Abstract
Sea cucumber (bêche-de-mer, Echinodermata: Holothuroidea) is one of the top internationally traded seafood varieties. Besides its direct nutritional benefits, it is continuously used in the traditional medicine in different areas and cultures in the world. This world-wide interest triggered various issues related to stocks´ declining and risks of species extinction. For these reasons, the current study was designed to provide molecular tools for accurate discrimination between two sea cucumber species that prevail the Mediterranean of these echinoderms in Egypt, that are Holothuria polii and H. sanctori. The power of three gene markers, i.e., 16S rDNA, 28S rDNA, and Histone H3 in achieving accurate DNA-based identification, as well as elucidating clear phylogenetic and genetic diversity differences between those two species was assessed. Among the three genes, 16S rDNA showed the highest potentials as genetic and phylogenetic species discrimination marker. Both 28S rDNA and H3 exhibited the least number of holothuroid reference sequences in the GenBank database. For genetic diversity within each species population, 16S rDNA exhibited the best potentials, followed by H3. 28S rDNA showed no genetic polymorphism at all. Moreover, the collective data of both H3 and 16S rDNA suggested a possible role of asexual reproduction behavior in H. sanctori in the reduction of genetic diversity, as a possible response to overfishing. Hence, the current research can recommend the simultaneous application of both 16S rDNA and H3 as accurate markers for genetic discrimination among H. polii, H. sanctori and other different holothuroid species.
References
Ahmed, M.I. (2016) Induced spawning in the sea cucumber Holothuria atra from the Egyptian coast of the Red Sea. Egyptian Journal of Aquatic Biology and Fisheries, 20 (4), 9–15. https://doi.org/10.21608/ejabf.2016.11173
Ahmed, Q., Ali, Q.M. & Macdonald, A.H. (2021) Identification of Sea cucumber Holothuria (Lessonothuria) pardalis (Selenka, 1867) and Holothuria (Semperothuria) cinerascens (Brandt, 1835) (Family-Holothuriidae) based on morphological and mitochondrial DNA evidence and phylogenetic analysis from Karachi Coast, Pakistan. International Journal of Environment and Geoinformatics, 8 (1), 57–64. https://doi.org/10.30897/ijegeo.820617
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) Basic local alignment search tool. Journal of Molecular Biology, 215 (3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Arndt, A., Marquez, C., Lambert, P. & Smith, M.J. (1996) Molecular Phylogeny of Eastern Pacific Sea Cucumbers (Echinodermata: Holothuroidea) Based on Mitochondrial DNA Sequence. Molecular Phylogenetic and Evolution, 6, 425–437. https://doi.org/10.1006/mpev.1996.0091
Aydin, M. (2016) Sea cucumber (Holothuroidea) species of Turkey. Türk Denizcilik ve Deniz Bilimleri Dergisi, 2 (1), 49–58.
Bordbar, S., Anwar, F. & Saari, N. (2011) High-Value Components and Bioactives from Sea Cucumbers for Functional Foods-A Review. Marine Drugs, 9 (10), 1761–1805. https://doi.org/10.3390/md9101761
Borrero-Pérez, G.H., Perez-Ruzafa, A., Marcos, C. & Gonzalez-Wangueemert, M. (2009) The taxonomic status of some Atlanto-Mediterranean species in the subgenus Holothuria (Echinodermata: Holothuroidea: Holothuriidae) based on molecular evidence. Zoological Journal of the Linnean Society, 157 (1), 51–69. https://doi.org/10.1111/j.1096-3642.2009.00529.x
Borrero-Pérez, G.H., Gómez-Zurita, J., González-Wangüemert, M., Marcos, C. & Pérez-Ruzafa, A. (2010) Molecular systematics of the genus Holothuria in the Mediterranean and Northeastern Atlantic and a molecular clock for the diversification of the Holothuriidae (Echinodermata: Holothuroidea). Molecular Phylogenetics and Evolution, 57 (2), 899–906. https://doi.org/10.1016/j.ympev.2010.08.019
Borrero-Pérez, G.H. & Vanegas-González, M.J. (2019) Holothuria (Mertensiothuria) viridiaurantia sp. nov. (Holothuriida, Holothuriidae), a new sea cucumber from the Eastern Pacific Ocean revealed by morphology and DNA barcoding. ZooKeys, 893, 1–19. https://doi.org/10.3897/zookeys.893.36013
Bronstein, O. & Kroh, A. (2019) The first mitochondrial genome of the model echinoid Lytechinus variegatus and insights into Odontophoran phylogenetics. Genomics, 111 (4), 710–718. https://doi.org/10.1016/j.ygeno.2018.04.008
Bronstein, O., Kroh, A. & Haring, E. (2016) Do genes lie? Mitochondrial capture masks the Red Sea collector urchin’s true identity (Echinodermata: Echinoidea: Tripneustes). Molecular Phylogenetics and Evolution, 104, 1–13. https://doi.org/10.1016/j.ympev.2016.07.028
Byrne, M., Rowe, F. & Uthicke, S. (2010) Molecular taxonomy, phylogeny and evolution in the family Stichopodidae (Aspidochirotida: Holothuroidea) based on COI and 16S mitochondrial DNA. Molecular Phylogenetics and Evolution, 56 (3), 1068–1081. https://doi.org/10.1016/j.ympev.2010.04.013
Clouse, R., Janies, D. & Kerr, A.M. (2005) Resurrection of Bohadschia bivittata from B. marmorata (Holothuroidea: Holothuriidae) based on behavioral, morphological, and mitochondrial DNA evidence. Zoology, 108 (1), 27–39. https://doi.org/10.1016/j.zool.2004.07.007
Colgan, D.J., McLauchlan, A., Wilson, G.D.F., Livingston, S.P., Edgecombe, G.D., Macaranas, J., Cassis, G. & Gray, M.R. (1998) Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology, 46 (5), 419–437. https://doi.org/10.1071/ZO98048
Conand, C. (1996) Asexual reproduction by fission in Holothuria atra: Variability of some parameters in populations from the tropical Indo-Pacific. Oceanologica Acta, 19 (3–4), 209–216.
Dafni, J. (1983) A new subspecies of Tripneustes gratilla from the northern Red Sea (Echinodermata: Echinoidea: Toxopneustidae). Israel Journal of Ecology and Evolution, 32 (1), 1–12.
Darriba, D., Taboada, G.L., Doallo, R. & Posada, D. (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods, 9 (8), 772. https://doi.org/10.1038/nmeth.2109
Dolmatov, I.Y. (2014) New data on asexual reproduction, autotomy, and regeneration in holothurians of the Order Dendrochirotida. Russian Journal of Marine Biology, 40 (3), 228–232. https://doi.org/10.1134/S1063074014030031
Dupont, S., Ortega-Martínez, O. & Thorndyke, M. (2010) Impact of near-future ocean acidification on echinoderms. Ecotoxicology, 19 (3), 449–462. https://doi.org/10.1007/s10646-010-0463-6
Fernandes, T.J.R., Amaral, J.S. & Mafra, I. (2020) DNA barcode markers applied to seafood authentication: an updated review. Critical Reviews in Food Science and Nutrition, 61 (22), 3904–3935. https://doi.org/10.1080/10408398.2020.1811200
Fisher, W., Schneider, M. & Bauchot, M.L. (1987) Guide FAO d’Identification des Espèces pour les Besoins de la Pêche Méditerranée et Mer Noire – Zone de Pêche 37, Vol. 1. Végétaux et Invertébrés. Organisation des Nations Unies pour l’Alimentation et l’Agriculture. Available from: http://www.fao.org/docrep/009/x0170f/x0170f00.htm (accessed October 2021)
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299.
Funk, D.J. & Omland, K.E. (2003) Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annual Review of Ecology, Evolution, and Systematics, 34 (1), 397–423. https://doi.org/10.1146/annurev.ecolsys.34.011802.132421
Galal-Khallaf, A., Ardura, A., Mohammed-Geba, K., Borrell, Y.J. & Garcia-Vazquez, E. (2014) DNA barcoding reveals a high level of mislabeling in Egyptian fish fillets. Food Control, 46, 441–445. https://doi.org/10.1016/j.foodcont.2014.06.016
Gao, Q.-F., Wang, Y., Dong, S., Sun, Z. & Wang, F. (2011) Absorption of different food sources by sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea): evidence from carbon stable isotope. Aquaculture, 319 (1–2), 272–276. https://doi.org/10.1016/j.aquaculture.2011.06.051
Gilliland, P.M. (1993) The skeletal morphology, systematics and evolutionary history of holothurians. Special Papers in Palaeontolgy, 47, 1–147.
Harmon, M. (2005) The Position of the Ophiuroidea within the Phylum Echinodermata. Available from: https://scholarcommons.usf.edu/etd/2916 (Accessed October 2021).
Hart, M.W. & Podolsky, R.D. (2005) Mitochondrial DNA phylogeny and rates of larval evolution in Macrophiothrix brittlestars. Molecular Phylogenetics and Evolution, 34 (2), 438–447. https://doi.org/10.1016/j.ympev.2004.09.011
Hasan, M.H. (2019) Destruction of sea cucumber populations due to overfishing at Abu Ghosoun area, Red Sea. The Journal of Basic and Applied Zoology, 80 (1), 1–10. https://doi.org/10.1186/s41936-019-0074-6
Hoareau, T.B. & Boissin, E. (2010) Design of phylum‐specific hybrid primers for DNA barcoding: addressing the need for efficient COI amplification in the Echinodermata. Molecular Ecology Resources, 10 (6), 960–967. https://doi.org/10.1111/j.1755-0998.2010.02848.x
Honey-Escandon, M., Laguarda-Figueras, A. & Solis-Marin, F.A. (2012) Molecular phylogeny of the subgenus Holothuria (Selenkothuria) Deichmann, 1958 (Holothuroidea: Aspidochirotida). Zoological Journal of the Linnean Society, 165 (1), 109–120. https://doi.org/10.1111/j.1096-3642.2011.00794.x
Honey-Escandon, M. & Solis-Marin, F. (2018) A revision of Holothuria (Halodeima) kefersteinii (Selenka, 1867) and the revival of Holothuria inornata Semper, 1868 from sea cucumbers collected in Mexico and Central America. Zootaxa, 4377 (2), 151–177. https://doi.org/10.11646/zootaxa.4377.2.1
Ismail, S.H. (2013) DNA Barcoding, Phylogenetic and Systematics of Holothuroidea (sea Cucumbers) in Malaysian Waters Based on 16s RRNA and Cytochrome Oxidase I (COI) Genes. Available from: http://eprints.usm.my/46241/1/Siti%20Hasmah%20Binti%20Ismail24.pdf (accessed October 2021)
Kamarudin, K.R., Hashim, R. & Usup, G. (2010) Phylogeny of sea cucumber (Echinodermata: Holothuroidea) as inferred from 16S mitochondrial rRNA gene sequences. Sains Malaysiana, 39, 209–218.
Kamarudin, K.R., Rehan, M.M., Noor, H.M., Ramly, N.Z. & Rehan, A.M. (2016) 16S rRNA Barcoding technique for species identification of processed sea cucumbers from selected malaysian markets. Journal of Science and Mathematics Letters, 4, 10–23.
Kamarudin, K.R., Rehan, M.M. & Rehan, A.M. (2019) Species identification of processed sea cucumbers from Malaysian market based on concatenated gene sequences of mitochondrial rRNA genes. Maritime Technology and Research, 1 (2), 28–44. https://doi.org/10.33175/mtr.2019.146259
Kim, S.W., Kerr, A.M. & Paulay, G. (2013) Colour, confusion, and crossing: resolution of species problems in Bohadschia (Echinodermata: Holothuroidea). Zoological Journal of the Linnean Society, 168 (1), 81–97. https://doi.org/10.1111/zoj.12026
Lafay, B., Smith, A. & Christen, R. (1995) A combined morphological and molecular approach to the phylogeny of asteroids (Asteroidea: Echinodermata). Systematic Biology, 44 (2), 190–208. https://doi.org/10.2307/2413706
Lawrence, A.J., Ahmed, M., Hanafy, M., Gabr, H., Ibrahim, A. & Gab-Alla, A.A.F.A. (2005) Status of the sea cucumber fishery in the Red Sea-the Egyptian experience. FAO Fisheries Technical Paper, 79–90.
Letunic, I. & Bork, P. (2019) Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Research, 47 (W1), W256–W259. https://doi.org/10.1093/nar/gkz239
Littlewood, D.T. (1994) Molecular phylogenetics of cupped oysters based on partial 28S rRNA gene sequences. Molecular Phylogenetics and Evolution, 3 (3), 221–229. https://doi.org/10.1006/mpev.1994.1024
Littlewood, D.T.J., Smith, A.B., Clough, K.A. & Emson, R.H. (1997) The interrelationships of the echinoderm classes: Morphological and molecular evidence. Biological Journal of the Linnaean Society, 61 (3), 409–438. https://doi.org/10.1111/j.1095-8312.1997.tb01799.x
Maggi, C. & Gonzalez-Wanguemert, M. (2015) Genetic differentiation among populations in the Western Mediterranean Sea: potential effects from its fishery and current connectivity. Mediterranean Marine Science, 16 (3), 489–501. https://doi.org/10.12681/mms.1020
Mahdy, A.A., Omar, H.A., Nasser, S.A.M., Abd El-Wakeil, K.F. & Obuid-Allah, A.H. (2019) Community structure of Echinoderms in littoral zone of the Red Sea Coast of Egypt. Egyptian Journal of Aquatic Biology and Fisheries, 22 (5), 483–498. https://doi.org/10.21608/ejabf.2019.26814
Miller, A.K., Kerr, A.M., Paulay, G., Reich, M., Wilson, N.G., Carvajal, J.I. & Rouse, G.W. (2017) Molecular phylogeny of extant Holothuroidea (Echinodermata). Molecular Phylogenetics and Evolution, 111, 110–131. https://doi.org/10.1016/j.ympev.2017.02.014
Mortensen, T.H. (1943) A Monograph of the Echinoidea, Vol. 3, No. 2 Camarodonta. I. Orthopsidae, Glyphocyphidae, Temnopleuridae and Toxopneustidae-Atlas. CA Reitzel, Copenhagen, vii + 553 pp.
Moussa, R. & Wirawati, I. (2018) Observations on some biological characteristics of Holothuria polii and Holothuria sanctori from Mediterranean Egypt. International Journal of Fisheries and Aquatic Studies, 6 (3), 351–357.
O’Loughlin, P.M., Paulay, G., VandenSpiegel, D. & Samyn,Y. (2007) New Holothuria species from Australia (Echinodermata: Holothuroidea: Holothuriidae), with comments on the origin of deep and cool holothuriids. Memoirs of Museum Victoria, 64, 35–52. https://doi.org/10.24199/j.mmv.2007.64.5
Omran, N.E., Salem, H.K., Eissa, S.H., Kabbash, A.M., Kandeil, M.A. & Salem, M.A. (2020) Chemotaxonomic study of the most abundant Egyptian sea-cucumbers using ultra-performance liquid chromatography (UPLC) coupled to high-resolution mass spectrometry (HRMS). Chemoecology, 30 (1), 35–48. https://doi.org/10.1007/s00049-019-00296-y
Palumbi, S., Martin, A., Romano, S., McMillan, W.O., Stice, L. & Grabowski, G. (1991) The simple fool’s guide to PCR, Ver. 2.0. University of Hawaii, Honolulu, 47 pp.
Perseke, M., Bernhard, D., Fritzsch, G., Brümmer, F., Stadler, P.F. & Schlegel, M. (2010) Mitochondrial genome evolution in Ophiuroidea, Echinoidea, and Holothuroidea: insights in phylogenetic relationships of Echinodermata. Molecular Phylogenetics and Evolution, 56 (1), 201–211. https://doi.org/10.1016/j.ympev.2010.01.035
Purcell, S.W., Lovatelli, A., Vasconcellos, M. & Ye, Y. (2010) Managing sea cucumber fisheries with an ecosystem approach. FAO Fisheries and Aquaculture Technical Paper, 50. Available from: https://hdl.handle.net/10535/6237 (accessed October 2021)
Purcell, S.W., Mercier, A., Conand, C., Hamel, J.F., Toral-Granda, M.V., Lovatelli, A. & Uthicke, S. (2013) Sea cucumber fisheries: global analysis of stocks, management measures and drivers of overfishing. Fish and Fisheries, 14 (1), 34–59. https://doi.org/10.1111/j.1467-2979.2011.00443.x
Rambaut, A., Drummond, A.J., Xie, D., Baele, G. & Suchard, M.A. (2018) Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7. Systematic Biology, 67 (5), 901–904. https://doi.org/10.1093/sysbio/syy032
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., Larget, B, Liu, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61 (3), 539–542. https://doi.org/10.1093/sysbio/sys029
Roskov, Y., Ower, G., Orrell, T., Nicolson, D., Bailly, N., Kirk, P.M., Bourgoin, T., DeWalt, R.E., Decock, W., Nieukerken, E.van., Zarucchi, J. & Penev, L. (2019) Species 2000 & ITIS Catalogue of Life, 2019 Annual Checklist. Species 2000: Naturalis, Leiden, the Netherlands. ISSN 2405-884X. Available from: www.catalogueoflife.org/annual-checklist/2019 (accessed 12 October 3021)
Rozas, J., Ferrer-Mata, A., Sanchez-DelBarrio, J.C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S.E. & Sanchez-Gracia, A. (2017) DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Molecular Biology and Evolution, 34 (12), 3299–3302. https://doi.org/10.1093/molbev/msx248
Safari, R., Qasemi, S.A., Rezvani Gilkalaee, S. & Qafari, H. (2020) Population structure of Holothuria leucospilota in the north of Persian Gulf and Oman Sea using DNA sequencing method. Aquaculture Sciences, 7 (2), 42–51.
Sicuro, B., Piccinno, M., Gai, F., Cesarina, A.M., Danieli, A., Daprà, F. & Mioletti, S. (2012) Food Quality and Safety of Mediterranean Sea Cucumbers Holothuria tubulosa and Holothuria polii in Southern Adriatic Sea. Asian Journal of Animal and Veterinary Advances, 7 (9), 851–859. https://doi.org/10.3923/ajava.2012.851.859
Soliman, T., Fernandez-Silva, I. & Reimer, J.D. (2016a) Genetic population structure and low genetic diversity in the over-exploited sea cucumber Holothuria edulis Lesson, 1830 (Echinodermata: Holothuroidea) in Okinawa Island. Conservation Genetics, 17 (4), 811–821. https://doi.org/10.1007/s10592-016-0823-8
Soliman, T., Takama, O., Fernandez-Silva, I. & Reimer, J.D. (2016a) Extremely low genetic variability within and among locations of the greenfish holothurian Stichopus chloronotus Brandt, 1835 in Okinawa, Japan. PeerJ, 4, e2410.
https://doi.org/10.7717/peerj.2410
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30 (12), 2725–2729. https://doi.org/10.1093/molbev/mst197
Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22 (22), 4673–4680. https://doi.org/10.1046/j.1365-294X.2003.01954.x
Toral-Granda, V. (2008) Population status, fisheries and trade of sea cucumbers in Latin America and the Caribbean. Sea cucumbers. A global review of fisheries and trade. FAO Fisheries and Aquaculture Technical Paper, 516, 213–229.
Uthicke, S. & Benzie, J.A.H. (2003) Gene flow and population history in high dispersal marine invertebrates: mitochondrial DNA analysis of Holothuria nobilis (Echinodermata: Holothuroidea) populations from the Indo-Pacific. Molecular Ecology, 12 (10), 2635–2648. https://doi.org/10.1046/j.1365-294X.2003.01954.x
Vergara-Chen, C., Gonzalez-Wanguemert, M., Marcos, C. & Perez-Ruzafa, A. (2010) Genetic diversity and connectivity remain high in Holothuria polii (Delle Chiaje 1823) across a coastal lagoon-open sea environmental gradient. Genetica, 138 (8), 895–906. https://doi.org/10.1007/s10709-010-9472-x
Ward, R.D., Holmes, B.H. & O’Hara, T.D. (2008) DNA barcoding discriminates echinoderm species. Molecular Ecology Resources, 8, 1202–1211. https://doi.org/10.1111/j.1755-0998.2008.02332.x
Weber, A.A.T., Stöhr, S. & Chenuil, A. (2019) Species delimitation in the presence of strong incomplete lineage sorting and hybridization: Lessons from Ophioderma (Ophiuroidea: Echinodermata). Molecular Phylogenetics and Evolution, 131, 138–148. https://doi.org/10.1016/j.ympev.2018.11.014
Wen, J. & Zeng, L. (2014) Use of species-specific PCR for the identification of 10 sea cucumber species. Chinese Journal of Oceanology and Limnology, 32 (6), 1257–1263. https://doi.org/10.1007/s00343-015-4050-9
Yamana, Y., Hayashibara, T., Yamamoto, M. & Setiamarga, D.H. (2019) First observation of the “double-faced X-framed cup ossicle” extracted from a deep sea holothurian in Japan. Zoosymposia, 15 (1), 203–211. https://doi.org/10.11646/zoosymposia.15.1.21