 0.0 12.5 12.5 12.3 12.3 12.0 11.8 12.0 12.0 12.3 12.8 12.5 10.3 10.1 13.8 13.8 12.0 11.8 11.8 11.6 11.6 12.3 12.1 11.8 12.0 11.8 9.8 0.4 2.3 Perez-Portela et al. (2013) 35 KF791887 A.1 Bacas Marina, Panama 0.0 11.8 11.8 12.0 0.0 12.0 12.0 11.8 11.8 11.6 11.8 11.5 11.5 12.3 12.3 12.0 10.3 10.1 13.3 13.3 11.5 11.3 11.8 11.6 11.6 11.8 12.1 12.1 12.0 12.3 9.8 0.4 2.4 0.4 Perez-Portela et al. (2013) 36 KF791888 A.1 Bacas Marina, Panama 0.0 12.0 12.0 12.3 0.0 12.3 12.3 12.0 12.0 11.8 11.6 11.8 11.8 12.1 12.5 12.3 10.1 9.8 13.5 13.5 11.8 11.5 11.6 11.3 11.3 12.0 11.8 11.8 11.8 12.0 9.6 0.2 2.1 0.2 0.2 Perez-Portela et al. (2013) 37 KF791889 A.1 Bacas Marina, Panama 0.0 12.6 12.6 12.8 0.0 12.8 12.8 12.6 12.6 12.3 11.6 12.3 12.3 12.1 13.1 12.8 10.1 9.8 14.0 14.0 12.3 12.0 11.6 11.3 11.3 12.6 11.8 11.8 12.3 12.0 9.6 0.2 2.1 0.6 0.6 0.4 Perez-Portela et al. (2013) 38 KF791890 A.1 Bacas Marina, Panama 0.0 13.3 13.3 13.6 0.0 13.6 13.6 13.3 13.3 12.6 11.3 13.1 13.1 11.8 13.3 13.1 10.3 10.1 14.8 14.8 13.1 12.3 11.3 11.1 11.1 13.3 11.6 11.6 13.1 11.8 9.8 1.3 2.8 1.7 1.7 1.5 1.5 Perez-Portela et al. (2013) 39 KF791891 A.1 Bacas Marina, Panama 0.0 13.1 13.1 13.3 0.0 13.3 13.3 13.1 13.1 12.3 11.6 12.8 12.8 12.1 13.0 12.8 10.6 10.3 14.5 14.5 12.8 12.0 11.6 11.3 11.3 13.1 11.8 11.8 12.8 12.0 10.1 1.5 3.0 1.5 1.5 1.3 1.7 0.2 Perez-Portela et al. (2013) 40 KF791896 B Kanagawa, Japan 0.0 22.6 22.6 22.9 0.0 22.9 22.9 22.6 22.6 22.6 21.2 22.3 22.9 21.2 23.2 22.9 20.3 20.9 22.0 23.7 22.6 22.6 21.2 20.9 20.9 22.6 21.5 21.2 22.3 21.2 20.6 18.9 19.8 19.0 19.5 19.2 18.7 20.0 20.3 Perez-Portela et al. (2013) 41 KF791897 B Kanagawa, Japan 0.0 19.8 19.8 20.1 0.0 20.1 20.1 19.8 19.8 19.8 20.1 19.5 20.1 20.2 20.4 20.1 20.4 20.9 19.3 20.9 20.7 20.7 20.1 19.8 19.8 19.8 20.4 20.1 20.1 20.1 20.6 19.8 20.6 19.3 19.3 19.5 19.5 20.9 20.6 2.1 Perez-Portela et al. (2013) 42 KF791898 B Kanagawa, Japan 0.0 14.3 14.3 14.5 0.0 14.5 14.5 14.3 14.3 14.8 18.0 14.0 14.5 18.0 14.8 14.5 16.6 18.0 14.0 15.5 15.3 15.6 18.0 17.7 17.7 14.3 18.3 18.0 14.5 18.0 17.7 17.9 19.0 17.4 17.4 17.7 17.7 19.0 18.7 8.0 5.7 Perez-Portela et al. (2013) 43 KF791876 C Hout Bay, South Africa 0.0 23.3 23.3 23.6 0.0 23.6 23.6 23.3 23.3 23.6 24.4 23.0 23.0 24.5 23.9 23.6 21.2 21.2 21.8 22.7 23.6 23.3 24.4 24.1 24.1 23.3 24.7 24.4 23.0 23.8 21.5 21.2 22.0 20.6 21.2 20.9 21.5 22.6 22.3 21.8 22.7 24.5 Perez-Portela et al. (2013) 44 KF791877 C Bastimentos Is., Panama 0.0 22.8 22.8 23.0 0.0 23.0 23.0 22.8 22.8 23.3 22.7 22.5 22.5 22.4 23.3 23.1 20.7 20.1 19.9 22.1 23.1 22.8 22.7 22.4 22.4 22.8 23.0 22.7 22.5 22.1 20.4 21.8 22.0 21.2 21.8 21.5 22.0 23.2 22.9 21.9 22.8 23.9 6.9 Perez-Portela et al. (2013) 45 KF791878 C Port Alfred, South Africa 0.0 24.2 24.2 24.5 0.0 24.5 24.5 24.2 24.2 24.8 23.9 23.9 23.9 23.6 24.8 24.6 21.6 21.3 21.3 23.3 24.6 24.2 23.9 23.6 23.6 24.2 24.2 23.9 23.9 23.3 21.6 23.2 23.5 22.6 23.2 22.9 23.5 24.7 24.4 22.2 23.1 24.3 7.4 1.3 Perez-Portela et al. (2013) 46 KF791879 C Port Alfred, South Africa 0.0 23.0 23.0 23.3 0.0 23.3 23.3 23.0 23.0 23.6 23.0 22.7 22.7 22.7 23.6 23.3 20.9 20.4 20.1 22.4 23.3 23.0 23.0 22.7 22.7 23.0 23.2 23.0 22.7 22.4 20.7 22.0 22.3 21.5 22.0 21.7 22.3 23.5 23.2 22.1 23.0 24.2 7.1 0.2 1.5 Perez-Portela et al. (2013) 47 KF791892 D Bastimentos Is., Panama 0.0 20.5 20.5 20.8 0.0 20.8 20.8 20.5 20.5 20.5 20.4 20.2 20.8 20.4 21.1 20.8 18.2 17.7 20.0 21.6 20.8 20.8 20.4 20.1 20.1 20.5 20.7 20.4 20.2 19.9 18.5 19.2 20.6 19.2 19.8 19.5 19.5 20.1 20.3 20.8 22.8 23.7 19.3 18.8 19.6 19.0 Perez-Portela et al. (2013) 48 KF791893 D Bastimentos Is., Panama 0.0 20.5 20.5 20.8 0.0 20.8 20.8 20.5 20.5 20.5 20.4 20.2 20.8 20.4 21.1 20.8 18.5 17.9 20.0 21.3 20.8 20.8 20.1 20.1 19.8 20.5 20.7 20.1 20.2 19.9 18.8 19.2 20.6 19.2 19.8 19.5 19.5 20.0 20.3 20.7 22.8 23.7 19.5 19.0 19.9 19.3 0.2 Perez-Portela et al. (2013) 49 D. gelatinosa KX650792 0.0 18.6 18.6 18.8 0.0 18.8 18.8 18.6 18.6 18.0 16.9 18.3 18.3 17.0 18.6 18.3 16.6 15.6 18.8 19.3 18.3 17.7 16.9 16.7 16.7 18.6 17.2 17.2 18.3 17.5 15.8 11.0 9.3 11.0 11.0 10.8 11.3 11.8 11.5 25.5 25.5 23.8 25.0 25.6 25.6 25.9 26.2 26.2 Unpublished 50 D. multifidum KX650796 0.0 25.7 25.7 25.4 0.0 25.4 25.4 25.7 25.7 25.0 22.4 25.4 25.4 22.5 25.7 25.4 23.6 22.7 26.3 24.8 25.4 25.1 22.7 22.1 22.4 25.7 22.7 22.7 25.4 22.2 23.6 23.9 23.3 24.2 24.5 24.2 24.2 23.9 24.2 32.8 34.0 31.4 30.7 31.0 30.4 31.3 30.5 30.5 27.2 Unpublished 51 D. simile KU221218 0.0 25.6 25.6 25.8 0.0 25.8 25.8 25.6 25.6 25.8 24.3 25.3 25.3 24.3 25.5 25.3 23.1 23.1 26.4 26.7 25.9 25.6 24.6 24.6 24.9 25.6 24.6 24.9 25.3 24.3 23.4 22.8 23.7 23.4 23.4 23.1 23.1 23.4 23.7 23.3 25.7 27.1 23.5 23.5 23.3 23.8 23.3 23.3 27.0 31.8 Da Silva Oliveira et al. (2017) 52 D. spongiforme KF309624 0.0 26.5 26.5 26.8 0.0 26.8 26.8 26.5 26.5 26.5 25.7 26.5 26.2 26.3 26.5 26.2 23.5 23.5 26.8 27.7 25.9 25.9 26.0 25.4 25.7 26.5 26.0 26.0 26.2 25.4 22.6 21.2 21.5 21.2 21.8 21.5 21.5 21.5 21.8 21.1 24.0 24.0 26.4 26.1 26.4 26.4 22.9 23.0 26.8 32.6 22.7 López-Legentil et al. (2015) 53 D. swamiensis KX650797 0.0 19.1 19.1 19.3 0.0 19.3 19.3 19.1 19.1 18.3 17.1 18.8 18.8 17.7 19.1 18.8 16.1 15.3 20.7 20.7 18.8 18.0 17.1 16.9 16.9 19.1 17.4 17.4 18.8 17.7 15.0 7.5 8.7 7.5 7.5 7.3 7.7 6.1 5.9 25.1 25.5 25.2 26.7 26.4 27.9 26.7 24.6 24.6 16.8 30.0 27.9 25.8 Unpublished 54 Diplosoma sp. MH258861 0.0 17.7 17.7 18.0 0.0 18.0 18.0 17.7 17.7 17.7 17.7 17.4 17.4 18.0 17.7 17.4 16.3 17.1 18.2 18.8 17.7 18.0 18.0 17.4 17.7 17.7 18.0 18.0 17.4 17.7 16.9 16.6 17.3 16.0 16.6 16.3 16.8 17.1 16.8 15.8 18.0 20.4 18.3 18.0 19.2 18.3 18.3 18.3 21.7 26.4 20.3 20.2 21.4 Unpublished 55 Diplosoma sp. MH258862 0.0 17.7 17.7 18.0 0.0 18.0 18.0 17.7 17.7 17.7 17.7 17.4 17.4 18.0 17.7 17.4 16.3 17.1 18.2 18.8 17.7 18.0 18.0 17.4 17.7 17.7 18.0 18.0 17.4 17.7 16.9 16.6 17.3 16.0 16.6 16.3 16.8 17.1 16.8 15.8 18.0 20.4 18.3 18.0 19.2 18.3 18.3 18.3 21.7 26.4 20.3 20.2 21.4 0.0 Unpublished 56 Didemnum vexillum KM259616 0.0 33.1 33.1 33.5 0.0 33.5 33.5 33.1 33.1 32.5 31.8 32.8 32.8 31.8 33.1 32.8 31.5 31.8 32.8 32.5 33.8 33.1 32.2 31.5 31.8 33.1 32.2 32.5 32.8 32.5 31.8 29.0 28.7 29.6 29.6 29.3 29.3 29.3 29.6 30.3 31.8 34.8 35.8 36.1 36.9 36.5 35.1 35.1 31.8 38.9 29.3 38.7 34.8 29.0 29.0 Smith et al. (2015)
COI, cytochrome c oxidase subunit I.
164 Anim. Syst. Evol. Divers. 37(2), 160-164