J. Mar. Biol. Ass. U.K. (2004), 84,1077^1084 Printed in the United Kingdom Mitochondrial DNA sequence evidence supporting the recognition of a new North Atlantic Pseudostichopus species (Echinodermata: Holothuroidea) O O P Francisco A. Sol|¤s-Mar|¤n*$, David S.M. Billett , Joanne Preston and Alex D. Rogers *Coleccion Nacional de Equinodermos, Laboratorio de Sistematica y Ecologia de Equinodermos, Instituto Ciencias del Mar y O Limnologia, Universidad Nacional Autonoma de Mexico, Apdo. Post. 70-305, CP. 04510, Mexico City. Southampton Oceanography P Centre (SOC), School of Ocean and Earth Sciences (SOES), Empress Dock, Southampton, SO14 3ZH, UK. British Antarctic Survey, High Cross, Madingley Road, Cambridge, CB3 0ET, UK. $Corresponding author, e-mail: [email protected] A new species of the synallactid sea cucumber genus Pseudostichopus is described, P. aemulatus sp. nov., based on genetic (DNA sequences of the mitochondrial gene Cytochrome Oxidase I [COI] gene) and morphological characters. A comparative molecular study with two other species of the same genus (P. villosus and P. mollis) and from a di¡erent family (Isostichopus fuscus) was carried out in order to clarify its taxonomic identity. The nucleotide distance between P. aemulatus sp.nov.andP. villosus and P. mollis is su⁄cient to support distinct species status. The estimated di¡erence in the number of amino acids, coded for by a partially sequenced COI gene, within the species of the family Synallactidae ranged from 4 to 18. The phylogenetic analysis clearly supports separate species status of these sympatric morphotypes, as indicated by the morphological analysis. INTRODUCTION ossicle morphology of Pseudostichopus species are ambig- Holothurians are amongst the most conspicuous uous and frequently contradictory. Di¡erences in general elements of the deep-sea megafauna. Many of their body shape and internal anatomy between Atlantic Pseudostichopus specimens are often small and again may morphological characters show a high degree of variation simply represent intraspeci¢c variation. some of which may arise as a result of artefacts of methods In this study we aimed to clarify the morphological used for collection and preservation of specimens. The identity of some of the north-east Atlantic Pseudostichopus deep-sea species of the family Synallactidae are typical of species, and to augment the morphological data with this situation. This family contains 19 controversial DNA sequences of the mitochondrial gene Cytochrome described genera including: Amphigymnas, Bathyplotes, Oxidase I (COI) gene. The data indicate that the Benthothuria, Hansenothuria, Mesothuria, Paroriza, Pelopatides, smaller, common morphotype of Pseudostichopus from the Pseudostichopus, Synallactes, Zygothuria, Allopatides, Bathyzona, Porcupine Abyssal Plain is a distinct species and that in Capheira, Dendrothuria, Filithuria, Galatheathuria, Perizona, Pseudothuria, Scotothuria. The genus Pseudostichopus The¤el, this case what has been interpreted as intraspeci¢c varia- 1886 is one of the oldest established synallactid taxa and tion within species is interspeci¢c variation between known and cryptic species of holothurians. The systematic its species also demonstrate a high level of putative intra- implications of this for deep-sea and shallow-water speci¢c variation. holothurians are discussed. Many synallactid holothurians were collected by the RRS ‘Discovery’ and RRS ‘Challenger’ from the deep-sea £oor of the Porcupine Abyssal Plain, north-east Atlantic MATERIALS AND METHODS during a number of European Union co-sponsored research programmes between 1991 and 1999, including Sampling the BENGAL project (High-resolution temporal and Pseudostichopus specimens were examined from 14 spatial study of the BENthic biology and Geochemistry of samples taken on the Porcupine Abyssal Plain to the south- a north-eastern Atlantic abyssal Locality) (Billett & Rice, west of Ireland, north-east Atlantic, ranging from 4764 to 2001). The samples included numerous specimens of 4849 m in depth (Table 1). Rice (1992, 1996, 1997), Billett P. v i l l o s u s The¤el, 1886, and many individuals of a smaller (2000) and Billett & Rice (2001) provide descriptions of Pseudostichopus morphotype (Billett et al., 2001). the study area, and the sampling strategy and gear used. Whether this smaller morphotype represented onto- In addition to these abyssal north-east Atlantic speci- genetic variation amongst one of the other Atlantic mens, Isostichopus fuscus was collected using SCUBA diving Pseudostichopus (P. depressus He¤rouard, 1902, P. lapidus from a shallow-water site (15m depth) in the East Paci¢c, He¤rouard, 1923, P. marenzelleri He¤rouard, 1923, P. occultatus o¡ the coast of Jalisco, Mexico (218N) in the year 2000. Marenzeller, 1893 and P. v i l l o s u s The¤el) or whether it Specimens used in the morphological analysis were represented a previously unrecognized taxon was uncer- ¢xed in 4^8% bu¡ered formalin for at least 24 hours and tain. Previous descriptions of the external features and then transferred to 70% ethanol. Specimens collected for Journal of the Marine Biological Association of the United Kingdom (2004) Downloaded from https:/www.cambridge.org/core. Open University Library, on 28 Jan 2017 at 14:21:44, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S002531540401046Xh 1078 F.A. Sol|¤s-Mar|¤n et al. DNA sequence of a new Pseudostichopus species Table 1. List of stations used in the study. RRS ‘Discovery’ cruises 222(2), 226, 237 and RRS ‘Challenger’ cruises 79, 142 (Rice, 1992, 1996, 1997; Billett, 2000). Position start Position end No. of Station number Date Latitude (N) Longitude (W) Latitude (N) Longitude (W) Depth (m) specimens 52701#42 24-05-91 48852.700 16838.500 48851.200 16828.500 4849^4843 85 12930#46 09-09-96 48847.210 16843.310 48849.490 16832.620 4837^4841 53 12930#78 16-09-96 48853.040 16830.490 48850.030 16841.920 4836^4840 82 13078#29 04-04-97 48856.200 16822.770 48847.350 16833.230 4844^4847 183 13627#10 30-09-98 48853.060 16842.060 49802.000 16853.030 4835^4837 68 54901#5 28-04-99 48844.870 16840.530 48848.160 16836.240 4835^4838 97 54901#7 29-04-99 48847.450 16848.880 48850.820 16846.040 4836^4838 2 54901#9 30-04-99 48846.890 16841.590 48850.580 16836.360 4837^4841 111 54903#1 03-05-99 49832.090 15856.020 49828.110 15856.520 4810^4817 545 54905#1 04-05-99 50832.650 16857.770 50828.660 16859.430 4764^4786 72 Total: 1298 molecular analyses were placed immediately in chilled (approximately 0.012% units/ml of Boeghringer water on-board ship and transferred to a temperature Mannheim, Cat. no. 1373-196). These preparations were controlled room (48C). Individuals were dissected to incubated for 2 h at 558C in an incubator and then a obtain a sample of longitudinal muscle from the body standard phenol/chloroform-isoamyl alcohol extraction wall, which was immediately placed in 99% ethanol. was carried out with precipitation of DNA by 2:1 ice-cold 100% ethanol plus 1:10 3M sodium acetate. Polymerase chain reactions (PCRs) were carried out in 20 ml total Identi¢cation volume using sterile water, and contained 160 mM each Taxonomic identi¢cation using external and internal dNTP, 10 mM Tris^HCL, pH 8.3, 40 mM KCL, 2 mM anatomy was carried out prior to DNA analysis, based on MgCl2,1mM each primer, 1 unit Taq-polymerase, and 10 original descriptions and keys. Total length (TL), as to 30 ng template DNA. The primers used to amplify the indicated in the results, was measured from the tip of the 30 end of the COI gene and their position in the mitochon- anterior part of the body to the posterior end. Width (W) drial map of the sea urchin Strongylocentrotus purpuratus was measured from the widest part of the body at the mid- (Stimpson, 1857) are shown in Table 2 (Jacobs et al., ventral region. For each set of measurements, the 1988; Arndt et al., 1996). maximum, minimum and median values were recorded. Polymerase chain reaction was performed using either a All measurement values are in millimetres. The specimens Perkin-Elmer 480 or a Hybaid PCR-Express thermocycler were deposited in the collections of the Natural History and comprised a 948C/4 min initial denaturing step Museum, London (NHM); the National Museum of followed by 30 cycles of 948C/1min, 508C/1min, and Natural History, Smithsonian Institution,Washington, DC, 728C/1min. A ¢nal elongation step of 728C/10min was USA (USNM); the Zoological Museum, Copenhagen, used. The ‘oil overlay’ and ‘hot lid’ methods worked Denmark (ZMC); Echinoderm National Collection, equally well. Products were visualized on a 1.5% agarose Universidad Nacional Autonoma de Mexico, Mexico City, gel stained with ethidium bromide. Polymerase chain (ICML-UNAM) and the Discovery Collections, South- reaction products were puri¢ed with Qiagen Qiaquick ampton Oceanography Centre, UK (SOC-DC). PCR puri¢cation columns (Cat. no. 28106), according to manufacturer’s guidelines. Cycle-sequencing reactions of 10 ml were prepared using Perkin-Elmer BigDye Termi- Polymerase chain reaction and sequencing nator Ready Reaction mixes (Cat. no. 4303152), using the Three species were screened in the present study, two manufacturer’s guidelines. The products of the cycle- Pseudostichopus species and Isostichopus fuscus (Ludwig, sequencing reactions were puri¢ed using Qiagen DyeEx 1875).The latter was used as outgroup for the phylogenetic Spin kits (Cat. no. 63104) and sequences visualized using analysis. DNA was extracted through digestion of a small a Perkin-Elmer ABI 377 automated DNA sequencing piece of muscle (approximately 200 mg) in 100 mM Tris^ machine. All samples were double-checked by reverse HCL, pH 8.0, 1.25% SDS, and 390 ng/ml proteinase K sequencing. Table 2.