Investigating the variability of the morphological characters classically used to describe species of Opheodesoma (Echinodermata: Holothuroidea: Synaptidae) Laura J. Kenyon UFID: Redacted Senior Honors Thesis Advisor: Dr. Gustav Paulay Florida Museum of Natural History, University of Florida, Gainesville, FL 32611-7800 USA ABSTRACT: Opheodesoma is one of the genera belonging to the sea cucumber family Synaptidae, found in shallow tropical waters. There are 11 species names currently used. The species described were differentiated based on morphological characters exclusively. However, Opheodesoma specimens exhibit a lot of variation in their morphology and species names have been revised throughout the past century. Due to the extent of the variation observed, and because the species were described from a limited number of individuals, it is probable that some of the currently described species represent intra-specific variation. We hypothesize that morphological characters previously used are too variable to distinguish between species. A phylogenetic tree based on a portion of the mitochondrial marker CO1 sequenced from 40 specimens reveals three genetically distinct groups. We tested several morphological characters to see if they co-varied with the mtDNA data. We did not find diagnostic morphology to distinguish between the two closest related groups, supporting our hypothesis. If further investigations are unable to distinguish the two groups, they must represent a single species. However, the third genetically distinct group did have morphological characters that co-varied with the mtDNA sequence data and appears to be an un-described species. KEYWORDS: phylogenetics, COI, sea cucumber, Synaptinae, Micrournae 2 INTRODUCTION: Opheodesoma belongs to Apodida, a unique lineage of sea cucumbers. Apodida is sister to all other sea cucumbers and is unified by the lack of tube feet, respiratory trees, and radial water canals (Kerr 2001). Apodida contains three families: Chiridotidae, Myriotrochidae, and Synaptidae. Of these, synaptids are the most diverse in shallow water habitats and are only found on reefs or hard bottoms. This monophyletic group is distinguished from the others by the presence of anchor and anchor plate ossicles (calcium deposits). Synaptidae contains 11 genera belonging to two subfamilies and includes Opheodesoma (Clark 1907). Opheodesoma is part of Synaptinae, the subfamily of Synaptidae found in tropical waters (Clark 1907). Opheodesoma belongs in the subgroup Micrournae which also includes Synapta, Polyplectana, Synaptula, and Euapta (Heding 1928). These genera are united by their ossicles. The anchor ossicles have smooth arms and minute knobs at the vertex and the anchor plates contain a bridge around the base. There are also miliary granules dispersed around the body wall (Heding 1928). Micrournae have a distinct form of ciliated funnel (Heding 1928). Ciliated funnels are small (70 to 150 µ) funnel-shaped bodies made of connective tissue that are ciliated and reside in the mesenteries (Clark 1907). Micrournae is found in shallow warm waters. It includes the longest sea cucumber in existence, Synapta maculata, which can reach over 3 meters, yet also contains species that only reach a few millimeters. They generally contain 12-15 tentacles like other synaptids, but Polyplectana is characterized by having around 25 tentacles (Clark 1924). Micrournae reproductive physiology is also variable, ranging from hermaphrodism to sexual dimorphism and from free-spawning to viviparity (Clark 1924). They are deposit-feeders and are known to 3 occasionally form large (up to 1,000 animals) aggregations where an abundance of food is available (Berrill 1965). Micrournae is an important and dominant group on coral reefs and are often encountered by divers. Opheodesoma is one of the 5 monophyletic clades in Micrournae and is sister to Euapta. It is differentiated from Euapta by the presence of numerous Polian vesicles, which are part of the water-vascular system (WVS) in sea cucumbers, and the presence of numerous madreporites, which also function in the WVS. Opheodesoma is further differentiated from Euapta by the notable constriction of the base of Opheodesoma anchor plates. The current species names of Opheodesoma are still poorly understood. The genus was first described by Fisher in 1907 and contains 11 currently used names based strictly on morphological characters. There are over 14 available names. Currently used names are often disagreed upon. For instance, O. mauritiae and O. africana were used until they became junior synonyms to O. grisea (Cherbonnier 1988). However, O. mauritiae was used in a 2003 publication (Samyn 2003). O. ramispicula and O. australiensis were both described by Heding in 1931, but O. ramispicula was later synonymized with O. australiensis (Clark 1946). The presently accepted species of Opheodesoma are characterized by body wall coloration, webbing between the digits of the tentacles, thickness of the cartilaginous ring, the presence of rods (ossicles) in the oral disc, the presence of rods in the tentacles, and the color of the calcareous ring (Table 1). Other characters, less consistently used, include the thickness of the body wall, the shape of the tentacles, the distribution of miliary granules, the size of the anchors, and the size of the anchor plates. The lack of clarity in the species descriptions and the tendency for species names to be revised throughout history is due to the high amount of intra- 4 and inter-specific variability in morphology. Previous authors have recognized this variability but lacked other ways to differentiate organisms (Clark 1907). Also, many of these characters are subjective and difficult to interpret. In addition, previous species descriptions have often been based on only one or a few specimens. Thus, the amount of variation possible within a single species or within a single specimen has not been explored. As a result, we are unclear of the valid species present. We must expand this investigation past just morphological data. With modern genetic technology burgeoning, we propose to incorporate molecular data with morphology. The use of genetic data will provide insight to the current valid species present. Since the mitochondrial maker Cytochrome Oxidase I (COI) has been widely used in Echinoderms, including many genera of sea cucumber, we propose the use of this maker in Opheodesoma (Ward 2008). As a mitochondrial marker, COI is expected to evolve at a faster rate than most nuclear markers, and can provide resolution among closely related organisms. A combination of COI data with morphology will reveal the extent of the variability seen in classically used characters. The purpose of our investigation is to first see how many genetically distinct groups are present in the Florida Museum of Natural History collection of over 40 specimens, and then to assess the variability of morphology within these genetic groups. Given the extent of the variation recorded in historical descriptions, we hypothesize that the amount of variation seen within genetically distinct groups will parallel or exceed the amount of variation seen between groups. This would suggest that the characters classically used are too variable to describe species. 5 METHODS: We studied 40 specimens from the Florida Museum of Natural History collections. We examined many of these specimens for the characters historically used in classification. We also included a new morphological trait to examine. We tested to see if the morphology covaried with COI data. Methods for the morphological investigation: Cartilaginous ring: We did not mark the thickness of the cartilaginous ring. The anterior portion of the animal must be dissected, with tissue removed, in order to see the cartilaginous ring. It is invasive and can destroy the preserved specimen. Heding marked the ring as either “voluminous” or “not voluminous or wanting” with no specific measurements, making the character state very subjective (Heding 1928). The trait is known to be highly variable in a single species and can grow with age (Clark 1924). Thus it would not be useful in this study. Anchors and Anchor Plates: We decided not to mark the size of the anchors or anchor plates. The sizes of both of these ossicle types are variable and overlap among specimens (Clark 1924). Also, anchors and anchor plates grow throughout development, making size designation an unreliable character (Clark 1924). Body wall coloration: Striped/Not Striped. We only examined live photos since preservation can alter coloration (Cherbonnier 1951). We were only able to examine 15 out of the 40 specimens, due to the lack of live photos available. We determined if the body wall had longitudinal striping present or absent. If the animal was blotched with different colors, or had small horizontal bands, it was marked as having no striping. 6 Webbing between digits: Present/Absent. We marked 23 specimens. Webbing exists between the digits (of the tentacles) as a thin membrane. This is hard to see if the animal is contracted or not-well preserved. Specimens that were too contracted or did not have well preserved digits were excluded from the examination. Examination requires extensive microscopy due to the minute size of the digits. This trait is continuous, thus we only marked specimens where the presence or absence was obvious. To maintain a consistent marking strategy, one person alone went through each of the specimens to mark the trait; thus, ambiguity associated with different definitions of webbing would be avoided. Rods in the oral disc: Present/Absent. We examined a piece of oral disc tissue under a light-microscope. If the tissue was not thin enough to detect ossicles, the tissue was dissolved in bleach, washed three times with de-ionized water, dried, and mounted onto a viewing slide with Euperal. The slides were then analyzed for ossicles. We marked 12 specimens. Rods in the tentacles: Present/Absent. We marked 29 organisms. We isolated a piece of tissue from the tentacle and examined it with a microscope. Like with the oral disc, if the tissue was not transparent enough to see through, we prepared a permanent slide.