Research Article |
Corresponding author: Giomar Helena Borrero-Pérez ( giomarborrero@gmail.com ) Academic editor: Alexander Martynov
© 2019 Giomar Helena Borrero-Pérez, María Juliana Vanegas-González.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Borrero-Pérez GH, Vanegas-González MJ (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
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Holothuria (Mertensiothuria) viridiaurantia sp. 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.
16S, COI, Chocó, Colombia, Echinodermata, Cabo Marzo, Gulf of Cupica, Gulf of Tribugá, Holothuroidea, mtDNA, morros, riscales, rocky reefs
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 (
Holothuria (Mertensiothuria) Deichmann, 1958, one of the 18 Holothuria subgenera, was reviewed by
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 (
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 (
Maps showing the geographic distribution of Holothuria (Mertensiothuria) viridiaurantia sp. nov. A, B Detailed distribution in Chocó, Colombia; the triangle indicates the only locality were H. (Mertensiothuria) hilla specimens were collected C Wider distribution of H. (M.) viridiaurantia sp. nov. showing other localities from Panama and Mexico where the species have been identified through photographs, and the locality in India from where the GenBank sequence KP780302 originated. Colombian localities are represented by exact coordinates, and all other localities were derived from specific localities mentioned in the literature.
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 (
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) (
Specimens of Holothuria (Mertensiothuria) viridiaurantia sp. nov. from Colombia and GenBank sequences analysed for the partial cytochrome oxidase subunit 1 (COI) and/or 16S genes. Sequence-Voucher Location column includes the origin of the sequences according to the GenBank references where they have been generated; sequences from this study include the catalogue number at the MHNMC-INVEMAR (INV EQU). An asterisk (*) in the species column indicates changes in the GenBank ID, where the sequences were previously identified as H. (Mertensiothuria) hilla.
Species | GenBank Accession number | Sequence-Voucher, Location | Reference | |
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COI | 16S | |||
H. (M.) viridiaurantia sp. nov. | MK477997 | MK477991 | Colombia (Pacific) Holotype (INV EQU4309) | This study |
MK477998 | – | Colombia (Pacific) Paratype (INV EQU4312) | This study | |
– | MK477992 | Colombia (Pacific) Paratype (INV EQU4234) | This study | |
H. (M.) viridiaurantia sp. nov.* | JN207616 | JN207515 | Mexico |
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KP780302 | – | India | Deepa and Bijukumar, unp. | |
H. (M.) hilla | MK477996 | MK477994 | Colombia (Pacific) (INV EQU4311) | This study |
– | MK477993 | Colombia (Pacific) (INV EQU4310) | This study | |
KX874337 | KX856783 | Mariana Islands, Guam |
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– | EU822442 | – | Uthicke and Byrne, unp. | |
– | FJ223856 | Malaysia |
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– | FJ223864 | Malaysia |
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H. (M.) leucospilota | JN207617 | JN207541 | Marshall Islands (Majuro) |
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KC405566 | KY986424 | Pangkor Island, Malaysia |
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KY986417 | KY986423 | Pangkor Island, Malaysia |
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KC405565 | KY986422 | Pangkor Island, Malaysia |
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H. (T.) impatiens | MK477999 | MK477995 | Colombia (Pacific) (INV EQU4236) | This study |
JN207632 | JN207526 | Mexico (Pacific) |
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H. (T.) aff. impatiens | – | FJ223857 | Malaysia |
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H. (T.) arenicola | JN207608 | JN207556 | Florida (USA) |
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Isostichopus fuscus | MK477908 | MK477869 | Panama (Pacific) IfTa210 | This study |
Genus Holothuria Linnaeus, 1767
Subgenus Mertensiothuria Deichmann, 1958
Holothuria (Mertensiothuria) hilla
Holotype
: INV EQU4309, one specimen, total length 70 mm; collected in La Viuda rocky reef, Gulf of Cupica, northern Chocó, Colombia (6°37.9812'N, 77°29.985'W), by G. Borrero, 24Oct 2016; at 15 m depth under rocks and attached; GenBank nucleotide sequences COI MK477997 and 16S MK477991 (Fig.
Type specimens of Holothuria (Mertensiothuria) viridiaurantia sp. nov. (A–F) and comparative material of Holothuria (Mertensiothuria) hilla (G–J). A Dorsal and ventral view of the alive holotype of H. (M.) viridiaurantia sp. nov. from Gulf of Cupica, Northern Chocó, Colombia (INV EQU4309, L = 70 mm) B calcareous ring and C stone canal and madreporite of the Holotype D detail of the preserved holotype tentacles E paratype from Cabo Marzo; Northern Chocó, Colombia (INV EQU4234, L = 35 mm) F smallest paratype from Gulf of Tribugá, Northern Chocó, Colombia (INV EQU4312, L = 25 mm) G, H alive specimen of H. (M.) hilla from Cabo Marzo, Northern Chocó, Colombia, and detail of tentacles in the preserved specimen (INV EQU4310, L = 100 mm) I, J preserved specimen of H. (M.) hilla, same locality as G, H and detail of tentacles (INV EQU4311, L = 65 mm). Scale bars: 2 mm (B, C); 1 cm (E, F, I).
Holothuria (Mertensiothuria) hilla
: INV EQU4245, one specimen, total length 75 mm; collected in Piedra de Rodrigo rocky reef, Cabo Marzo, northern Chocó, Colombia (6°47.0346'N, 77°41.6148'W), by M.J. Vanegas, 25 April 2016; at 19 m depth under rocks; INV EQU4310, four specimens, total length 70–100 mm; by G. Borrero, 26 Oct 2016; same locality, depth, and habitat as previous; GenBank nucleotide sequences 16S MK477993 (Fig.
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.
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.
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.
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.
Ossicles
: Dorsal and ventral body wall include similar tables and buttons, with dorsal tables taller and dorsal buttons larger than ventral (Table
Ossicle comparison between Holothuria (Mertensiothuria) viridiaurantia sp. nov. and Holothuria (Mertensiothuria) hilla. A Holotype of H. (M.) viridiaurantia sp. nov. (INV EQU4309, L = 70 mm) B H. (M.) hilla (INV EQU4311, L = 65 mm); showing ossicle set from dorsal body wall (tables, buttons) and ventral body wall (tables, buttons); grey squares indicated in the images are presented enlarged below each image. Scale bar: 100 µm.
Ossicles comparison between Holothuria (Mertensiothuria) viridiaurantia sp. nov. and Holothuria (Mertensiothuria) hilla. A Holotype of H. (M.) viridiaurantia sp. nov.(INV EQU4309, L = 70 mm B H. (M.) hilla (INV EQU4311, L = 65 mm); showing ossicle set from dorsal papillae (tables, buttons, rods), dorsal papillae tip (showing the plate and small rods at the tip) and tube feet (tables, buttons and supporting plates); grey squares indicated in some images are presented enlarged below each image. Scale bar: 100 µm.
Ossicles comparison between Holothuria (Mertensiothuria) viridiaurantia sp. nov. and Holothuria (Mertensiothuria) hilla. A Holotype of H. (M.) viridiaurantia sp. nov. (INV EQU4309, L = 70 mm) B H. (M.) hilla (INV EQU4311, L = 65 mm); showing end plates from tube feet and ossicle set from tentacles (large and small rods) and longitudinal muscles (C's and O's ossicles). Scale bars: 100 µm (except A upper).
Comparison of ossicle size in Holothuria (Mertensiothuria) viridiaurantia sp. nov. holotype, juvenile paratype, and H. (M.) hilla of similar body size.
Characteristics | H. (M.) viridiaurantia sp. nov. Paratype INV EQU4312 | H. (M.) viridiaurantia sp. nov. Holotype INV EQU4309 | H. (M.) hilla INV EQU4311 | |||
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L = 25 mm | L = 70 mm | L = 65 mm | ||||
Range (µm) | Average | Range (µm) | Average | Range (µm) | Average | |
Dorsal body wall | ||||||
Buttons length | 88–119 | 99 | 79–115 | 101 | 47–70 | 61 |
Tables disc diameter | 62–84 | 73 | 60–81 | 68 | 51–75 | 59 |
Tables height | 56–68 | 62 | 43–54 | 49 | 35–45 | 41 |
Width spires | 14–15 | 15 | 23–31 | 29 | 21–26 | 23 |
Ventral body wall | ||||||
Buttons length | 78–104 | 90 | 82–108 | 94 | 50–73 | 61 |
Tables disc diameter | 54–80 | 63 | 57–71 | 63 | 47–61 | 53 |
Tables height | 45–56 | 51 | 40–49 | 44 | 34–44 | 39 |
Width spires | 9–17 | 14 | 22–29 | 25 | 16–20 | 18 |
Dorsal papillae | ||||||
Buttons, Buttons-like plates length | 80–111 | 99 | 106–130 | 117 | 64–91 | 79 |
Tables disc diameter | 51–95 | 74 | 60–82 | 71 | 48–66 | 58 |
Tables height | 58–71 | 64 | 42–62 | 52 | 43–47 | 45 |
Width spires | 19–28 | 25 | 30–42 | 36 | 20–25 | 22 |
Rods | 97–168 | 143 | 167–203 | 187 | 102–152 | 129 |
Plates | – | - | 99–134 | 118 | 104–107 | 106 |
Small Rods | – | - | 37–58 | 48 | 43–67 | 57 |
Ventral tube feet | ||||||
Buttons, Buttons-like plates length | 93–116 | 105 | 91–140 | 110 | 52–84 | 70 |
Tables disc diameter | 54–75 | 63 | 53–73 | 61 | 41–57 | 51 |
Tables height | 46–54 | 50 | 41–51 | 45 | 34–43 | 38 |
Width spires | 15–23 | 18 | 23–31 | 28 | 17–24 | 21 |
Plates length | 66–122 | 96 | 105–133 | 117 | 76–127 | 96 |
Plates width | 46–83 | 65 | 72–129 | 116 | 68 –99 | 84 |
End Plates | 391–401 | 396 | 578–581 | 580 | 570–584 | 577 |
Tentacles | ||||||
Large Rods Length | 132–259 | 197 | 185–261 | 221 | 166–267 | 227 |
Large Rods Width | 21–39 | 29 | 33–150 | 75 | 10–27 | 18 |
Small Rods Length | 37–91 | 56 | 58–107 | 74 | 37–65 | 47 |
Longitudinal muscle | ||||||
C-O shape ossicles length | NA | NA | NA | NA | 13–33 | 24 |
Paratypes
: Juveniles, 35 and 25 mm long, 12 and 8 mm wide respectively (Fig.
Ossicles of the juvenile paratype of Holothuria (Mertensiothuria) viridiaurantia sp. nov. (INV EQU4312, L = 25 mm). A Dorsal body wall (tables, buttons) B ventral body wall (tables, buttons) C dorsal papillae (tables, buttons, rods) D tube feet (tables, buttons, supporting plates) E tube feet (end plate) F tentacles (large and thick rods, small rods). Scale bars: 100 µm.
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).
Holothuria (Mertensiothuria) viridiaurantia sp. nov. is known and confirmed along the Eastern Pacific from Mexico (as Holothuria (Mertensiothuria) hilla,
Holothuria (Mertensiothuria) viridiaurantia sp. nov. is associated with rocky bottoms from the intertidal to 15 m depth (
As the specimens of Holothuria (Mertensiothuria) viridiaurantia sp. nov. were previously assigned to H. (M.) hilla, the conservation status of this species must be considered. Currently H. (M.) hilla is included in the IUCN Red List in the category of Least Concern, and in addition, it is classified as a low-value species (about USD3 kg-1 dried in the Philippines) among commercially important sea cucumbers of the world (
The new species was previously assigned to Holothuria (Mertensiothuria) hilla (
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.
Bayesian inference tree of mitochondrial genes of the specimens analysed for the present study. A COI B 16S. The numbers on the nodes indicate Neighbour Joining (bootstrap %) / Maximum Likelihood (bootstrap %) / Bayesian posterior probability. Hyphen (-) indicates nodes not supported in some trees. Sequences from Colombia obtained in this study include the catalogue number at the MHNMC - INVEMAR (INV EQU); GenBank ascension number is included for the other sequences (see Table
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).
Species | 1 | 2 | 3 | 4 | 5 | 6 | |
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1 | H. (M.) viridiaurantia sp.nov. | 1.3 / 0.5 | 12.5 | 11.8 | 15.7 | 13.5 | 32.6 |
2 | H. (M.) hilla | 16.7 | 13.8 / 7.9 | 13.9 | 19.4 | 16.3 | 33.9 |
3 | H. (M.) leucospilota | 15.6 | 17.7 | 1.9 / 1.6 | 19.4 | 17.2 | 34.8 |
4 | H. (T.) impatiens | 21.3 | 21.5 | 21.8 | 0.9/ 8.8 | 15.5 | 33.2 |
5 | H. (T.) arenicola | 18.0 | 19.9 | 17.6 | 19.4 | nc / nc | 34.3 |
6 | Isostichopus fuscus | 24.4 | 22.5 | 24.0 | 24.5 | 20.7 | nc / nc |
We thank researchers who participated in the project “Caracterización biótica de los arrecifes rocosos (Riscales y Morros) en el Chocó Norte, Pacífico Colombiano” for helping during collection of the specimens; Angelica Fernández and Sandy Caldera (MHNMC, INVEMAR) for their collaboration in the molecular lab work; Janneth Beltrán (LABSIS, INVEMAR) for the elaboration of the maps; Crystal Kelehear-Graham for her diligent proofreading of this paper. External reviewers for their suggestions to improve the manuscript. This work was supported by INVEMAR through the projects “Caracterización Biótica de los Arrecifes Rocosos (Riscales y Morros) en el Chocó Norte, Pacífico Colombiano 2016–2018” and “Obtención del código de barras del ADN (Barcoding) para los grupos de fauna y flora marinos– 2016, 2017”; both under the agreement with the Ministry of Environment and Sustainable Development of Colombia, Code: BPIN: 2014011000405 y 2017011000113. INVEMAR contribution No. CTRB – 1235.