Sea cucumbers of the genus Stichopus Brandt, 1835 (Holothuroidea, Stichopodidae) in Straits of Malacca with description of a new species

Abstract Five sea cucumber species including one new species of the genus Stichopus are reported from the shallow coral reefs of Straits of Malacca. The new species Stichopus fusiformiossa has unusual fusiform spicules in the tentacles, which are not found in the other species of the genus. Pseudo-tables and large perforated plates are newly recorded for Stichopus hermanni Semper, 1868 and Stichopus vastus Sluiter, 1887, respectively.


Introduction
Recent revisions on the holothuroid taxonomy have resulted in some taxonomical changes and introduction of several new species in the genus Stichopus Brandt, 1835 of the family Stichopodidae (Rowe and Gates 1995;Massin 1999;Massin et al. 2002;Byrne et al. 2010). Outer morphology of Stichopus is somehow very deceptive and similar interspecifically (Clark 1922;Massin et al. 2002), and variable intraspecifically. Field identification by their external appearances proved to be difficult and identification using spicules is essential (Clark 1922;Clark and Rowe 1971;Massin et al. 2002;Massin 2007). To enhance the understanding of classification within the family Stichopodidae, Moraes et al. (2004) introduced chemotaxonomic approach while Byrne et al. (2010) and Uthicke et al. (2010) have employed molecular sequence data.
One of the earliest comprehensive records on the diversity and distribution of holothurians in Malaysian waters was done by Ridzwan and Che Bashah (1985). Then, Zulfigar et al. (2008) produced a field guide to sea cucumbers in shallow water and coral reefs in Malaysia. However, taxonomic studies of the genus Stichopus in Malaysia were scarce and done only at the southern part of the South China Sea (Siti et al. 1999;Massin et al. 2002). Massin et al. (2002) described two new species, Stichopus rubermaculosus and Stichopus ocellatus there. The genus Stichopus is one of the dominant genera in tropical shallow waters which is an important fishery commodity. Although Straits of Malacca is a major area for stichopodid fisheries, there has not been a comprehensive taxonomic study done on stichopodids in that area. Furthermore, the genus Stichopus is taxonomically very confusing due to their similarity between each species in outer appearance and the presence of variations (Massin et al. 2002). In this study, detailed morphological descriptions were done on the species of the genus Stichopus including a new species found from the shallow reefs of the Straits of Malacca.

Material and methods
Sea cucumbers were collected from the shallow coral reef areas of Pulau Payar (6°26'2.7"N, 99°40'54.8"E), Pulau Songsong (5°48'31.2"N, 100°17'38.0"E) and Pulau Sembilan (4°1'46.8"N,100°32'39.7"E) in the Straits of Malacca as shown in Figure 1. The sampling areas were situated in highly sedimented waters of the Straits of Malacca (Chua et al. 2000) with poor reef framework formation (Pillai and Scheer 1974). All sampling areas exhibited similar shallow reef flat at depths about 10-15m with gradual slope of sandy substrate extending to 30m depth. SCUBA diving was employed in collecting specimens using wandering transect covering an area of about 150-200 m 2 , .up to 30 m water depth during day and night. The sea cucumber specimens were fixed in absolute ethanol for two weeks and stored in 70% ethanol. Spicules were extracted from the tissues of the dorsal body, tip of the papillae, tentacles, and tube feet. The tissue were dissolved using commercial bleach and spicules were then washed several times with distilled water before transferring them to a glass slide to be observed under microscope. Pencil drawing of the spicules were done using a drawing tube attached to the microscope. The pencil drawings were then traced on a tracing paper using fine technical pens and digitized by scanning. All specimens were deposited at Marine Science Laboratory, Universiti Sains Malaysia (USM/MSL).

Stichopodidae Haeckel, 1896
Stichopus Brandt, 1835 Type species. Stichopus chloronotus Brandt, 1835 Diagnosis. Peltate-shaped tentacles at ventral mouth with quadrangular shaped body. Flattened ventral sole with tube feet in ambulacra area. Papillae conspicuous. No cuvierien tubules and no anal teeth or traceable papillae around the cloacal opening. Gonads in two tufts, one at each sides of the dorsal mesentery. Spicules primarily tables, branched rods and C-shaped rods (Clark and Rowe 1971).

Stichopus hirotai
Spicules: Dorsal body wall with tables, C-shaped rods, and S-shaped rods ( Fig.  3A-C). Tables abundant in dorsal body wall; base smooth with four large central perforations and 4-10 smaller peripheral holes; four pillars forming spires, joined with one crossbeam; tip of each pillar spiny. C-shaped rods smooth with sharp endings; some being irregular in form. S-shaped rods derived from C-shaped rods present. Dorsal papillae bearing C-shaped rods, S-shaped rods, elongated rods, and tables ( Fig. 3D-G). Tables in dorsal papillae with large disc, four central perforations, and multiple peripheral holes; pillars joined by one, sometimes incomplete, crossbeam; tip of pillars with multiple large spines. Ventral tube feet bearing plates, thick rods, tables, and C-shaped rods ( Fig. 3H-J). Large plates in ventral tube feet having distinctive larger central perfo-  A C-shaped rods from the dorsal body B S-shaped rods from the dorsal body wall C tables from the dorsal body wall D table with large base from the dorsal papillae E C-shaped rods form the dorsal papillae F S-shaped rods from the dorsal papillae; G rosettes from the dorsal papillae H tables from the tube feet I large rods with central perforations from the tube feet J C-shaped rods from the tube feet K perforated plates from the tube feet L reduced tables from the tube feet M rods from the tentacles N tables from the tentacles. rations; numerous smaller peripheral holes distributed over plates. Other smaller plates in ventral tube feet having perforations similar in size to each other. Edges of plates in ventral tube feet jagged but less pointy and sharp. Tables in ventral tube feet having reduced pillars or no pillar; one crossbeam incompletely joining all pillars; four central perforations with less peripheral holes compared to tables from dorsal body. Rods in ventral tube feet with 0-3 (mostly one) perforation(s) at central extension (Fig. 3I). Tentacles with tables and rods. Tables in tentacles reduced to only disk without pillars, with four central perforations surrounded by smaller peripheral holes; similar to tables in dorsal body wall (Fig. 3F). Rods in tentacles in various sizes, with spiny and rough surface; smaller rods straight while larger rods slightly curved (Fig. 3L).
Remarks. Stichopus chloronotus is fairly easy to be identified in situ due to the bright, distinctive green-blue colouration on the body with yellowish coloured papillae ( Fig. 2A), although there are some colour variations in the papillae across different localities (Massin et al. 2002). This species is distinguished from the other congeneric species also by its smooth body.
Large spicules with elaborated sculpted surface in the tentacles reported from Madagascar by Cherbonnier (1988) were not found in our specimens. This type of spicule is also missing from other descriptions by Théel (1886), Massin (1996) and Massin et al. (2002).
Distribution. This is a common species found throughout the Indo-West Pacific area (Clark and Rowe 1971). Type locality. Philippines. Description. External morphology: Large body with quadrangular cross-section with four distinctive sides; firm, rugose, and having thick folding surfaces. Uniformly greyish brown on dorsal side; light brown to yellowish on ventral body with an orange patch spreading from mid ventral body to anterior ventral body. Two rows of small and short papillae on dorso-lateral edges; papillae absent on ventro-lateral edges; tip of papillae brown; base grey-coloured. Numerous, smaller, brown-tipped papillae spreading across dorsal body. Laterally, papillae being lesser in number and density. Ring of minute papillae surrounding oral opening. Tube feet numerous in ambulacra areas. Central ambulacrum occupying more rows of tube feet compared to other two am- bulacra areas. Narrow interambulacra areas without tube feet. Twenty peltate-shaped tentacles. Anus terminal.

Stichopus herrmanni Semper, 1868
Spicules: Dorsal body mainly tables, C-shaped rods, rosettes, and pseudo tables ( Fig. 5A-D). Table spicules in dorsal body have rounded to quadrangular in shape bases, with four central perforations and numerous peripheral holes; three or four short pillars forming spires connected by a cross beam; tip of pillars with thorny crown endings (Fig. 5A). Rosettes in dorsal body abundant with various shapes and sizes; simple to complex bifurcation on both ends (Fig. 5C). Pseudo tables in dorsal body have four pillars extending from reduced base; no disk formed at base (Fig. 5D). Papillae consist of tables, C-shaped rods, and rosettes ( Fig. 5E-G). Base of tables in papillae with rough rims; four pillars with multiple spines on the tip erected from disc; four central holes on the disc with 2-3 peripheral holes. C-shaped rods and rosettes in papillae similar to those in dorsal body. Tube feet have large perforated plates, rods, and reduced tables ( Fig. 5H-J). Perforated plates in tube feet in rectangular and square shapes, jagged and pointy rims (Fig. 5H). Rods in tube feet with central extended perforations (Fig. 5J); surface covered with spinelets. Reduced tables of tube feet have base with four central perforations and 5-8 peripheral holes; reduced pillars liken knobs formed at central of base; rim of base being smooth (Fig. 5I). Tentacles containing rods of different sizes with rough surfaces covered with spinelets; slightly bended ( Fig. 5K).
Remarks. Stichopus herrmanni is originally described as a subspecies of Stichopus variegatus before Rowe and Gates (1995) reclassify and accorded S. herrmanni a species status. Stichopus herrmanni is closely related to Stichopus monotuberculatus (Quoy & Gaimard, 1833) but the papillae of S. herrmanni are clearly smaller and less conspicuous than those of S. monotuberculatus. Massin et al. (2002) noted that rosette spicules in S. herrmanni were highly variable in size and abundance. The presence of pseudo-tables found in the dorsal body of our S. herrmanni specimens was the first to be reported for the genus Stichopus. Pseudo-tables has only been found in Thelenota within the family Stichopodidae (Cherbonnier and Féral 1984;Cherbonnier 1988;Massin and Lane 1991;Massin 1999). It is not certain whether this spicule is commonly occurring in this species of different localities. Pseudo-tables can be used to distinguish S. herrmanni from other congeners if they are consistently present in specimens from other localities.
Distribution. Throughout the Indo-West Pacific (Clark and Rowe 1971). Type locality. Society Islands, French Polynesia Description. External morphology: Body slightly sub quadrangular in crosssection, with firm and rough surface; wrinkles on mid-dorsal area. Dominantly dark brown and yellowish in colour, with sporadic whitish-brown patches (Fig. 6). Papillae scattered across dorsal surface with larger papillae along dorso-and ventro-lateral areas; base of papillae dark grey, apex white. Ring of small papillae surrounding oral opening. Tube feet restricted in three ambulacra areas, with mid ambulacrum being wider with more rows of tube feet but a gap of lesser tube feet density in middle area of mid ambulacrum. Cream-white background colour with patches of brown dots across ventral body. Twenty peltate tentacles. Anus terminal.

Stichopus horrens Selenka, 1867
Spicules: In dorsal body, numerous tables and C-shaped rods spicules. Tables in dorsal body with four central perforations, 9-21 peripheral holes around smooth-surfaced base; four pillars with moderate heights forming spires joined with one crossbeam, tip of spires with large spines (Figure 7A-B). Spicules in dorsal papillae comprised of tackliked tables, rods, C-shaped rods, and perforated plates (Fig 7C-F). Four pillars erected and fused at tips forming tall spire from middle of base (Fig. 7C). Large rods have rough surface and tiny spines on surface, especially at both ends; central perforations can be elaborate or simple (Fig. 7E). Perforated plates in papillae have jagged rims and 6-12 irregular holes; smaller in size than those of tube feet. Tube feet with large rods, multiperforated plates, and tables ( Fig. 7G-I). Large rods have central plate with perforations; rod surfaces rough and covered with spinelets (Fig. 7G). In tentacles, spicules consist of rods in different size and thickness (Fig. 7J). All rods have rough surface and spinelets; slight curves in larger rods while smaller appeared straight.
Remarks. This species exhibits diverse body colours in specimens from different localities. Clark (1922) noted specimens from the Hawaiian Islands were dark olivegreen, mottled with deep brownish-green in colour, which were not seen in specimens from the Torres Strait. Domantay (1953) remarked that the colour of this species changes with age. The species S. horrens look very similar to S. fusiformiossa sp. n. but are readily distinguishable by the presence of tack-like spicules on the papillae of S. horrens and the absence of fusiform spicules on the tentacles in S. horrens. Detailed characters distinguishing these species are provided in the section describing S. fusiformiossa sp. n.
Distribution. Society Islands, Galapagos, Indian Ocean, Bay of Bengal, South China Sea, Southern Japan, Papua New Guinea, Philippines, Australia, Hawaii to New Caledonia. Type locality. Java, Indonesia. Description. External morphology: Body slightly quadrangular in cross-section, without distinct edges. Body surface smooth and tough. Black deep depression lines on dorsal body; dorsal background brown colour with numerous darker brown strips transverse and encircles the base of papillae. Two rows of large papillae with very low protrusion on dorsal body. Tube feet only in ambulacral areas; median tube feet twice wider than those in other two ambulacral areas; narrow interambulacral areas clearly separates each ambulacrum area. Reddish brown background colour on ventral side with lighter yellowish-brown on interambulacral areas. Ventral mouth with 18-20 peltate tentacles surrounded by ring of minute papillae at oral opening (Fig. 8). A Large tables from the dorsal body wall B rosettes from the dorsal body wall C C-shaped rods from the dorsal body wall D reduced tables from the dorsal body wall E rods with large perforated central plate from the tube feet F multiperforated plates from the tube feet G tables from the tube feet H C-shaped rods from the tube feet I tables from the dorsal papillae J large reduced tables from the dorsal papillae K C-shaped and S-shaped rods from the dorsal papillae L rosettes found from the dorsal papillae M large perforated plates from the dorsal papillae N rods from the tentacles O C-shaped rods from the tentacles Q tables from the tentacles. Spicule: Tables, C-shaped rod, and rosette spicules present in dorsal body (Fig. 9A-D). Tables in dorsal body with large quadrangular base, four central perforations, and 15-27 smaller peripheral holes; four pillars forming spire from the central of the base with a cross beam connecting them; tip of pillars with multiple thorny spines (Fig. 9A). Reduced tables in dorsal body having similar structure to the base of tables but lack pillars; slightly raised surface on the central (Fig. 9D). Spicule of papillae consists of tables, reduced tables, C-shaped rods, rosettes, and perforated plates (Fig. 9I-M). Tables in papillae similar to those in dorsal body; some with larger thorns on pillar apexes (Fig. 9I). Some C-shaped rods were modified liken S-shaped rods with protrusion at the middle body (Fig. 9K). In ventral tube feet, centrally-perforated rods, perforated plates, tables, and C-shaped rods (Fig. 9E-H) present. Centrally-perforated rods in tube feet have conspicuous large central plate and multiple perforations of different sizes; surface rough and covered with spinelets (Fig. E). Oval-to square-shaped perforated plates; rim were not smooth and jagged (Fig. 9F). Tables in tube feet have small base disc with four main central holes and 5-8 peripheral holes; four pillars erected from central of base with thorny crowns on the tip, but do not extend out of base (Fig. 9G). Small C-shaped rods in tube feet (Fig. 9H). Spicules of tentacles consists of rods, perforated plates, tables, and C-shaped rods (Fig. 9N-Q). Curved rods in tentacles in multiple different sizes; rough surface covered with spinelets especially dense at both ends. Perforated plates in tentacles spiny, rough surface covered with spinelets; some likens dichotomous rods (Fig. 9M). Tables and C-shaped rods in tentacles similar to ones found in the papillae (Fig. 9O, Q).

Stichopus vastus Sluiter, 1887
Remarks. Stichopus vastus is the only species in Stichopus that have elaborate reticulated stripes covering the dorsal body with different density and intensity (Fig.  8). Massin (1999) and Massin et al. (2002) demonstrated this coloration varies for specimens from across different localities. The black transverse line in the area between bulges is also unique to this species. Massin et al. (2002) mentioned S-shaped rods were found in the dorsal body in small specimens, and the absent in larger specimens (>160 mm in body length). C-shaped rods in the tentacles of the present specimens were also mentioned by Cherbonnier and Féral (1984) but not by Massin (1999) and Massin et al. (2002). Prominent large perforated plates found in the dorsal papillae have not been reported from this species and they look like dichotomous branching rods found in the tentacles.

Stichopus fusiformiossa
Description. External morphology: Body quadrangular in cross-section with slight rounded four sides. Body wall firm, rugose, and wrinkled surface; variouslysized tiny warts regularly-arranged on dorsal body (Fig. 10). Dorsal body brown in background colour, with patchy beige areas and black patches; latter usually running from anterior to posterior ends and concentrated in middle part of body, and absent in some specimens. Ventral body wall light beige background with two orange, narrow lines spreading from oral to aboral. Large dorsal papillae 0.5-1.0 cm in width at base in the holotype, but highly variable between specimens, more or less arranged in two continuous rows; smaller dorsal papillae scattered on dorsal body; colour of dorsal papillae black, with apex always white. Two rows of large (about 2 cm), white-coloured papillae aligned and arranged in straight line along ventro-lateral edges. Minute pa-  tiple peripheral perforations; four pillars erected from base forming a spire connected by a cross beam, with spines at the tip (Fig. 11A). Some tables in dorsal body having smaller base with less peripheral perforations; pillars connected incompletely with a crossbeam, tip of spire without crown of spines (Fig. 11D). Tables in dorsal body sometimes with pillars reduced to knobs and disc with four central perforations and limited peripheral perforations (Fig. 11B). Papillae consisting large tables, reduced tables, rosettes, C-shape rods, X-shaped rods, and rods ( Fig. 13A-I). Large tables in papillae with multiperforated base disc; four pillars forming spire connected with a crossbeam, tip of pillar very spiny (Fig. 13A). Reduced tables in papillae larger compared to reduced tables in dorsal body (Fig. 13B). Rosettes in papillae form simple curving to extensive bifurcations at both ends (Fig. 13D); smaller rosettes more complex in bifurcations (Fig. 13G). C-shaped rods in papillae simple, some modified to S-shaped rods (Fig. 13E). X-shaped rods in papillae have bifurcate endings in each arms; some with five arms (Fig. 13H). Rods in papillae with rough surface (Fig. 13I). Spicules in tube feet mainly large rods with perforated central plate, multiperforated plates, and tables ( Fig. 12E-G). Surface of rods in tube feet rough, covered with spinelets; numbers and sizes of perforations on central plates of tube feet vary between rods. Tables in tube feet simpler compared to tables in dorsal body and papillae; tip of pillars less spinous and forming small crowns. Spicules in the tentacles consisting of fusiform spicules and rods (Fig. 11A, B). Fusiform spicules spindle-like in shape with dense spines interconnected to each other forming some hollow spaces in between (Fig. 14B). Rods in tentacles rough, slightly curved, and densely covered with spinelets at both ends.
Remarks. This new species looks very much like S. horrens in its body colouration. They both have similar, grey-brown background with irregular grey and black spots in colour. The live specimens of this species do not have very long papillae as observed in S. horrens reported by Rowe and Gates (1995) and Massin et al. (2002). Stichopus fusiformiossa also lacks tack-like table spicules in the papillae which are the definitive taxonomic feature for S. horrens. The prominent white and black colour of the papillae is distinctive to separate S. fusiformiossa from S. rubermaculosus with the red-coloured papillae, S. quadrifasciatus that has brown to red papillae tip, and from S. chloronotus with yellow-coloured papillae. Furthermore, S. quadrifasciatus has four transverse black-grey bands on the dorsal body wall, which are not seen in S. fusiformiossa. The sporadic arrangement of papillae on the dorsal body of S. fusiformiossa do not have consistent nor specific patterns as similarly observed in S. horrens in this study. However, the two rows of large, black papillae with white tip, arranged in a straight line along ventro-lateral edges are not seen in any other species.
Stichopus fusiformiossa lacks C-shaped rods in the dorsal body, which is commonly seen in the other Stichopus species. Reduced tables are found in the dorsal body in S. fusiformiossa and S. herrmanni, but the former lacks rosettes and C-shaped spicules that are present in the dorsal body of S. herrmanni. The C-shaped rods in the papillae of S. fusiformiossa are strongly modified and do not resemble to any spicules observed in the other Stichopus species except the C-shaped rods of S. chloronotus (Theel 1886;Sluiter 1887;Ludwig 1887;Mitsukuri 1912). But the colouration and arrangement of papillae easily separate S. fusiformiossa from S. chloronotus.
The X-shaped rods in the papillae of S. fusiformiossa have been only found in S. variegatus (now S. herrmanni) by Cherbonnier (1988). The X-shaped rods of S. variegatus (now S. herrmanni) are derived from rosettes (Cherbonnier 1988). Careful examination of his drawings (Cherbonnier 1988: fig. 62H) suggested that X-shaped rods of S. fusiformiossa were more rigid and angled compared to the curvy and slender ones of S. variegatus (now S. herrmanni).
The thick rods with rough surfaces seen in S. fusiformiossa are commonly found in the tentacles of any other species of Stichopus. Fusiform and spindle-liked spicules instead are rare and only shared with S. variegatus (now S. herrmanni) (Cherbonnier 1947, fig . C). Since S. variegatus had been separated to either S. herrmanni and S. monotuberculatus by Rowe and Gates (1995), the presence of fusiform and spindle-like spicules can be accorded to S. fusiformiossa as a character differentiating it from both S. herrmanni and S. monotuberculatus because both S. herrmanni and S. monotuberculatus lack the presence of this spicule. A reexamination of specimens of Cherbonnier (1947) collected from the Gulf of Oman, Madagascar, and the Red Sea are necessary to establish the correct species name of those specimens.
Etymology. The new specific name is a compound descriptive name from the combination of adjective-noun derived from the Latin words of fusiform (fusiformis) and bone (ossa). The name is nominative, neuter, and plural; referring to the distinctive fusiform and spindle-liked spicules found in the tentacles.