Expansion of the genus Massinium (Holothuroidea: Thyonidae) to the American continent and description of a new species

Introduction: The genus Massinium includes 11 species, most from the Indo Pacific Ocean, and had not previously being reported from the American continent. Objective: To present the new record of the genus Massinium and describe a new species of this genus. Methods: Material collection was done by SCUBA-diving to depths of a maximum of 9 m. Results: Massinium ocumichoensis sp. nov. is described from seven specimens that extends the range of the genus to the American continent (Guerrero, Michoacan and Jalisco, Mexico). The new species lives in sandy-rocky substrata, from 2 to 16 m deep. It is distinguished from its congeneric species by a combination of morphological characters: mid-dorsal slightly bigger pseudobuttons (40-60 μm), and tables in the body wall. A taxonomic key for distinguishing the species of Massinium is provided. Conclusions: The geographic range of the genus Massinium is extended to the Mexican Pacific with M. ocumichoensis sp. nov.

genus Massinium Samyn & Thandar, 2003 was included in the subfamily Semperiellinae Heding & Panning, 1954by Thandar (2017, this genus was erected to accommodate sea cucumbers which have the posterior processes of their tubular calcareous ring distally joined to form a ring-like structure around the esophagus and body wall with granuliform rods and/or rosette-shaped deposits and tables. Samyn and Thandar (2003) and Samyn, Thandar, and Van-denSpiegel (2010) included six species in the genus: the Australian endemic M. granulosum Samyn, Thandar and VandenSpiegel, 2010; the New Caledonian endemic M. albicans Samyn, Thandar and VandenSpiegel, 2010; the central Indo-Pacific M. magnum (Ludwig, 1882), the southern African endemics M. arthroprocessum (Thandar, 1989), M. maculosum Samyn & Thandar, 2003 and M. dissimilis (Cherbonnier, 1988)  There are no previous records of the genus on the American continent waters. Seven specimens were collected along the Mexican Pacific, representing a new record of the genus and a new species.

MATERIALS AND METHODS
During recent surveys of the echinoderm fauna of the Mexican Pacific, seven specimens belonging to this new species were found. Collection was done by SCUBA-diving to depths of a maximum of 9 m. Specimens were anaesthetized in a solution of 4 % magnesium chloride and seawater. After properly labelled, fixation of the specimens was done with 70 % ethyl alcohol. All measurements were obtained from fixed specimens. Ossicles were extracted from the body wall (anterior, medium and posterior region) introvert and tentacles. The tissue was dissolved in fresh household bleach (5-6.5 %) in centrifuge tubes. After centrifugation at 1 000 rpm for 10 min, bleach was pipetted off and the ossicles were rinsed and centrifuged with distilled water that was pipetted off afterwards. The same process was done with 70, 80, and 95 % ethanol. Absolute ethanol was added to the ossicles, and finally a small aliquot was taken and placed to dry on a cylindrical double-coated conductive carbon tape stub. Then it was sputter coated with gold 2.5 kV in the ionizer Polaron E3000 for 3 min and photographed using a Hitachi S-2460N scanning electron microscope (SEM). Photographs of the holotype specimen were taken using a Leica DFC490 digital camera, and a Leica Z16 APOA stereomicroscope. The examined material is deposited in the ICML-UNAM and LACM.  Grube, 1840 Family Thyonidae Panning, 1949(sensu Smirnov, 2012 Diagnosis: Dendrochirotida with 10-20 tentacles. Tube feet scattered all over body or restricted to the radii. Calcareous ring segmented. Ring often tubular, forked processes of radial segments long, prominent. Inter-radial segments often strongly elongated basally fused with processes of radial segments; both calcareous ring segments, and processes subdivided into many pieces. Ossicles: tables with two or four pillars and/or plates; sometimes baskets present (modified from Smirnov, 2012).

Subfamily Semperiellinae
Heding & Panning, 1954 Diagnosis: (modified from O'Loughlin et al., 2012 andSmirnov, 2012). A polytentaculate subfamily of Thyonidae with 15-20 tentacles. Tube feet lying along radii or scattered over entire body. Calcareous ring well developed, often tubular, radial and inter-radial plates always broken into a mosaic of pieces, posterior processes of radial and often the inter-radial plates sub-divided, and processes of radial and sometimes the inter-radial plates joining posteriorly, the latter forming a ribbon-like ring surrounding the water vascular ring. Body wall ossicles 2-4 pillared tables or their derivatives.
Genus Massinium Samyn & Thandar, 2003 Diagnosis: (modified from O'Loughlin et al., 2014). Slim, elongated body, sometimes semi-spherical forms; anterior and posterior parts of the body narrow, with oral and anal dorsal orientations; 20 dendritic tentacles arranged in two circles of ten large outer and ten small inner (proximal peri-oral); calcareous ring elongated, tubular, with both radial and inter-radial plates fragmented into a mosaic of pieces, and posterior prolongations linked distally to form inter-radial oval non-calcified spaces beneath the water vascular ring; polian vesicles from one to four; ossicles variably include granuliform rods, rosettes, pseudobuttons and tables; table spires with one or two or three or reduced pillars.
Massinium ocumichoensis sp. nov. Diagnosis: Small-sized (60-74 mm TL), U-shaped thyonid with bloated mid-body and relatively short anterior and posterior ends. Anus surrounded by ten small papillae, variously developed. Tube feet scattered over entire body. Ossicles of body wall two pillared tables and pseudobuttons, often with minute perforations. Introvert ossicles include pseudobuttons, tables and rosettes. Tentacles of outer ring with smooth, slightly curved rods, perforated at the ends, external tips of the outer tentacles ring devoid of ossicles, tentacles of inner ring with rods and rosettes. Body color off-white to grey, mottled with dark brown, sometimes black; tentacle dendritic branches black, trunks transparent with black moles.

Etymology:
The name ocumichoensis refers to Ocumicho, Michoacan (same state as the type locality), a famous small town where folk artisans create colorful clay magical devils and beasts.
Description of holotype: Specimen entire, well preserved, dissected (Fig. 1A). Mouth anterior, anus posterior, slightly developed anal papillae; body wall firm, leathery, rather thin (0.8-1 mm), slightly rough to the touch. Body form cylindrical, slightly contracted, bloated, with narrow anterior and posterior ends. Introvert well extended, attached to the main body. Length of specimen along ventral surface 74 mm; along dorsal surface 64 mm; height of mid-body approximately 18 mm; anterior and posterior ends approximately 20 and 18 mm long; introvert 8 mm long. Coloration of body and introvert beige brown with brown small moles. Tube feet of body wall beige, numerous, small, mostly retracted, uniformly scattered over entire body, very small suckers; close cover ventrally and around mouth and anus; tube feet of introvert brownish, darker proximally, aligned in radial areas in two welldefined rows, absent in distal 3 mm. Tentacles 20, ten large, ten small, arranged in two rows (Fig. 1B); outer tentacles, when alive, 35-40 mm long, black tips, middle part shaft whitish with black moles; when preserved the tentacles are highly contracted (9 mm). Calcareous ring short (10 mm), tubular, composite; both radial and interradial plates broken into a mosaic; radial plates elongated, with a shallow central notch, central part constricted and posterior part divided in two short tails, inter-radial plates triangular pointed anteriorly, large oval un-calcified posterior section closed distally by non-calcified link; one madreporite, well calcified, stone canal scalloped and attached to one extension of the interradial plate posterior processes of the calcareous ring (Fig. 1C); four polian vesicles (Fig. 1C); respiratory trees extending the length of the coelom.
Ossicles in mid-body wall are large pseudobuttons (40-60 μm) ( Fig. 2A), and tables sparse dorsally (Fig. 2B) table disc outlines irregularly round, typically four larger central and some small outer perforations, margin smooth or spinous, discs up to 42 μm wide; tables frequently with two pillars, low or residual, few thick blunt apical spines. Anterior and posterior body wall areas with small pseudobuttons (15-30 μm) ( Fig. 2A). Oral disc with abundant tables with discs up to 35 μm long, irregularly oval, many perforations, margins smooth; spires short, up to 10 μm tall, two pillars, rarely single, one to two median perforations, short apical spines typically splayed (Fig.   2B). Peri-anal body wall with abundant tables similar to those in the mid-body wall; some rods with distal perforations, rods up to 80 μm long; anal papillae with small multi-layered anal scales about 45 μm long. Introvert with pseudobuttons, tables and rosettes (Fig. 2C,  2D, 2E). Tube feet with endplates, up to 40-45 μm diameters, margin thick (Fig. 2F), with support ossicles as smooth rods with perforated ends (Fig. 2G). Tentacles of outer ring with abundant simple slightly curved rods perforated at the ends and granuliform rod ossicles; simple rods up to 50 μm long with perforated ends; granuliform rods up to 55 μm long with short rod widened distally with many small perforations created by dendritic branch fusing (Fig. 2H); abundant well-elaborated rosettes (Fig. 2I). External tips of the outer tentacles ring devoid of ossicles, tentacles of inner ring with rods and rosettes.
Body live color off-white to grey, mottled with dark brown, sometimes black; tentacle dendritic branches black, trunks transparent with black moles; color preserved off white to pale beige, tube feet beige, tentacles black, trunks off-beige to black (Fig. 1B).

Description of paratypes:
The paratypes are from 40 to 74 mm long. The ossicles of the body wall and other parts of the body are similar to those in the holotype. The calcareous ring in 40-50 mm TL specimens is small and the mosaic is very difficult to observe. Preserved specimens color varies from light to dark grey. Some specimens have light beige body and beige to brown tube feet. Ecology: This species lives buried deeply in sand (10-15 cm deep), under boulders and rocks exposing its tentacle crown only when feeding. Upon slightest disturbance, even shading, the tentacle crown and the introvert are swiftly retracted into the body leaving no trace of the animal.
M. ocumichoensis sp. nov. is distinguished from other species in the genus by the combination of morphological characters shown in the taxonomic key.  Ethical statement: authors declare that they all agree with this publication and made significant contributions; that there is no conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements. All financial sources are fully and clearly stated in the acknowledgements section. A signed document has been filed in the journal archives. Introducción: El género Massinium incluye 11 especies, la mayoría del Océano Indo-Pacífico, y no se había reportado previamente en el continente americano. Objetivo: Presentar el nuevo registro del género Massinium y describir una nueva especie de este. Métodos: La recolección de material se realizó mediante buceo a una profundidad máxima de 9 m. Resultados: Massinium ocumichoensis sp. nov. se describe a partir de siete ejemplares que extiende el rango de distribución del género al Continente Americano (Guerrero, Michoacán y Jalisco, México). La nueva especie vive en sustratos arenosos-rocosos, de 2 a 16 m de profundidad. Se distingue de sus especies congenéricas por una combinación de caracteres morfológicos: pseudobotones de la zona dorsal media ligeramente más grandes (40-60 μm) y tablas en la pared del cuerpo. Se proporciona una clave taxonómica para distinguir las especies de Massinium. Conclusiones: El rango geográfico del género Massinium se extiende al Pacífico mexicano con M. ocumichoensis sp. nov. Palabras clave: Echinodermata; Dendrochirotida; biodiversidad; taxonomía; nuevo registro.