Additions to the Morphology of the Cucumariidae. I. Intraspecific Variation of the Retractor Muscle in Parathyone braziliensis (Verrill, 1868) and Thyonidium seguroensis (Deichmann, 1930) (Holothuroidea: Dendrochirotida)

Intraspecific variation of the gross morphology of the retractor muscle was studied in 60 specimens of Parathyone braziliensis and 17 specimens of Thyonidium seguroensis. Specimens were dissected to expose the retractor muscle or were analyzed by micro-computed tomography. Early expectations that the point of origin of the retractor muscle at the longitudinal muscle relative to the midpoint of the body length was age-related and that the split of the retractor muscle into separate muscle bands could be used as taxonomic characters to separate species were not supported by anatomical evidence.


Introduction
The muscular system of Holothuroidea is subdivided into somatic and visceral. The visceral musculature is composed of a coelomic epithelium or mesothelium, whilst the somatic musculature comprises large muscles with different organizations and functions (Hyman 1955). The somatic muscles are arranged into five longitudinal muscle bands that extend from the posterior to the anterior end of the body wall occupying radial positions. The longitudinal bands extend posteriorly along the bulb toward the calcareous ring, until they finally attach to the anterior process in the anterior notch of the calcareous ring (Martins et al. 2021). The circular muscles are embedded in the body wall (Ludwig 1889(Ludwig -1892Hyman 1955;Hendler et al. 1995).
In Dendrochirotida and a few Molpadida (Molpadia Cuvier 1817), there are in addition five retractor muscles that run from the longitudinal muscles across the coelom and attaches to the corresponding radial plates of the calcareous ring. These muscle groups are responsible for locomotion based on peristalsis (longitudinal and circular) and the retraction of the tentacles and introvert (longitudinal and retractors) ( Fig. 1) (Hyman 1955;Heffernan and Wainwright 1974).
Contrary, the retractor muscle departs from the longitudinal muscle posteriorly to the midpoint of the body length in Oloughlinia discovery (Heding, in Heding and Panning  In none of these accounts, however, illustrations were provided as support for the descriptive material nor were the observations complemented by information on variations based on adequate sample size. In order to fill knowledge gaps on the intraspecific variation of the gross morphology of the retractor muscle, 60 specimens of the cucumariid Parathyone braziliensis (Verrill 1868) and 17 specimens of Thyonidium seguroensis (Deichmann 1930) were dissected to expose the retractor muscle or were analyzed by micro-computed tomography. The observations and illustrations of the variation in form, point of origin and attachments of the retractor muscle to the calcareous ring form the base of this report.  (Verrill 1868). a Diagram of the muscular system, calcareous ring and tentacles, lateral view: LMyellow (longitudinal muscle); RM-pink (retractor muscle); CR-gray (calcareous ring). b1 Retractor muscle type 1 and calcareous ring, ventral view (MZUSP 1143, 40 mm). b2 Detail of retractor muscle type 1, ventral view (MZUSP 2109, 30 mm). b3 Diagram of the retractor muscle type 1, lateral view. c1 Retractor muscle type 2, circular muscles (CM) and calcareous ring, ventral view (MZUSP1094, 30 mm). c2 Detail of retractor muscle type 2, ventral view (MZUSP 1383, 50 mm). c3 Diagram of the retractor muscle type 2, lateral view. DIS, PROX, distal and proximal ends, respectively

Material and Methods
Information on size, form and point of origin of the retractor muscle for specimens of Parathyone braziliensis (Verrill 1868) and Thyonidium seguroensis. provided in Tables 1 and  2 respectively. Measurements (given in millimeters, mm) refer to length from mouth to anus. All the specimens were preserved in ethanol prior to measurements being made. Specimens were dissected and examined under a Zeiss stereoscopic microscope accoupled with software Axio vision V 4.8.
The dermal ossicles were sampled from different body regions (i.e. dorsal, ventral, introvert, tentacles and tube feet), liberated with household bleach after being washed five times in distilled water and then, five times in absolute ethanol. To confirm the identity of specimens, a number of ossicle samples were mounted on slides with Entellan Merck ® for permanent storage and then examined and measured under optical microscope. Micrometric data were obtained from 30 measurements for each ossicle category. Other ossicle samples were dried and mounted on metal stubs with double-sided tape, coated with gold and observed with a LEO 440 Scanning Electron Microscope (SEM). Selected specimens were used for illustrations (Figs. 1, 2, and 3).

Image Acquisition
In order to scan the calcareous ring, whole and non-dissected specimens were placed in a straw-shaped plastic container sealed in both ends using parafilm and narrow enough to prevent movement while not squeezing the specimen. Scanning was performed using a microfocus X-ray µCT system (GE phoenix v|tome|x, Wunstorf, Germany). The scanning parameters were the following: detector size of 2,014 × 2,024 pixels, source voltage 50-60 kV, current 85-300 µA, voxel size 2.6-21.1 µm, 1,000-1,500 images; X-ray projection images were recorded at 1000 ms of time exposure per image. To improve image resolution, a multiscan of the whole sample was produced based on three individual scans.

3D Reconstruction and Software
Raw data reconstruction was performed using the systemsupplied software phoenix datos|x reconstruction v. 2.3.0 (General Electric Measurement & Control Solutions, Wunstorf, Germany). Three-dimensional visualization, segmentation and the analysis of the reconstructed data was performed using VGStudio MAX 2.2.3 (Volume Graphics GmbH, Heidelberg, Germany). Plates were prepared using Adobe Photoshop CS and Adobe Illustrator CS (Adobe Systems, San Jose, U.S.A.).

Diffusion-based Staining
The internal organs and their connections to the calcareous ring were observed by submerging the entire body of a specimen of Parathyone braziliensis in a solution containing 0.3% phosphotungstic acid (PTA) for two hours, following Metscher (2011). The specimen was then removed from the staining solution, placed into the plastic tube and µCT-scanned using the same parameters as the unstained specimens. PTA staining does not alter the specimen coloration.
A diagram depicting the muscular system circular, longitudinal and retractor muscle and its respective terminology is given in the Fig. 1. Otherwise, the following abbreviations and acronyms were used: AP, Anterior processes; Spec(s), specimen(s); SEM, Scanning Electron Microscope; Prox, proximal; Dis, distal; CR, calcareous ring; LM, longitudinal muscle; RM, retractor muscle and CM circular muscle. The studied specimens were deposited at the Museu de Zoologia, Universidade Federal da Bahia, Salvador, Brazil (UFBA) and Museu de Zoologia, Universidade de São Paulo, São Paulo, Brazil (MZUSP).

Parathyone braziliensis (Verrill 1868)
Remarks The intraspecific variations in the gross morphology of the RM fall into two types: RM type 1. Retractor muscle single, short, tapering both proximally and distally, splitting from LM anteriorly to midpoint of body length; proximal end attached to radial plate of CR (Fig. 1b1-b3). RM type 2. Retractor muscle single, long, tapering distally, proximal end distinctly widened, spatuliform, splitting from LM posteriorly to midpoint of body length; proximal end attached to both radial and interradial plates of CR (Fig. 1c1-c3).
Young specimens (ranging from 10 to 25 mm) were consistent in possessing only RM type 1. However, as size increases specimens ranging from 30 to 60 mm possessed RM type  1 3 2 in addition of type 1. Indeed, in 33 specimens ranging from 30 to 60 mm, the RM splits from LM anteriorly to the midpoint of the body length (RM type 1), whereas in 17 specimens (also ranging from 30 to 60 mm), the RM splits from LM posteriorly to the midpoint of the body length (RM type 2). In no specimens was the point of origin of the RM about at the midpoint of the body length in P. braziliensis (Table 1).

Thyonidium seguroensis (Deichmann 1930)
Remarks The LM can be of single or double-type in Thyonidium seguroensis; single and double LMs occur with single and double RMs, respectively. Hyman (1955: 134, 140) had already mentioned without further specifics that "When the longitudinal bands are paired, the retractors may also be paired" in Dendrochirotida and a few Molpadonia" [Molpadida] and Apoda. Fig. 2 a- In Thyonidium seguroensis the RM always splits from LM anteriorly to the midpoint of the body length, whether it be of single-or double-type. Single and double RMs are both broad widened and spatuliform proximally, ending in an acute tip that attaches to the radial plate of CR. Whilst single-type RMs split as a slender band from LM (Table 2; Fig. 3b, c), double-type RM's are spatuliform at the point of origin from LM, then narrow abruptly to form slender bands, which will ultimately fuse together before reaching to the CR (Table 2; Fig. 3d-f).
Although both LM and RM were of single-type in three young specimens with 15 mm, 25 mm and 40 mm, the occurrence of single-and double-type LMs/RMs does not seems to be age-related in T. seguroensis. Indeed, among 15 specimens ranging from 60 to 180 mm, eight were of singletype and seven of double type (Table 2).

Discussion
The findings reported here are reminders of how incomplete is the knowledge of the muscular system in Holothuroidea. The dendrochirotids encompass almost 50% of the living holothuroid species and are known to have the more developed and complex calcareous rings among all Holothuroidea. The calcareous ring is the is point of insertion for the longitudinal and retractor muscles among other roles (Hyman 1955) and, therefore, it should not constitute surprise that the diversity of the retractor muscle is greater than previously recorded in Dendrochirotida. Ludwig (1889Ludwig ( -1892 noted that the retractor muscle arises posteriorly to the midbody in young forms of Cucumaria frondosa (Gunnerus 1767) (Cucumariidae) and surmised that the point of origin of the RM from the LM was age-related, a deduction not supported by the dissection of young forms of P. braziliensis and T. seguroensis in which the retractor muscle arises anteriorly from the midbody.
Early findings also fed expectations that the relative length of the retractor muscle (i.e. the point of origin of the retractor muscle at the longitudinal muscle relative to the midpoint of the body length) and the split of the RM into separate muscle bands could be used as taxonomic characters to separate species (e.g. Ludwig 1889Ludwig -1892Heding and Panning 1954). However, the intraspecific variations found here in Parathyone braziliensis and T. seguroensis did not confirm these initial expectations.