Vol. XXIX Issue 2
Article 3

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><!-- [et_pb_line_break_holder] --><html xmlns="http://www.w3.org/1999/xhtml"><!-- [et_pb_line_break_holder] --><head><!-- [et_pb_line_break_holder] --><meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><!-- [et_pb_line_break_holder] --><title>Documento sin título</title><!-- [et_pb_line_break_holder] --></head><!-- [et_pb_line_break_holder] --><!-- [et_pb_line_break_holder] --><body><!-- [et_pb_line_break_holder] --><p align="right"><font size="3" face="Arial, Helvetica, sans-serif"><strong>ARTÍCULOS ORIGINALES</strong></font></p><!-- [et_pb_line_break_holder] --><p><font size="4" face="Arial, Helvetica, sans-serif"><strong>Morphological and molecular characterization of</strong> <!-- [et_pb_line_break_holder] --> <strong><em>Isostichopus </em>sp. in the Colombian Caribbean sea</strong></font></p><!-- [et_pb_line_break_holder] --><p><i><font size="3" face="Arial, Helvetica, sans-serif"><strong>Caracterización morfológica y molecular de Isostichopus</strong> <strong>sp. en el mar Caribe Colombiano</strong></font></i></p><!-- [et_pb_line_break_holder] --><p> </p><!-- [et_pb_line_break_holder] --><p><b><font size="3" face="Arial, Helvetica, sans-serif">Vergara, W.<SUP>1</sup>, Agudelo, V.<SUP>1</sup>, Castro, L.<SUP>2</sup>, R. Rodríguez A.<SUP>1*</sup>, Eeckhaut, I.<SUP>3</sup></font></b></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><SUP>1</sup> Aquaculture Laboratory, Grupo de Investigación y Desarrollo Tecnológico en Acuicultura, Fisheries Engineering Program. <!-- [et_pb_line_break_holder] --> Universidad del Magdalena. Cra. 32 No 22–08, Santa Marta, Colombia.<br /><!-- [et_pb_line_break_holder] --> <SUP>2</sup> Grupo de Investigación en Evolución, Sistemática y Ecología Molecular. Biology Program. <!-- [et_pb_line_break_holder] --> Universidad del Magdalena. Cra. 32 No. 22 - 08, Santa Marta, Magdalena.<br /><!-- [et_pb_line_break_holder] --> <SUP>3</sup> Laboratoire de Biologie des organismes marins et Biomimétisme, Umons, 7000 Mons, Belgique.<br /><!-- [et_pb_line_break_holder] --> * Corresponding author: <a href="mailto:arodriguezf@unimagdalena.edu.co">arodriguezf@unimagdalena.edu.co</a></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>Fecha de recepción</b>: 23/07/2018<br /><!-- [et_pb_line_break_holder] --> <b>Fecha de aceptación de versión final</b>: 07/12/2018</font></p><!-- [et_pb_line_break_holder] --><hr /><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>ABSTRACT</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><em>Isostichopus </em>sp. (Aspidochirotida: Stichopodidae) are sea cucumbers widely distributed in the Caribbean Sea. Amongst them, <em>Isostichopus</em><!-- [et_pb_line_break_holder] --> <em>badionotus </em>is one of the most harvested species. It shows a wide range of morphotypes widespread in the Caribbean region including different<!-- [et_pb_line_break_holder] --> habitats (muddy, sandy and rocky bottoms, and sea grass beds). In Colombia, three morphotypes can be distinguished; two of them live in sea grass<!-- [et_pb_line_break_holder] --> beds while the third one is found on rocky substrates. The present study describes the morphological characteristics of these morphotypes and<!-- [et_pb_line_break_holder] --> analyzes their genetic structures through 16S rDNA and COI data. Our phylogenetic analyses show that the morphotype living on rocky substrates<!-- [et_pb_line_break_holder] --> is morphologically and genetically distinct from the two other morphotypes and might not correspond to <em>I. badionotus</em>, the only species of the<!-- [et_pb_line_break_holder] --> <em>Isostichopus </em>genus previously reported for this region.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>Key words</b>: Cucumbers; <em>COI</em>;<em> 16S rDNA</em>;<em> Isostichopus badionotus</em></font>.</p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>RESUMEN</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><em>Isostichopus </em>sp. (Aspidochirotida:Stichopodidae) son pepinos de mar ampliamente distribuidos en el mar Caribe. Entre ellos, <em>Isostichopus badionotus</em><!-- [et_pb_line_break_holder] --> es una de las especies mas cosechadas. Muestra un amplio rango de morfotipos extendidos en la region caribena, incluyendo diferentes habitats<!-- [et_pb_line_break_holder] --> (fondos barrosos, arenosos y rocosos, y bancos de pastos marinos). En Colombia, se pueden distinguir tres morfotipos; dos de ellos viven en los<!-- [et_pb_line_break_holder] --> bancos de pastos marinos mientras que el tercero se halla sobre sustratos rocosos. En el presente estudio se describen las caracteristicas morfologicas<!-- [et_pb_line_break_holder] --> de esos morfotipos y se analizan sus estructuras geneticas mediante datos de 16S rDNA y COI. Nuestros analisis filogeneticos muestran que el<!-- [et_pb_line_break_holder] --> morfotipo que vive sobre sustratos rocosos es morfologica y geneticamente distinto de los otros dos morfotipos y puede no corresponder a <em>I.</em><!-- [et_pb_line_break_holder] --> <em>badionotus</em>, la unica especie del genero <em>Isostichopus </em>previamente reportada para esta region.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>Palabras clave</b>: Pepinos; <em>COI</em>;<em> 16S rDNA</em>;<em> Isostichopus badionotus</em></font>.</p><!-- [et_pb_line_break_holder] --><hr /><!-- [et_pb_line_break_holder] --><p> </p><!-- [et_pb_line_break_holder] --><p><b><font size="3" face="Arial, Helvetica, sans-serif">INTRODUCTION</font></b></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">Sea cucumbers are over-exploited in the three main<!-- [et_pb_line_break_holder] --> oceans, and started to be recently targeted in the Caribean<!-- [et_pb_line_break_holder] --> sea (Purcell <em>et al</em>., 2012). <em>Isostichopus badionotus </em>is one of the<!-- [et_pb_line_break_holder] --> sea cucumber species widespread in the Caribbean region<!-- [et_pb_line_break_holder] --> (Guzman <em>et al</em>., 2003), ranging from North Carolina to<!-- [et_pb_line_break_holder] --> northern Brazil. In addition, this species has been previously<!-- [et_pb_line_break_holder] --> reported in the east to the middle-Atlantic and in the Gulf<!-- [et_pb_line_break_holder] --> of Guinea (western Africa) (Hendler <em>et al.</em>, 1995). It is<!-- [et_pb_line_break_holder] --> found at depths from 0-55m (Miller and Pawson, 1990). <em>I.</em><!-- [et_pb_line_break_holder] --> <em>badionotus </em>is a common shallow water species that inhabits<!-- [et_pb_line_break_holder] --> rocky bottoms, as well as sea grass beds (Hendler <em>et al</em>.,<!-- [et_pb_line_break_holder] --> 1995). In Colombia, there is an illegal, unregulated and<!-- [et_pb_line_break_holder] --> unquantified fishery (Toral-Granda, 2008), and it is of<!-- [et_pb_line_break_holder] --> potential commercial interest in Florida, Puerto Rico and<!-- [et_pb_line_break_holder] --> the U.S. Virgin Islands (Bruckner, 2006).<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> At present, Colombian Caribbean Sea cucumbers<!-- [et_pb_line_break_holder] --> have not been well studied (Rodriguez <em>et al</em>., 2013;<!-- [et_pb_line_break_holder] --> Agudelo and Rodriguez, 2015) and there are many gaps<!-- [et_pb_line_break_holder] --> in their knowledge especially related to their taxonomic<!-- [et_pb_line_break_holder] --> characterization (Honey-Escandon <em>et al.</em>, 2012; Smirnov,<!-- [et_pb_line_break_holder] --> 2012). The advances in this regard allowed the reporting<!-- [et_pb_line_break_holder] --> of around 44 species, most of which have been captured<!-- [et_pb_line_break_holder] --> by exploratory fishing, and identified mainly as deep-sea<!-- [et_pb_line_break_holder] --> species (Caycedo, 1978; Borrero <em>et al</em>., 2003; Borrero-Perez<!-- [et_pb_line_break_holder] --> <em>et al</em>., 2012; Toral-Granda, 2008; Toral-Granda <em>et al</em>., 2008).<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> Since sea cucumbers are an important source of animal<!-- [et_pb_line_break_holder] --> proteins, studies in Colombia are beginning to develop<!-- [et_pb_line_break_holder] --> the conditions for their production under controlled<!-- [et_pb_line_break_holder] --> environments (Rodriguez <em>et al</em>., 2013; Agudelo and<!-- [et_pb_line_break_holder] --> Rodriguez, 2015; Vergara and Rodriguez, 2015; 2016).<!-- [et_pb_line_break_holder] --> In this way, part of the present team studied the gonad<!-- [et_pb_line_break_holder] --> morphology and the larval development of a species,<!-- [et_pb_line_break_holder] --> <em>Isostichopus </em>sp. aff <em>badionotus</em>, including its spawning period<!-- [et_pb_line_break_holder] --> (Agudelo-Martinez and Rodriguez-Forero, 2017).<!-- [et_pb_line_break_holder] --> According to Guzman and Guevara (2002) and Toral-<!-- [et_pb_line_break_holder] --> Granda (2008), there are three species of <em>Isostichopus</em><!-- [et_pb_line_break_holder] --> including <em>I. badionotus</em>, <em>I. fuscus</em>, and <em>I. macroparentheses</em><!-- [et_pb_line_break_holder] --> distributed in Central and South America. However, the<!-- [et_pb_line_break_holder] --> observation of various morphotypes inside the species<!-- [et_pb_line_break_holder] --> groups suggests the occurrence of more than three species<!-- [et_pb_line_break_holder] --> for this region. In Colombia, three morphotypes can be<!-- [et_pb_line_break_holder] --> distinguished in <em>I. badionotus</em>. The morphotypes studied<!-- [et_pb_line_break_holder] --> are similar because they all have a robust body, a mouth<!-- [et_pb_line_break_holder] --> located ventrally, twenty pelleted tentacles, anus in terminal<!-- [et_pb_line_break_holder] --> position, gonads in the form of tufts, podias or ambulatory<!-- [et_pb_line_break_holder] --> feet distributed in three rows, one or two poly vesicles,<!-- [et_pb_line_break_holder] --> all these, distinctive characteristics of the Stichopodidae<!-- [et_pb_line_break_holder] --> family. Preliminary phylogenetic analyses showed two<!-- [et_pb_line_break_holder] --> morphotypes that cluster together and one that is well<!-- [et_pb_line_break_holder] --> separated from the others. Two of them live in sea grass<!-- [et_pb_line_break_holder] --> beds while the third is found on rocky substrates. Also,<!-- [et_pb_line_break_holder] --> histological studies revealed a different structure of the<!-- [et_pb_line_break_holder] --> body wall. The present study describes the morphological<!-- [et_pb_line_break_holder] --> characteristics of these morphotypes and analyzes their<!-- [et_pb_line_break_holder] --> genetic structures through <em>16S </em>rDNA and <em>COI </em>data.<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> This research can be very useful to increase the<!-- [et_pb_line_break_holder] --> knowledge about the taxonomy status of some Colombian<!-- [et_pb_line_break_holder] --> sea cucumbers and to find out the morpho-anatomical and<!-- [et_pb_line_break_holder] --> genetic differences between species of genus <em>Isostichopus</em>,<!-- [et_pb_line_break_holder] --> from the native populations of the Santa Marta, Caribbean<!-- [et_pb_line_break_holder] --> Sea. This information will also be useful for conservation<!-- [et_pb_line_break_holder] --> and management purposes.</font></p><!-- [et_pb_line_break_holder] --><p><b><font size="3" face="Arial, Helvetica, sans-serif">MATERIAL AND METHODS</font></b></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Collection of animals</em><!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> During one year, two hundred sea cucumbers were<!-- [et_pb_line_break_holder] -->purchased from local artisanal fishermen in Rodadero<!-- [et_pb_line_break_holder] -->Bay (11° 13′ 22.73″ N - 74° 13′ 32.59″ W), Airport Bay<!-- [et_pb_line_break_holder] --> (11° 07′ 10″ N - 74° 13′ 50″ W), and Taganga Bay (11° <!-- [et_pb_line_break_holder] --> 16′ 03.4″ N - 74° 11’ 32.3″ W), located in Santa Marta,<!-- [et_pb_line_break_holder] --> Magdalena, Colombia (<a href="#fig1">Figure 1</a>) and transferred to the<!-- [et_pb_line_break_holder] --> Aquaculture Laboratory (Universidad del Magdalena,<!-- [et_pb_line_break_holder] --> Colombia). Most of the material was collected in shallow<!-- [et_pb_line_break_holder] --> waters (1 to 20 m depth). One hundred and thirty sea<!-- [et_pb_line_break_holder] --> cucumbers were collected between Rodadero and<!-- [et_pb_line_break_holder] --> Airport bays while seventy were obtained from the bay<!-- [et_pb_line_break_holder] --> of Taganga. Individuals from the three morphotypes were<!-- [et_pb_line_break_holder] --> weighed with an analytical scale Ohaus (0.001 g), and<!-- [et_pb_line_break_holder] --> their length was measured with a standard measuring<!-- [et_pb_line_break_holder] --> board (mm). Photographs of the dorsal and ventral sides<!-- [et_pb_line_break_holder] --> of the body were taken by using digital camera (Cannon<!-- [et_pb_line_break_holder] --> EOS, Rebel XTi). Besides the external and internal<!-- [et_pb_line_break_holder] --> anatomical observations, skin samples, tentacles and podia<!-- [et_pb_line_break_holder] --> were taken for reviewing ossicles. Samples were dissolved<!-- [et_pb_line_break_holder] --> in 10% sodium hypochlorite for 10 min and rinsed with<!-- [et_pb_line_break_holder] --> water as described by Lambert (1985), and examined<!-- [et_pb_line_break_holder] --> microscopically through slide preparations. For this, the<!-- [et_pb_line_break_holder] --> animals were anesthetized by immersion in sea water with<!-- [et_pb_line_break_holder] --> ice and then sacrificed. The characterization was made<!-- [et_pb_line_break_holder] --> by comparison with Tommasi (1969) and Purcell <em>et al.,</em><!-- [et_pb_line_break_holder] --> (2012). Our results showed that the specimens of the three<!-- [et_pb_line_break_holder] --> morphotypes came from two lineages of <em>Isostichopus </em>species<!-- [et_pb_line_break_holder] --> (<em>Isostichopus badionotus </em>and <em>Isostichopus </em>sp., characterized<!-- [et_pb_line_break_holder] --> in this report) that were recorded in the Center of<!-- [et_pb_line_break_holder] --> Biological Collections of Universidad de Magdalena (Santa<!-- [et_pb_line_break_holder] --> Marta, Magdalena, Colombia) (CBUMAG:ECH:00001,<!-- [et_pb_line_break_holder] --> CBUMAG:ECH:00002, CBUMAG:ECH:00003).<!-- [et_pb_line_break_holder] --> </font></p><!-- [et_pb_line_break_holder] --><p><a name="fig1" id="fig1"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig1.jpg" width="551" height="427" /><br /><!-- [et_pb_line_break_holder] --> Figure 1</b>. Marine and coastal areas of Colombia Caribbean Sea (modified by José Viillacob). Blue points in the<!-- [et_pb_line_break_holder] -->studied areas.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>DNA extraction, amplification and sequencing</em> <br /><!-- [et_pb_line_break_holder] --> Twenty-seven individuals were dissected and preserved in<!-- [et_pb_line_break_holder] --> absolute ethanol (99.5%). DNA extraction was performed<!-- [et_pb_line_break_holder] --> from up to 15 mg of muscle tissue using the DNeasy Blood &<!-- [et_pb_line_break_holder] --> Tissue Kit (QIAGEN). The quality of the DNA was verified<!-- [et_pb_line_break_holder] --> on a 1% agarose gel stained with GelRed (Biotium). PCR<!-- [et_pb_line_break_holder] --> amplification of the <em>COI </em>gene was performed using the<!-- [et_pb_line_break_holder] --> primers co1eF (5’-ATAATGATAGGAGGRTTTGG-3’)<!-- [et_pb_line_break_holder] --> and co1eR (5’-GCTCGTGTRTCTACRTCCAT-3’)<!-- [et_pb_line_break_holder] --> (Arndt <em>et al</em>., 1996), and of the <em>16S </em>gene with the<!-- [et_pb_line_break_holder] --> primers 16Sas (5’-CGCCTGTTTATCAAAAACAT-3’)<!-- [et_pb_line_break_holder] --> and 16Sbr (5’-CTCCGGTTTGAACTCAGATCA-3’).<!-- [et_pb_line_break_holder] --> PCR reactions were performed with 2 μL template<!-- [et_pb_line_break_holder] --> in a 25 μL volume with final concentrations of 2 mM<!-- [et_pb_line_break_holder] --> MgCl2, 5X PCR buffer (no MgCl2 BIOLINER), 0.4 μM<!-- [et_pb_line_break_holder] --> of each primer, 0.4 μM of each dNTP, and 2 units Taq<!-- [et_pb_line_break_holder] --> (BIOLASETM, BIOLINER). PCR amplifications were<!-- [et_pb_line_break_holder] --> performed under the following conditions: 1 min at 95<!-- [et_pb_line_break_holder] --> °C, followed by 35 cycles of 15 s at 95 °C, 1 min at 40 °C,<!-- [et_pb_line_break_holder] --> 1.5 min at 72 °C, and there was a final extension period<!-- [et_pb_line_break_holder] --> of 5 min at 72 °C. The PCR products were verified on a<!-- [et_pb_line_break_holder] --> 2% agarose gel stained with GelRed (Biotium). Following </font><font size="3" face="Arial, Helvetica, sans-serif">amplification, PCR products were purified using the<!-- [et_pb_line_break_holder] --> MACHEREY-NAGEL kit (NucleoSpinR Extract II), and<!-- [et_pb_line_break_holder] --> both strands were sequenced in both directions.<!-- [et_pb_line_break_holder] --></font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Sequence analyses and molecular phylogenetic analyses</em><br /> <!-- [et_pb_line_break_holder] --> Primer sequences were removed from the start and the end<!-- [et_pb_line_break_holder] --> of the obtained sequence and sequence ambiguities were<!-- [et_pb_line_break_holder] --> resolved by comparing the electropherograms using the<!-- [et_pb_line_break_holder] --> program BioEdit v. (Hall, 1999). After trimming,<!-- [et_pb_line_break_holder] --> forward and reverse sequences for each specimen were<!-- [et_pb_line_break_holder] --> assembled. Each assembled sequence was examined and<!-- [et_pb_line_break_holder] --> edited by hand, and each sequence was checked for stop<!-- [et_pb_line_break_holder] --> codons (in the case of the <em>COI </em>data file). Finally the<!-- [et_pb_line_break_holder] --> consensus sequence from each contig was verified using<!-- [et_pb_line_break_holder] --> the Blast tool on NCBI (www.ncbi.nlm.nih.gov). The<!-- [et_pb_line_break_holder] --> obtained sequences and downloaded sequences from<!-- [et_pb_line_break_holder] --> GenBank were aligned using the ClustalW algorithm<!-- [et_pb_line_break_holder] --> (Thompson <em>et al.</em>, 1997) in MEGA 6 (Tamura <em>et al</em>., 2013).<!-- [et_pb_line_break_holder] --> The downloaded sequences were, for the <em>COI </em>data set,<!-- [et_pb_line_break_holder] --> one sequence of <em>I. badionotus </em>(EU848276.1), one <em>COI</em> sequence of <em>Isostichopus </em>sp. (FJ971400.1), five sequences of <em>I.</em><!-- [et_pb_line_break_holder] --> <em>fuscus </em>(AF486424.1/28.1), three sequences of <em>S. herrmanni</em>,<!-- [et_pb_line_break_holder] --> and one sequence of <em>Holothuria leucospilota </em>(KC405565.1);<!-- [et_pb_line_break_holder] --> for the <em>16S </em>data set, one sequence of <em>I. macroparentesis</em> (AY338415.1), two sequences of <em>I. badionotus </em>(JN207495.1,<!-- [et_pb_line_break_holder] --> EU822435.1), six sequences of <em>I. fuscus </em>(AY153494.1,<!-- [et_pb_line_break_holder] --> AY153499.1), three sequences of <em>Stichopus herrmanni</em> (EU822451.1, EU856636.1, FJ223863.1), and one<!-- [et_pb_line_break_holder] --> sequence of <em>Holothuria leucospilota </em>(JQ657266.1). The <em>COI</em> final alignment was of 665 bp, and the <em>16S </em>final alignment<!-- [et_pb_line_break_holder] --> was of 508 bp. Sequences were submitted to GenBank<!-- [et_pb_line_break_holder] --> under accession numbers KX383967-KX384018.<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> Sequence divergences were calculated using the K2P<!-- [et_pb_line_break_holder] --> distance model (Kimura, 1980) following the barcoding<!-- [et_pb_line_break_holder] --> approach suggested by Hebert <em>et al</em>. (2003; 2004).<!-- [et_pb_line_break_holder] --> Phylogenetic analyses were made performing a Neighbour-<!-- [et_pb_line_break_holder] --> Joining (NJ) tree with a distance matrix generated by using<!-- [et_pb_line_break_holder] --> MEGA 6 (Tamura <em>et al</em>., 2013). Bootstrap values were<!-- [et_pb_line_break_holder] --> obtained by making 1,000 replicates. Bayesian analyses<!-- [et_pb_line_break_holder] --> were also performed using MrBayes (Ronquist and<!-- [et_pb_line_break_holder] --> Huelsenbeck, 2003), by considering the best substitution<!-- [et_pb_line_break_holder] --> models for each region using MrModelTest 3.7 (Nylander,<!-- [et_pb_line_break_holder] --> 2004). Two independent runs of 2,000,000 generations<!-- [et_pb_line_break_holder] --> were performed, with trees sampled every 100 generations,<!-- [et_pb_line_break_holder] --> 25% of the trees were discarded as burning. Convergence<!-- [et_pb_line_break_holder] --> was validated by the standard deviation of split frequencies<!-- [et_pb_line_break_holder] --> (<0.01), by plotting the likelihood values over time, and<!-- [et_pb_line_break_holder] --> by using the sump command in MrBayes. The percentage<!-- [et_pb_line_break_holder] --> of trees recovering a particular clade was used as a measure<!-- [et_pb_line_break_holder] --> of that clade’s posterior probability (Huelsenbeck and<!-- [et_pb_line_break_holder] --> Ronquist, 2001).</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><b>RESULTS</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Morphological analyses</em><!-- [et_pb_line_break_holder] --> <em><br /><!-- [et_pb_line_break_holder] --> Morphotype I</em><!-- [et_pb_line_break_holder] --> <em><br /><!-- [et_pb_line_break_holder] --> Description</em><!-- [et_pb_line_break_holder] --> Seventy<br /> <!-- [et_pb_line_break_holder] --> adults with average weight 232.4±44.7 g,<!-- [et_pb_line_break_holder] --> one specimen of 1 kg. Body is elongated, cylindrical,<!-- [et_pb_line_break_holder] --> completed in rounded ends. The species presents varieties<!-- [et_pb_line_break_holder] --> in body colorations (brown, reddish, yellow), size and in<!-- [et_pb_line_break_holder] --> the ending of the body edges (<a href="#fig2">Figure 2</a>).</font></p><!-- [et_pb_line_break_holder] --><p><a name="fig2" id="fig2"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig2.jpg" width="583" height="657" /><br /><!-- [et_pb_line_break_holder] --> Figure 2</b>. <em>Isostichopus badionotus. </em>a) Morphotype I. Typical morphotype, solid beige in the dorsal side, which is<!-- [et_pb_line_break_holder] --> covered with dark chocolate round warts. b) Morphotype II. 1) Orange sea cucumber. The dorsal side is<!-- [et_pb_line_break_holder] --> covered by orange warts that end in a dark color. 2) Dark sea cucumber surrounded by yellow round warts. 3)<!-- [et_pb_line_break_holder] -->Ambulacral feet organized in three rows with podias that end in a dark color.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"> Specimens have brown coloration and numerous<!-- [et_pb_line_break_holder] --> conical warts colouring brown. The dorsal side of the<!-- [et_pb_line_break_holder] --> body presents a variable number of rectangular shape<!-- [et_pb_line_break_holder] --> protuberances caused by depressions at the corners. This<!-- [et_pb_line_break_holder] --> morphotype has an organization similar to four rows of<!-- [et_pb_line_break_holder] --> low projections with small central spots that give it an<!-- [et_pb_line_break_holder] --> appearance of breasts. In the two dorso-lateral margins<!-- [et_pb_line_break_holder] --> there is only one row of warts and two more in the ventrolateral<!-- [et_pb_line_break_holder] --> body. The ambulacral feet are of brown color with<!-- [et_pb_line_break_holder] --> three lines of longitudinal podiums.<!-- [et_pb_line_break_holder] --> Calcareous ring composed of projections in the form<!-- [et_pb_line_break_holder] --> of pyramid and tower by way of a wheel, with a marked<!-- [et_pb_line_break_holder] --> invagination at the base, with two small mountains or<!-- [et_pb_line_break_holder] --> bumps on its outer edge. A respiratory tree and one or<!-- [et_pb_line_break_holder] --> two poly vesicles are present. No presence of tubules of<!-- [et_pb_line_break_holder] --> Cuvier. Gonads are placed in clusters, coloring whitecreamy,<!-- [et_pb_line_break_holder] --> depending on the maturity gonad stage. Mature<!-- [et_pb_line_break_holder] --> from July to November. Digestive tract is two times longer<!-- [et_pb_line_break_holder] --> than body length, cylindrical beige, which occupies three<!-- [et_pb_line_break_holder] --> times the length of their body.<!-- [et_pb_line_break_holder] --> Ossicles: Towers, canes, in the shape of s and c, in badges<!-- [et_pb_line_break_holder] --> perforated (<a href="#fig3">Figure 3</a>).</font></p><!-- [et_pb_line_break_holder] --><p><a name="fig3" id="fig3"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig3.jpg" width="545" height="520" /><br /><!-- [et_pb_line_break_holder] --> Figure 3</b>. <em>Isostichopus badionotus ossicles</em>. a) Perforated tables, b) larger plates, c), d) and e) C-shaped rods, f)<!-- [et_pb_line_break_holder] -->S-shaped rods.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Habitat (or ecology)<br /><!-- [et_pb_line_break_holder] --></em><!-- [et_pb_line_break_holder] --> These specimens have been found in Colombia Caribbean<!-- [et_pb_line_break_holder] --> Sea in Rodadero beach (11° 12′ 25.5″ N - 74° 13′ 54.8″<!-- [et_pb_line_break_holder] --> W), Airport beach (11° 09′ 20.2″ N - 74° 13′ 56.6″ W), and<!-- [et_pb_line_break_holder] --> mainly in Taganga bay (11° 15′ 53.0″ N - 74° 11′ 32.0″ W).<!-- [et_pb_line_break_holder] --> They inhabit sandy bottoms and have cryptic behavior. They<!-- [et_pb_line_break_holder] --> feed on particulate material that includes algae, bacteria and<!-- [et_pb_line_break_holder] --> marine sediment. Mature from July to November.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Morphotype II</em><!-- [et_pb_line_break_holder] --> <em><br /><!-- [et_pb_line_break_holder] --> Description</em><!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> Adults with average weight 470.4±162.8 g, one<!-- [et_pb_line_break_holder] --> specimen of 1 kg. Body is elongated, cylindrical, completed<!-- [et_pb_line_break_holder] --> in rounded ends. These specimens present varieties in<!-- [et_pb_line_break_holder] --> colorations (brown, reddish, yellow), size and in the ending<!-- [et_pb_line_break_holder] --> of the edges (<a href="#fig2">Figure 2</a>).<!-- [et_pb_line_break_holder] --> The body is light brown and on its surface has tiny pale<!-- [et_pb_line_break_holder] --> spots with small, almost imperceptible dark papillae. They<!-- [et_pb_line_break_holder] --> showed elongated body, cylindrical or fusiform, robust,<!-- [et_pb_line_break_holder] --> with blunt edges. The ventral region is flat and the dorsal</font> <font size="3" face="Arial, Helvetica, sans-serif">region is concave. The ventro-lateral margin is devoid of<!-- [et_pb_line_break_holder] --> fleshy projections. The podiums are dark coffee arranged<!-- [et_pb_line_break_holder] --> in three longitudinal rows, the middle one being thicker<!-- [et_pb_line_break_holder] --> than those of the lateral ones. They have dark brown<!-- [et_pb_line_break_holder] --> coloration with tiny conical pinnules, which colouring<!-- [et_pb_line_break_holder] --> reddish-brown, whose endings are yellow in colour (<a href="#tab1">Table<!-- [et_pb_line_break_holder] --> 1</a>). The mouth position is ventral and the anus, terminal.<!-- [et_pb_line_break_holder] --> Eighteen to twenty peltates tentacles and anal opening<!-- [et_pb_line_break_holder] --> without tooths. Presence of tube feet arranged in three<!-- [et_pb_line_break_holder] --> rows, which become six rows when observed in the water.<!-- [et_pb_line_break_holder] -->Their terminal colour is brown (<a href="#fig2">Figure 2</a>).</font></p><!-- [et_pb_line_break_holder] --><p><a name="tab1" id="tab1"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 1</b>. Comparative characters among sea cucumber species <em>Habitat (or ecology)</em> These specimens have been found in the Colombia<!-- [et_pb_line_break_holder] --> Caribbean Sea, mainly in Taganga bay (11° 15′ 53.0″ N<!-- [et_pb_line_break_holder] --> - 74° 11′ 32.0″ W), inhabiting sandy bottoms. They feed<!-- [et_pb_line_break_holder] --> on particulate material that includes algae, bacteria and<!-- [et_pb_line_break_holder] -->marine sediment. Mature from July to November.</font><br /><!-- [et_pb_line_break_holder] --><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04tab1.jpg" width="521" height="453" /></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">Calcareous ring composed of projections in the form<!-- [et_pb_line_break_holder] --> of pyramid and tower by way of a wheel, with a marked<!-- [et_pb_line_break_holder] --> invagination at the base, with two small mountains or<!-- [et_pb_line_break_holder] --> bumps on its outer edge. A respiratory tree and one or<!-- [et_pb_line_break_holder] --> two poly vesicles without tubules of Cuvier are present.<!-- [et_pb_line_break_holder] --> Gonads are placed in clusters, coloring white-creamy,<!-- [et_pb_line_break_holder] --> depending on the maturity gonad stage. Mature from July<!-- [et_pb_line_break_holder] --> to November. Digestive tract long, three times longer than<!-- [et_pb_line_break_holder] --> body length cylindrical beige, which occupies three times<!-- [et_pb_line_break_holder] --> the length of their body.<!-- [et_pb_line_break_holder] --> Ossicles: Towers, canes, in the shape of s and c, in badges<!-- [et_pb_line_break_holder] --> perforated (<a href="#fig3">Figure 3</a>).</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Morphotype III</em><!-- [et_pb_line_break_holder] --> <em><br /><!-- [et_pb_line_break_holder] --> Description</em><!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> Adults with average weight around 192.9±61.6 g.<!-- [et_pb_line_break_holder] --> Body is elongated, cylindrical, completed in blunt ends.<!-- [et_pb_line_break_holder] --> The species presents varieties in colorations in the dorsal<!-- [et_pb_line_break_holder] --> region, which are cream-yellow with irregular spots: coffee,<!-- [et_pb_line_break_holder] --> orange or reddish. Its ventral region and the tube feet are<!-- [et_pb_line_break_holder] --> beige (<a href="#fig4">Figure 4</a>) (<a href="#tab1">Table 1</a>).<!-- [et_pb_line_break_holder] --> </font></p><!-- [et_pb_line_break_holder] --><p><a name="fig4" id="fig4"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig4.jpg" width="524" height="317" /><br /><!-- [et_pb_line_break_holder] --> Figure 4</b>. New proposed species. a) Variations in irregular spots. Trapezoidal-shaped body. 1cm bar scale. b). Variations in<!-- [et_pb_line_break_holder] --> body color. c) Close view of the peltates tentacles of <em>Isostichopus isabellae n </em>sp. d) Peltates tentacles contracted<!-- [et_pb_line_break_holder] --> of the new proposed species. Lateral view. e) Ambulacral feet organized in three rows (arrows) with most of the<!-- [et_pb_line_break_holder] -->podia ending in a cream color rather than in black (white line). Mouth (♦), pinules (*).</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">Mouth is ventral with 16-20, peltate tentacles, anus is<!-- [et_pb_line_break_holder] --> terminal without teeth (<a href="#fig4">Figure 4</a>). Presence of ambulacral<!-- [et_pb_line_break_holder] --> feet arranged in three rows. The dorsal region was slightly<!-- [et_pb_line_break_holder] --> curved while the ventro-lateral margin of the wall was<!-- [et_pb_line_break_holder] --> thick and provided with extensions called papillae.<!-- [et_pb_line_break_holder] --> The calcareous ring has a pyramid form and tower with<!-- [et_pb_line_break_holder] --> two small bumps on its top edge. Presence of respiratory<!-- [et_pb_line_break_holder] --> tree. Presence of poly (1-2) vesicles without tubules of<!-- [et_pb_line_break_holder] --> Cuvier. Gonads arranged in clusters, beige, green and<!-- [et_pb_line_break_holder] --> yellow-greenish coloration, depending on the maturity<!-- [et_pb_line_break_holder] --> gonad stage. Female gonads tubules reaching a diameter<!-- [et_pb_line_break_holder] --> of 405.07±248.19 μm. Male gonads with thinner tubules<!-- [et_pb_line_break_holder] --> which diameter was around of 272.18±56.37 μm. Mature<!-- [et_pb_line_break_holder] --> oocyte diameter: 125.24±13.11 μm. Cuvierian organ<!-- [et_pb_line_break_holder] --> absent. Long, cylindrical digestive tube of beige colour, with</font> <font size="3" face="Arial, Helvetica, sans-serif">reddish pharynx, twice the length of its body (<a href="#fig5">Figure 5</a>).<!-- [et_pb_line_break_holder] --> </font></p><!-- [et_pb_line_break_holder] --><p><a name="fig5" id="fig5"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig5.jpg" width="451" height="319" /><br /><!-- [et_pb_line_break_holder] --> <b>Figure 5</b>. Internal morphology of the new specie proposed. 1) Pigmented polian vesicle; 2) Respiratory<!-- [et_pb_line_break_holder] -->tree; 3) Gonads; 4) Intestine; 5) Internal muscle; 6) Skin.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">Ossicles: tables terminated in a crown, perforated by<!-- [et_pb_line_break_holder] --> four central holes, c and s shaped rods, rounded and larger<!-- [et_pb_line_break_holder] --> plates whit several holes (<a href="#fig6">Figure 6</a>).</font></p><!-- [et_pb_line_break_holder] --><p><a name="fig6" id="fig6"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig6.jpg" width="526" height="306" /><br /><!-- [et_pb_line_break_holder] --> <b>Figure 6</b>. Ossicles of the dorsal body wall of the new specie proposed. a) Perforated tables with a single central hole, four<!-- [et_pb_line_break_holder] --> smaller peripheral holes that end in crown as towers b) Rod with small holes, c) Rod of tentacles d) C-shaped rod,<!-- [et_pb_line_break_holder] --> e) Perforate round plates of the dorsal body wall, f) Panoramic view of ossicles samples: very small tables, larger<!-- [et_pb_line_break_holder] -->plates, rods some with holes.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Habitat (or ecology)</em><!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> It inhabits rocky and sandy and feeds on particulate<!-- [et_pb_line_break_holder] -->material that includes algae, bacteria and marine sediment.<!-- [et_pb_line_break_holder] -->The species have been found in Rodadero beach, Airport<!-- [et_pb_line_break_holder] -->beach, Taganga bay. Gonochoric species with bifurcated<!-- [et_pb_line_break_holder] -->gonad attached to dorsal mesentery. Mature from July to<!-- [et_pb_line_break_holder] -->November.<!-- [et_pb_line_break_holder] --></font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Remarks</em><br /> <!-- [et_pb_line_break_holder] --> Most of them are smaller in size than Morphotype I<!-- [et_pb_line_break_holder] --> and II. They have a trapezoidal-shaped body, which is thin<!-- [et_pb_line_break_holder] --> and rigid. They show dorsal irregular spots and different<!-- [et_pb_line_break_holder] --> colours that varies from coffee, to red, orange and beige,<!-- [et_pb_line_break_holder] --> depending on the individual. Conical pinnules. Ossicles<!-- [et_pb_line_break_holder] --> are similar to that other <em>Isostichopus</em>. Seasonal spawning<!-- [et_pb_line_break_holder] --> from July to November. </font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Sequence and phylogenetic analyses<br /><!-- [et_pb_line_break_holder] --></em><!-- [et_pb_line_break_holder] --> <a href="#tab2">Tables 2</a> and <a href="#tab3">3</a> show the divergent nucleotide<!-- [et_pb_line_break_holder] --> sequences of the <em>COI </em>and <em>16S </em>nucleotide alignments.<!-- [et_pb_line_break_holder] --> For the <em>COI </em>gene three haplotypes corresponding to the<!-- [et_pb_line_break_holder] --> three morphotypes where found (<a href="#tab2">Table 2</a>). The 16S gene<!-- [et_pb_line_break_holder] --> is more variable, as expected, and we found more than one<!-- [et_pb_line_break_holder] --> haplotype for morphotype III (<a href="#tab3">Table 3</a>). The results of our<!-- [et_pb_line_break_holder] --> phylogenetic analyses are presented in <a href="#fig7">Figure 7</a> and <a href="#fig8">Figure<!-- [et_pb_line_break_holder] --> 8</a>. Both the NJ and the Bayesian analyses, for both genes<!-- [et_pb_line_break_holder] --> (<em>COI </em>and <em>16S</em>), recovered the same tree topology.<!-- [et_pb_line_break_holder] --> </font></p><!-- [et_pb_line_break_holder] --><p><a name="tab2" id="tab2"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 2</b>. Comparison of Santa Marta <em>Isostichopus </em>COI sequences from the three found morphotypes and other sequences<!-- [et_pb_line_break_holder] --> available in Genbank. Numbers indicate divergent nucleotide positions along the alignment. Dots correspond to<!-- [et_pb_line_break_holder] -->conserve nucleotides. Only sequences representative of each different haplotype are shown.</font><br /><!-- [et_pb_line_break_holder] --><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04tab2.jpg" width="530" height="102" /></p><!-- [et_pb_line_break_holder] --><p><a name="tab3" id="tab3"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b>Table 3</b>. Comparison of Santa Marta <em>Isostichopus </em>16S sequences from the three found morphotypes and other sequences<!-- [et_pb_line_break_holder] --> available in Genbank. Numbers indicate divergent nucleotide positions along the alignment. Dots correspond to<!-- [et_pb_line_break_holder] -->conserve nucleotides. Only sequences representative of each different haplotype are shown.</font><br /><!-- [et_pb_line_break_holder] --><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04tab3.jpg" width="522" height="623" /><br /><!-- [et_pb_line_break_holder] --><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04tab3b.jpg" width="251" height="164" /></p><!-- [et_pb_line_break_holder] --><p><a name="fig7" id="fig7"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig7.jpg" width="509" height="415" /><br /><!-- [et_pb_line_break_holder] --> Figure 7</b>. Tree topology obtained by Neighbor-joining and Bayesian analyses derived from COI (cytochrome c oxidase I)<!-- [et_pb_line_break_holder] --> sequences showing two genetically distinct lineages of <em>Isostichopus </em>in Colombia. Sequences downloaded from<!-- [et_pb_line_break_holder] --> GenBank are coded with each accession number. Asterisks represent posterior probabilities/booststraps that<!-- [et_pb_line_break_holder] --> were > 0.90/90%, diamonds represent posterior probabilities/booststraps that were > 0.80/80%.<!-- [et_pb_line_break_holder] --></font></p><!-- [et_pb_line_break_holder] --><p><a name="fig8" id="fig8"></a></p><!-- [et_pb_line_break_holder] --><p align="center"><font size="2" face="Arial, Helvetica, sans-serif"><b><img src="https://sag.org.ar/jbag/wp-content/uploads/2019/11/xix_a04fig8.jpg" width="497" height="376" /><br /><!-- [et_pb_line_break_holder] --> Figure 8</b>. Tree topology obtained by Neighbor-joining and Bayesian analyses derived from <em>16S rRNA </em>sequences showing<!-- [et_pb_line_break_holder] --> two genetically distinct lineages of <em>Isostichopus </em>in Colombia. Sequences downloaded from GenBank are coded<!-- [et_pb_line_break_holder] --> with each accession number. Asterisks represent posterior probabilities/booststraps that were > 0.90/90%,<!-- [et_pb_line_break_holder] -->diamonds represent posterior probabilities/booststraps that were > 0.80/80%.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">The phylogram obtained by Neighbor-joining and<!-- [et_pb_line_break_holder] --> Bayesian analysis derived from <em>COI </em>sequences (<a href="#fig7">Figure<!-- [et_pb_line_break_holder] --> 7</a>) showed that the three morphotypes of the so-called <em>Isostichopus badionotus </em>species form two well separated<!-- [et_pb_line_break_holder] --> clades: one with all morphotype III (M3) individuals and<!-- [et_pb_line_break_holder] --> the other including all morphotypes I (MI) and II (MII).<!-- [et_pb_line_break_holder] --> The “morphotype III” clade also includes a sequence<!-- [et_pb_line_break_holder] --> retrieved from GenBank identified as <em>Isostichopus </em>sp.,<!-- [et_pb_line_break_holder] --> the sister group of the “morphotype III” clade <em>I. fuscus</em>.<!-- [et_pb_line_break_holder] --> The “morphotype I+II” clade also includes a sequence<!-- [et_pb_line_break_holder] --> retrieved from GenBank identified as <em>Isostichopus badionotus</em>.<!-- [et_pb_line_break_holder] --> This clade forms the sister group of the clade formed by<!-- [et_pb_line_break_holder] --> morphotypes III and <em>I. fuscus</em>.<!-- [et_pb_line_break_holder] --> The <em>COI </em>distance matrix indicated that the average<!-- [et_pb_line_break_holder] --> distance percentage for <em>Isostichopus </em>was 0.20%, while<!-- [et_pb_line_break_holder] --> the average between species distance value was 7.80%.<!-- [et_pb_line_break_holder] --> Comparisons with <em>S. herrmanni </em>sequences indicated that the<!-- [et_pb_line_break_holder] --> between genera distance average value was 16% (<a href="#tab2">Table 2</a>).<br /><!-- [et_pb_line_break_holder] --> For the <em>16S </em>distance matrix we obtained similar results,<!-- [et_pb_line_break_holder] --> however, the sequence of <em>I. macroparentheses </em>available in<!-- [et_pb_line_break_holder] --> Genbank is genetically very different from other <em>Isostichopus</em> sequences. In this case the average within species distance<!-- [et_pb_line_break_holder] --> percentage for <em>Isostichopus </em>was of 0.5%, while the average<!-- [et_pb_line_break_holder] --> between species distance value was of 13.78%. The<!-- [et_pb_line_break_holder] --> distance values between <em>I. macroparentheses </em>and the other <em>Isostichopus </em>species were higher than the distance values<!-- [et_pb_line_break_holder] --> obtained between the other <em>Isostichopus </em>species and <em>Stichopus herrmanni </em>(<a href="#tab3">Table 3</a>). One <em>16S </em>sequence of <em>I.</em><!-- [et_pb_line_break_holder] --> <em>badionotus </em>(JN207495.1) clusters within the “Morphotype<!-- [et_pb_line_break_holder] --> III” clade.</font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><b>DISCUSSION</b> </font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif">For years, there have been great controversy around the<!-- [et_pb_line_break_holder] --> categorization of species worldwide and Colombian sea<!-- [et_pb_line_break_holder] --> cucumber is no exception. Genetic studies are definitive<!-- [et_pb_line_break_holder] --> for the understanding of the taxonomic identification of<!-- [et_pb_line_break_holder] --> sea cucumbers. A species under a certain area can be easily<!-- [et_pb_line_break_holder] --> confused or mistakenly classified if only its macroscopic<!-- [et_pb_line_break_holder] --> characters are observed. There are pronounced differences<!-- [et_pb_line_break_holder] --> between the two lineages studied in this work. One of<!-- [et_pb_line_break_holder] --> the main features is that <em>I. badionotus </em>has strong chocolate<!-- [et_pb_line_break_holder] --> spots with well pronounced edge, while <em>Isostichopus</em> sp. (morphotype III) presents different brownish spots<!-- [et_pb_line_break_holder] --> distributed on the dorsal surface of the body. This<!-- [et_pb_line_break_holder] --> morphotype was characterized by having a trapezoidalshaped<!-- [et_pb_line_break_holder] --> body, which is in contrast to <em>I</em>. <em>badionotus </em>who<!-- [et_pb_line_break_holder] --> has the ventral region flat and the dorsal region concave. <em>Isostichopus </em>sp. (morphotype III) was found always in rocky<!-- [et_pb_line_break_holder] --> bottoms while <em>I. badionotus </em>was found in sandy bottoms<!-- [et_pb_line_break_holder] --> as was state by Purcell <em>et al</em>., (2012). Additionally, the<!-- [et_pb_line_break_holder] --> morphotype III digestive tract length, which occupies<!-- [et_pb_line_break_holder] --> twice the length of its body in contrast to three times the<!-- [et_pb_line_break_holder] --> length for <em>I. badionotus</em>. Our findings suggest morphotype<!-- [et_pb_line_break_holder] --> III, could be a different species of <em>Isostichopus</em>. </font></p><!-- [et_pb_line_break_holder] --><p><font size="3" face="Arial, Helvetica, sans-serif"><em>Genetic evidence for taxonomic divergence</em> <br /><!-- [et_pb_line_break_holder] --> Since its appearance the DNA barcoding approach has<!-- [et_pb_line_break_holder] --> been proposed as a strategy that could help on species<!-- [et_pb_line_break_holder] --> characterization and discovery by allowing taxonomists<!-- [et_pb_line_break_holder] --> to rapidly sort specimens and by highlighting divergent<!-- [et_pb_line_break_holder] --> taxa that may represent new species (Hebert and Gregory,<!-- [et_pb_line_break_holder] --> 2005). The increase in DNA-barcoding initiatives has<!-- [et_pb_line_break_holder] --> led to the realization that, for a significant proportion<!-- [et_pb_line_break_holder] --> of species, there is an actual correlation between genetic<!-- [et_pb_line_break_holder] --> divergence and taxonomic status (Hebert <em>et al</em>., 2004). For<!-- [et_pb_line_break_holder] --> the application of genetic barcodes we need to analyze<!-- [et_pb_line_break_holder] --> the genetic divergence between and within species (interand<!-- [et_pb_line_break_holder] --> intra-genetic distances), so that, for a given genetic<!-- [et_pb_line_break_holder] --> marker (typically the <em>COI </em>gene), the lowest interspecific<!-- [et_pb_line_break_holder] --> value does not overlap with the highest intraspecific value,<!-- [et_pb_line_break_holder] --> maintaining a gap also called the “barcoding-gap” (Hebert<!-- [et_pb_line_break_holder] --> <em>et al</em>., 2003). This threshold, depending on the organisms<!-- [et_pb_line_break_holder] --> and the study, seems to lie around 2-4% (Hebert <em>et al</em>.,<!-- [et_pb_line_break_holder] --> 2003; Meyer and Paulay, 2005). Although the <em>COI </em>has<!-- [et_pb_line_break_holder] --> proven to be a useful tool for the characterization of<!-- [et_pb_line_break_holder] --> species in the Holothuroidea (Uthicke <em>et al</em>., 2010), the<!-- [et_pb_line_break_holder] --> 16S rRNA gene has also been successfully tested for this<!-- [et_pb_line_break_holder] --> purpose (Kamarudin, 2015; Wen and Zeng, 2014). In this<!-- [et_pb_line_break_holder] --> study we evaluated the potential use of the <em>COI </em>and <em>16S</em><!-- [et_pb_line_break_holder] --> <em>rRNA </em>genes for the characterization of <em>Isostichopus </em>species<!-- [et_pb_line_break_holder] --> in Colombia.<!-- [et_pb_line_break_holder] --> <br /><!-- [et_pb_line_break_holder] --> In Uthicke´s <em>et al. </em>(2010) barcoding study with <em>COI</em>,<!-- [et_pb_line_break_holder] --> conspecific sequence variation averages of 1.3% (numbers<!-- [et_pb_line_break_holder] --> between 2.0 and 4.5%) and a congeneric average value of<!-- [et_pb_line_break_holder] --> 16.9% were obtained, showing a clear “barcoding gap”. In<!-- [et_pb_line_break_holder] --> our study, the average COI within-species distance value<!-- [et_pb_line_break_holder] --> was of 0.20%, while the average between-species distance<!-- [et_pb_line_break_holder] --> value was of 7.80%. We also obtained a clear “barcoding<!-- [et_pb_line_break_holder] --> gap”. Comparisons with <em>S. herrmanni </em>sequences indicated<!-- [et_pb_line_break_holder] --> that the average between-genera distance value was of<!-- [et_pb_line_break_holder] --> 16%. Both for the <em>COI </em>and the <em>16S </em>genes we found<!-- [et_pb_line_break_holder] --> larger genetic distances values between <em>Isostichopus </em>sp.<!-- [et_pb_line_break_holder] --> morphotype III and <em>I. badionotus </em>(morphotypes I and II)<!-- [et_pb_line_break_holder] --> than between either of those with <em>I. fuscus</em>. Unfortunalty<!-- [et_pb_line_break_holder] --> there is very little genetic information published in<!-- [et_pb_line_break_holder] --> Genbank for the group. Although we tried to include the<!-- [et_pb_line_break_holder] --> only 16S sequence available for <em>I. macroparentheses</em>, this<!-- [et_pb_line_break_holder] --> sequence clearly does not belong to this species. When<!-- [et_pb_line_break_holder] --> the sequence is compared to others in the dataset using<!-- [et_pb_line_break_holder] --> the Blast tool, it matches another Istichopodid, <em>Astichopus</em><!-- [et_pb_line_break_holder] --> <em>multifidus</em>. Our tree reconstructions (both genes) showed<!-- [et_pb_line_break_holder] --> the presence of two <em>Isostichopus </em>well differentiated<!-- [et_pb_line_break_holder] --> groups. These barcoding results, corroborated by the<!-- [et_pb_line_break_holder] --> morphological analyses, suggest that there might be two <em>Isostichopus </em>lineages or species in our Colombian samples.<!-- [et_pb_line_break_holder] --> Furthermore, although we initially thought that the<!-- [et_pb_line_break_holder] --> morphotype II could also be different to <em>I. badionotus </em>due<!-- [et_pb_line_break_holder] --> to its external morphology, as proposed in Vergara and<!-- [et_pb_line_break_holder] --> Rodriguez (2015), the genetic results revealed that it is<!-- [et_pb_line_break_holder] --> part of the variety of morphotypes of the <em>I. badionotus</em> species. Nevertheless, its external characteristics, as well as<!-- [et_pb_line_break_holder] --> the ossicles conformation directed the studies to analysis of<!-- [et_pb_line_break_holder] --> genetic sequences that allowed us to confirm the presence<!-- [et_pb_line_break_holder] --> of two lineages of <em>Isostichopus </em>in the Colombian Caribbean.<br /><!-- [et_pb_line_break_holder] --></font><font size="3" face="Arial, Helvetica, sans-serif"><em>Isostichopus </em>includes ten nominal Atlantic species: <em>I</em>. <em>assimilis</em>, <em>I</em>. <em>maculatus</em>, <em>I</em>. <em>acanthomela</em>, <em>I</em>. <em>badionotus</em>, <em>I</em>. <em>diaboli</em>, <em>I</em>. <em>errans</em>, <em>I</em>. <em>haytiensis</em>, <em>I</em>. <em>macroparentheses</em>, <em>I. moebii </em>and <em>I</em>. <em>xanthomela </em>(Heilprin, 1888). While in the present literature<!-- [et_pb_line_break_holder] --> only two Atlantic species are recognized, it is quite possible<!-- [et_pb_line_break_holder] --> that the morphotype III encountered here represents a<!-- [et_pb_line_break_holder] --> described species currently relegated into the synonymy of <em>I. badionotus</em>. In order to confirm the taxonomical status of<!-- [et_pb_line_break_holder] --> the Colombian <em>Isostichopus</em>, all these nominal species need<!-- [et_pb_line_break_holder] --> to be evaluated on the basis of their descriptions and types.<!-- [et_pb_line_break_holder] --> It is well known that studies that confirm the taxonomic<!-- [et_pb_line_break_holder] --> classification of the species are an essential tool for the<!-- [et_pb_line_break_holder] --> implementation of management plans and conservation<!-- [et_pb_line_break_holder] --> programs for sea cucumbers. This study can be a tool<!-- [et_pb_line_break_holder] --> to establish the plans that currently do not exist in the<!-- [et_pb_line_break_holder] --> country.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"><b>ACKNOWLEDGMENTS</b></font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif">This work was supported by Vicerrectoria de Investigacion<!-- [et_pb_line_break_holder] --> of the Universidad del Magdalena and Sistema General de<!-- [et_pb_line_break_holder] --> Regalias Grant (Gobernacion del Magdalena-Convenio<!-- [et_pb_line_break_holder] --> 090/2014-2015). Authors would like to thank Eng. Yahir<!-- [et_pb_line_break_holder] --> Mendoza Vanegas and fisherman Jorge Polo who helped<!-- [et_pb_line_break_holder] --> collecting holothurians and to the Grupo de Investigacion<!-- [et_pb_line_break_holder] --> y Desarrollo Tecnologico en Acuicultura (GIDTA). </font></p><!-- [et_pb_line_break_holder] --><p><b><font size="2" face="Arial, Helvetica, sans-serif">BIBLIOGRAPHY</font></b></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif">1. Agudelo-Martinez V., Rodriguez-Forero A. (2017)<!-- [et_pb_line_break_holder] --> Gametogenesis, spawning and larval development<!-- [et_pb_line_break_holder] --> of <em>Isostichopus </em>sp. aff <em>badionotus</em>. SPC Beche-de-mer<!-- [et_pb_line_break_holder] --> Information Bulletin 37: 65-74.</font></p><!-- [et_pb_line_break_holder] --><p><font size="2" face="Arial, Helvetica, sans-serif"> 2. Agudelo V.Y., Rodriguez A. 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