Holothuria (Mertensiothuria) viridiaurantia sp. nov. (Holothuriida, Holothuriidae), a new sea cucumber from the Eastern Pacific Ocean revealed by morphology and DNA barcoding

Abstract Holothuria (Mertensiothuria) viridiaurantiasp. nov. is described based on specimens from rocky reefs of northern Chocó in the Colombian Pacific Ocean; however, it also occurs along the Eastern Pacific Ocean from Mexico and Panama. Although specimens from Mexico and Panama were previously identified as Holothuria (Mertensiothuria) hilla Lesson, 1830 the new species is easily distinguished morphologically and via mtDNA. In terms of morphology, the species can be identified by its olive-green background and white-orange papillae and tentacles, larger tentacles with deep indentations and also by larger buttons on the dorsal and ventral body wall, papillae and tube feet; large, thick and rough tentacle rods, and the absence of ossicles in the longitudinal muscles. The new species is included in the subgenus Mertensiothuria considering molecular evidence.


Introduction
The family Holothuriidae Ludwig, 1894 currently includes 211 valid species, with the genus Holothuria Linnaeus, 1867 being the most diverse, containing 165 formally described species (WoRMS 2019a). Sixteen new Holothuria species have been described from different localities around the world since 2000; two of them from the Central and Tropical Eastern Pacific Honey-Escandón et al. 2011). The diversity of Holothuria will likely continue to grow considering some "cryptic" species currently recognised based on molecular evidence (COI mtDNA) and morphological characteristics, such as colouration, as reported for the Holothuria (Thymiosycia) impatiens complex (Michonneau 2015). In addition, the exploration of poorly known regions could generate new information on the diversity of holothurians and other marine organisms. In particular, an area that warrants further exploration is the north of the Colombian Pacific (Chocó), part of the Tumbes-Magdalena-Chocó biogeographical hotspot that is considered a mega-diverse area (Cortés 1997).
Among the species in this subgenus H. (M.) hilla Lesson, 1830 is the most widespread species, reported from the Red Sea to Madagascar and across the Indian Ocean and the Pacific Ocean to the Central and Tropical Eastern Pacific (Purcell et al. 2012). It is a common species in the Central and Tropical Eastern Pacific occurring in its common colour morph, comprised of a yellow background and white papillae (Samyn and Massin 2003;Purcell et al. 2012). However, specimens with different colours, such as an olive-green background and white-orange papillae, have been reported by several authors (Solís-Marín et al. 2009, Lam. 30A;Sotelo-Casas et al. 2015: fig. 2E; Molina et al. 2015: fig. 3C). Specimens with yellow and green colour patterns were collected in the Colombian Pacific Ocean in 2016, allowing comparison of the morphology and mitochondrial DNA. The purpose of this paper is to describe a new species of Holothuria from the Eastern Pacific and to indicate how it differs from Holothuria (Mertensiothuria) hilla.

Materials and methods
The specimens reviewed were collected as part of the project "Riscales", developed by the Instituto de Investigaciones Marinas y Costeras -INVEMAR (www.invemar.org.co), seeking to characterise the biodiversity of the rocky reefs (called locally "riscales" and "morros") located in northern Chocó in the Colombian Pacific Ocean. These ecosystems are important for regional fisheries and conservation (Díaz-Fahrenberger et al. 2016). Specimens were collected by hand using SCUBA diving at three rocky reefs between 10 and 15 m depth, during two sampling events in April and October 2016 (Fig. 1A, B). The specimens were placed in plastic bags with seawater, relaxed using magnesium chloride, fixed and preserved in 96% ethanol. They are deposited at the Museo de Historia Natural Marina de Colombia (MHNMC) -Makuriwa of INVEMAR (INV EQU).
External and internal morphology were reviewed to record standard data for sea cucumbers. Tissue from papillae, dorsal body wall, tube feet, ventral body wall, tentacles, and internal organs (longitudinal muscles, respiratory trees, tentacle ampullae, cloaca, and intestine) was removed and dissolved in fresh household bleach. Ossicles were observed and photographed using light microscopy and, at least, ten ossicles of each type were measured using the software ImageJ (Schneider et al. 2012). Type of ossicles, shape, and size were compared with those described by Lesson (1830) and Samyn and Massin (2003).
Ethanol-fixed tissues of the sea cucumbers collected during the project were processed to obtain sequences of the mitochondrial cytochrome oxidase I (COI) and 16S (large subunit) genes; in this paper, only the data of the specimens of interest are shown. Genomic DNA was extracted using the QIAGEN extraction kit (DNeasy Blood & Tissue Kit) and COI and 16S were amplified using the primers COIceF (ACTGCCCACGCCCTAGTAATGATATTTTTTATGGTNATGCC) and COIceR (TCGTGTGTCTACGTCCATTCCTACTGTRAACATRTG) (Hoareau and Boissin 2010) and 16SA (CGCCTGTTTATCAAAAACAT) and 16SB (CTCCGGTTT-GAACTCAGATCA) (Palumbi 1996). PCRs were carried out following the conditions described by Hoareau and Boissin (2010). PCR products were purified and sequenced using the BigDye 3.1 (Applied Biosystems) technology. The obtained nucleotide sequences were edited using Mega 7. We analysed a fragment of 443 bp of 16S genes (including gaps) and 439 bp of COI. Sequences of COI were translated into amino acids to ensure their integrity and accuracy. The sequences obtained in the present study were submitted to GenBank (Table 1) (Table 1). 16S sequences were aligned using the L-INS-i method implemented in MAFFT 6 (Katoh et al. 2002) and COI with Clustal W ( Thompson et al. 1994). Distances using Kimura 2 parameters correction were calculated and neighbour-joining trees were generate using Mega 7 (Kumar et al. 2016). Withingroup genetic differences were analysed on the species level also based on Kimura 2-parameter distances. The best substitution model was searched using the Akaike information criterion implemented in jMoldelTest (Posada 2008). Phylogenetic relationships were inferred using Bayesian Inference (BI) and Maximum Likelihood (ML).
BI was performed with MrBayes v. 3.2.6 (Ronquist and Huelsenbeck 2003) using unlinked GTR+G evolutionary model for each gen; the data set was run twice, using four Markov chains for ten million generations for each analysis to estimate posterior probabilities. ML analysis was performed in Mega 7; support was assessed in this case by 1000 bootstrap pseudoreplicates.  Diagnosis. Olive-green background with white-orange dorsal papillae, tube feet and tentacles; buttons >75 µm in length; large tentacles with deep indentations; tentacle rods thick, rough and with some perforations; longitudinal muscles without ossicles.

Order
Description. External appearance: medium-sized species, holotype preserved specimen 70 mm long and 21 mm wide; body loaf like (length < 4× diameter) length/width ratio 2.3. Body shape of living ex situ specimen cylindrical in cross-section ( Fig. 2A), tapering posteriorly and widening anteriorly, ending in a large crown of tentacles. Body wall soft and thin (2-3 mm thick). Anus terminal surrounded by small papillae. Mouth directed ventrally in live and preserved specimens, encircled by large papillae (Fig. 2A,  D). Large peltate tentacles 20; ca. 5-6 mm total length, and 4-5 mm width shield; with deep indentions 2-3 mm. Few large, long and slender conical papillae scattered on the dorsal surface, although a vague arrangement into four rows is observed, two of them are lateral, where they are a little larger; smaller papillae scattered among the largest. Ventral tube feet cylindrical, large and thick, densely distributed throughout the ventral surface.
Colour. Background of living specimens olive-green; base of the papillae is a light or whitish green that changes to orange from the middle to the ends, however, the tips of the papillae are whitish. Ventral surface similar to dorsum, with orange tube feet and white suckers; tentacles orange, same colour as papillae and tube feet ( Fig. 2A). Dark brownish green in preserved specimens with papillae, tube feet, and tentacles a dark yellow (Fig. 2D-F). Internal anatomy. Square radial plates in the calcareous ring, 3 mm wide and 3 mm high, with three anterior rounded processes, and posterior margin with shallow rounded indentation; interradial plates slender, 1.5 mm high and 2.5 mm wide, pointed anterior margin and rounded posterior margin (Fig. 2B). One free stone canal, 4 mm long, and a helicoidally madreporite, 4 mm long (Fig. 2C). Tubular tentacle ampullae, 3-4 mm long and striped coloured. Tube-like polian vesicle, 17 mm long. Longitudinal muscles pair flat, thinner in the middle of each pair, irregularly wide, 3-4 mm wide each band, or 2-2 mm wide, attached, with narrow free edges. Gonads absent. Cuvierian organ present. Right respiratory tree extending to anterior end; left respiratory tree attached to the intestine until the middle of the body.
Paratypes: Juveniles, 35 and 25 mm long, 12 and 8 mm wide respectively (Fig.  2E, F). External morphology different to the holotype, which is much larger at 70 mm long. Small dorsal papillae in the four main rows, as described for the holotype; and three rows of tube feet, two lateral and one in the middle of the ventral side which includes two irregular lines of pedicels (Fig. 2F). Dorsal and ventral body wall buttons are smaller in the juvenile, although there is not a considerable difference in size; however, in shape they are more rounded at the extremities and frequently present more than three pairs of holes (Table 2, Fig. 6A, B). Tables showed more changes during growth in comparison with buttons: the tables spire are taller and narrower, pointed-like without cross beam clearly noted, with few spines around the top; and the tables disc diameter is larger, with peripheral holes less in number and larger in size in the juvenile (Table 2,  Fig. 6A, B). Dorsal papillae and tube feet present similar pattern of change during growth in buttons and tables when comparing the juvenile with the holotype; however, tables in dorsal papillae and tube feet in the juveniles are less pointed-like and one cross beam is clearly noted in most of the tables in comparison with those from the dorsal and ventral body wall (Fig. 6C, D). In addition, rods in dorsal papillae are smaller in size; it was not possible to observe the small plates and rods at the very top of the papillae. Supporting plates and end plates in the tube feet are also smaller in the juvenile ( Table 2, Fig. 6D, E). Tentacle rods are not well developed in the paratype, being almost similar in length but less thick than those of the holotype, however, they are thicker than those in the H. (M.) hilla individual of 65 mm in length (    Etymology. From the Latin viridis (green) and aurantius (orange-coloured), referring to the living colour with olive-green background and orange-white papillae, tube feet, and tentacles (feminine).
Distribution. Holothuria (Mertensiothuria) viridiaurantia sp. nov. is known and confirmed along the Eastern Pacific from Mexico (as Holothuria  Molina et al. 2015) and Colombia (present study) (Fig. 1). However, a GenBank sequence of one specimen from Kerala coast, India (Accession number KP780302.1) suggests that the new species could have a wider geographical distribution across the Indian Ocean and the Pacific Ocean to the Central and Tropical Eastern Pacific, like H. (M.) hilla (Fig. 1C). However, it was not possible to review the specimen belonging to the sequence, so colouration and morphological characteristics described in the present paper should be reviewed and confirmed. Notably, images of green-coloured H. (M.) hilla from the Philippines are presented by Dolorosa et al. (2017;Fig . 2J).
Habitat. Holothuria (Mertensiothuria) viridiaurantia sp. nov. is associated with rocky bottoms from the intertidal to 15 m depth (Molina et al. 2015;present study). Specimens collected in Colombia were found attached under medium rocks, differing Remarks. The new species was previously assigned to Holothuria (Mertensiothuria) hilla Santos-Beltrán and Salazar-Silva 2011;Honey-Escandón et al. 2012;Molina et al. 2015;Sotelo-Casas et al. 2015), however there is no mention of the distinct and striking colouration of the specimens reported in those papers in comparison with H. (M.) hilla. Perhaps the identification of this species was based on the similar external appearance (shape of the body and papillae) and apparent similar ossicles at first sight; without regard to the colouration, which has been traditionally considered to be intra-specific variability in echinoderms. However, recent research demonstrates that it can be a diagnostic characteristic, for example in the species complex H. (T.) impatiens (Michonneau 2015); this subject requires careful and exhaustive study, especially the purpose of colouration in sea cucumbers (Clark 1922;Michonneau 2015). In this study, a detailed revision of specimens from the new species and H. (M.) hilla, showed not only the colouration as a diagnostic feature, but also the size and shape of the tentacles, which are larger and with deeper indentations in the new species (Fig. 2). In reference to the ossicles, although similar in shape at first sight, a detailed revision showed several diagnostic characteristics: 1) differences in the size of the complete ossicle sets from the dorsal and ventral body wall, dorsal papillae and tube feet; specifically, the tables are taller and thicker with wider discs and the buttons are larger in the new species, in both juvenile and large specimens (Table 2; Figs 3-6); size of the buttons is the most diagnostic trait for the species; 2) the size and shape of the tentacle rods, being wide (plate-like), thick and very rough, and with some perforations in the new species compared to slender rods in H. (M.) hilla (Table 2; (Table 2; Fig. 5A, B). In general, the morphological structures of the new species are thicker and stronger than those of H. (M.) hilla, which is a more delicate species. Among the morphological characteristics of the new species, the absence of ossicles in the longitudinal muscles, larger size of the perforated plates of the tube feet, and size and shape of the tentacle ossicles, match those considered by Samyn and Massin (2003) for excluding Holothuria arenacava and Holothuria platei from Mertensiothuria. However, the decision for including the new species in this subgenus was made based on the mtDNA evidence.  Table 1).

Molecular characteristics.
We obtained COI and 16S sequence data from three specimens of Holothuria (Mertensiothuria) viridiaurantia sp. nov. and two of Holothuria (Mertensiothuria) hilla from the rocky reef in northern Chocó, Colombia. Specimens of H. (M.) viridiaurantia sp. nov. from Colombia (type specimens) were recovered in a well-supported clade, separated from H. (M.) hilla for both, COI and 16S genes (Fig.  7). Two sequences, derived from one specimen from Mexico (GenBank Accession No. JN207616-COI and JN207515-16S) and one from India (KP780302-COI), were recovered in the same clade as type specimens from Colombia. However, different tree topologies for COI and 16S sequence data were recovered.  (Fig. 7B). Species from Thymiosycia subgenus appear separated from Mertensiothuria subgenus for both genes and all tree reconstruction methods (Fig. 7A, B). Evidence for species status of H. (M.) viridiaurantia sp. nov. comes from the COI and 16S genetic distances. Inter-specific distances between the two previously recognised Mertensiothuria species included in the analysis is 17.7% for COI and 13.9% for 16S; and distances between the new species and them are 16.7 and 15.6% for COI and 12.5 and 11.8% for 16S; inter-specific distances among species of Mertensiothuria and Thymiosycia showed larger values (Table 3). In addition, intra-specific distances for H. (M.) viridiaurantia sp. nov. were 1.31% for COI and 0.5% for 16S, the lowest values in all the species analysed. Intra-specific distances for H. (M.) hilla (13.8% for COI and 7.9% for 16S) could be showing a species complex, similar to what was described by Michonneau (2015) for H. (T.) impatiens, which is also recovered here with 8.8% for 16S, including one specimen identified as H. (T.) aff. impatiens (Table 3). Lower intraspecific distance for COI (0.9%) for H. (T.) impatiens is explained because the sequence for COI was not available for this specimen. There is, therefore, strong molecular evidence that H. (M.) viridiaurantia sp. nov. is an undescribed species different from H. (M.) hilla, a finding also supported by the morphological characteristics described previously. Table 3. Kimura 2 parameter distances (%) within specimens of Holothuria (Mertensiothuria) viridiaurantia sp. nov. and between the Holothuria species included in the analysis. COI distances are below diagonal and 16S distances above. The numbers in bold lettering along the diagonal represent average within species distances for COI and 16S (COI / 16S).