A new species of Ovabunda (Octocorallia, Xeniidae) from the Andaman Sea, Thailand with notes on the biogeography of this genus

Abstract A survey of xeniid octocorals was carried out in the waters off Southwestern Thailand in September, 2007. Microscopic investigation of the colonies revealed that three specimens belonged to the genus Ovabunda. Gross morphological examination is presented here accompanied by scanning electron micrographs of the sclerites. Molecular phylogenetic analysis showed identical genotypes at mtMutS, COI, and 28S rDNA for all three specimens and supports their generic assignment. Colony size and shape, sclerite size, and pinnule arrangement differ from nominal species of Ovabunda and thus a new species, O. andamanensis is introduced here. This work also presents a new eastern geographical record for the genus Ovabunda.

Here we describe a new species of Ovabunda from recent collections in the Andaman Sea. Additionally, we report the first record of this genus outside of the eastern Indian Ocean and Red Sea.

Collection and Morphological examination
All specimens were collected using SCUBA to a maximum depth of 10 meters. Photographs of living colonies were taken for each specimen. Specimens were fixed in 90% ethyl alcohol immediately after collection. The corals examined in this survey are deposited in the reference collection of the Phuket Marine Biological Center, Phuket, Thailand (PMBC).
Morphological examination of the preserved colonies was performed under a dissecting microscope at 20× power. Polyps were photographed; number of pinnule rows, pinnules along the outermost row and inner row when present for each specimen were recorded. Sclerites were prepared for light microscope examination (Janes 2008b); 20-25 sclerites were selected and measured to the nearest 0.001 mm on a compound microscope fitted with a Filar Micrometer. Scanning electron microscope (SEM) images of sclerites were made from preparations following the method given by Aharonovich and Benayahu (2011). Sclerites were sputter coated with a gold/palladium blend for 75 seconds. They were then examined using a FEI XL30 Environmental Scanning Electron Microscope (ESEM) at 10 kV and a Nova 200 NanoLab Focused Ion Beam (FIB) Microscope at 5 kV, 0.40 nA.

Molecular phylogenetic analyses
Extraction of DNA from ethanol-preserved tissue samples, PCR amplification, and sequencing of two mitochondrial (mtMutS, COI + igr1) and a nuclear (28S rDNA) gene followed the protocols published in McFadden et al. (2014a). Sequences were aligned to a reference set of published sequences for other xeniid taxa (Haverkort-Yeh et al. 2013;McFadden et al. 2014b) using the L-INS-i method in MAFFT (Katoh et al. 2005). Pairwise genetic distances (Kimura 2-parameter) among specimens were calculated using the DNADist program in PHYLIP v. 3.69 (Felsenstein 2005). MOTHUR v. 1.29 (Schloss et al. 2009) was used to cluster sequences into molecular taxonomic units (MOTUs) based on an average neighbour distance threshold of 0.3%, a value that has been shown previously to yield a high concordance between molecular and morphological identifications in other octocoral taxa including xeniids (McFadden et al. 2014b). Modeltest 3.0 (Posada and Crandall 1998) was used to select appropriate models of evolution for maximum likelihood analyses that were run for 100 bootstrap replicates using GARLI 2.0 (Zwickl 2006). The 28S rDNA and mitochondrial gene (mtMutS + igr1 + COI) datasets were analyzed separately, and in a combined analysis with different models of evolution applied to separate data partitions (mt genes: HKY+I+G; 28S: GTR+I+G). Bayesian analyses were run using MrBayes v. 3.2.1 (Ronquist et al. 2012) with the same data partitions. Analyses were run for 2,000,000 generations (until standard deviation of split partitions < 0.005) with a burn-in of 25% and default Metropolis coupling parameters (i.e., 2 runs, 4 chains (3 heated), sample frequency = 500 generations). Description. The holotype is comprised of multiple short, branched stalks sharing a common base attached to coral rock. In life, the holotype ( Fig. 1a) is thickly beset with monomorphic polyps which were observed to be non-pulsatile. The tentacles are cylindrical, slender and up to 2.0 cm long (Fig 1b). In a preserved state, the holotype consists of multiple stalks; two of the stalks are divided into two short branches (Fig. 2a). The stalks measure up to 8.0 mm tall and 5.0 mm wide at the base. A slightly convex capitulum is present at the distal end of the stalks from which moderately dense aggregations of polyps are growing. The polyp bodies are cylindrical, shrunken and measuring from their attachment at the capitulum to the base of the tentacle they are 1.5 mm long by 0.5 mm wide. Tentacles are slender, measuring up to 8.0 mm long by 0.2 mm wide with a blunt tip. There is one row of pinnules present on either side of the tentacle (Fig. 2b at arrow) with 17 to 19 pinnules in a row. The pinnules are barrellike with a rounded tip and slight taper at the end. They measure 0.2 to 0.3 mm long by 0.1 mm wide. There is an open space on the tentacles between adjacent pinnules measuring 0.1 to 0.15 mm wide, nearly equal to their width. Zooxanthellate.

Systematic section
Sclerites are present in all parts of the holotype. They are moderately dense in the polyps ( Fig. 3a-b) and fewer are found in the basal portion of the stalk. All of the sclerites are round to slightly oval or irregularly shaped spheroids (Fig. 4a). They measure from 0.010 to 0.018 mm in diameter on average with a few sclerites as large as 0.020 mm in maximal diameter and the smallest recorded at 0.005 mm in diameter. The sclerites are comprised of numerous, minute circular to egg-shaped corpuscular microscleres that quickly disassociate when extracted from the coral tissue with sodium hypochlorite. Rarely, a second form of sclerite can be found in the polyp tissue, which contains a solid calcite core coated with microscleres (Fig. 4b). The microscleres measure from 0.00035 to 0.00060 mm in diameter. SEM imaging revealed that they have a fine, granular surface ultrastructure (Fig. 4c) when viewed under moderately high magnification (×40,000). The ultrastructure of the surface is comprised of a series of ridges, furrows, and coarse nodules (Fig. 4d) when examined at 65,000 power. Most microscleres are intact but occasionally fractured ones are found (Fig. 4e). Broken microscleres reveal the presence of a cavity or cavities within. Further evidence of these can be seen in FIB imaging where the microsclere has been sliced longitudinally, demonstrating the depth of the furrows (Fig. 5a) and size of cavities (Fig. 5b) when magnified at 200,000 power.
Color. The preserved specimens are cream colored with whitish tentacles and light tan pinnules. Living colonies exhibit tan colored stalks; white polyp bodies and tentacles, and pinkish pinnules ( Fig. 1a-d).
Etymology. The name is derived from the collection location, the Andaman Sea. Distribution and ecology. This species was collected from Koh Doc Mai (Fig. 6) and Koh Phi Phi, Hin Bida located along the eastern coast of Phuket Island, Thailand. Colonies occur in low abundance, spaced 1-2 meters apart on small ledges of vertical walls growing among sea fans, corallimorpharians, and Dendronephthya sp. soft corals. Ovabunda andamanensis sp. n. has also been observed in situ in the Mergui Archipelago, Myanmar (T. Chanmethakul, personal observation).
Among species of Xenia, X. puerto-galerae Roxas, 1933 most closely resembles O. andamanensis sp. n. The holotype is described by Roxas (1933) as branched, measuring 20.0 mm tall and 8.0 mm in diameter with polyps comprised of thick tentacles that are proportionately small and "two rows of slender pointed pinnules, fifteen to seventeen in a row". The tentacles are 8.0 mm long by 1.0 mm wide at the base and pinnules measure 0.7 to 0.8 mm long by 0.2 to 0.3 mm wide. However, in the colony with two rows (PMBC 11862) in O. andamanensis sp. n., the stalks are smaller, 8.0 mm by 5.0 mm, the tentacles are narrower, 8.0 mm by 0.2 mm, the pinnules are smaller, 0.2 to 0.3 mm by 0.1 mm, and there are fewer pinnules (13 to 14). Most notable are the sclerites, which are described as "thin, oval discs 0.018 mm long and 0.018 to 0.0124 mm wide" in X. puertogalerae compared to the 0.010 to 0.018 mm in diameter sphere shaped sclerites observed in O. andamanensis sp. n. Unfortunately, the location of the holotype of X. puerto-galerae remains unknown so a direct SEM comparison of the sclerites could not be performed.

Molecular analysis
All three Ovabunda specimens in this collection had identical genotypes at mtMutS, COI + igr1 and 28S rDNA. Phylogenetic analyses placed them in a well-supported clade with all other species of Ovabunda as well as several species of Xenia from the Red Sea (Fig. 7). Within that clade, only four MOTUs were distinguished by an average genetic distance of 0.3% or greater. The three Thai specimens belonged to a MOTU that was separated from all other species by 0.5%. The two species of Xenia (X. umbellata, X. hicksoni) found within the clade belonged to a MOTU that was separated from Red Sea Ovabunda species by 0.4%. All Red Sea Ovabunda species belonged to just two MOTUs that were separated by 0.3%: the group of [USNM1201941, USNM1201943 and ZMTAUCO34077] and all others. Based on its sclerite size, colony size and form, pinnule arrangement, and unique phylogenetic position, we hereby designate a new species O. andamanensis sp. n. for our material.

Discussion
Octocorals are thought to exhibit their greatest species richness in the Indo-Malayan region consisting of Indonesia, the Philippines and New Guinea (Ekman 1953;Hoeksema Figure 7. Maximum likelihood reconstruction of family Xeniidae based on a partitioned analysis of mt-MutS, COI and 28S rDNA sequences (2255 bp). Numbers above nodes are bootstrap percentages (100 replicates) from ML analyses; numbers below nodes are Bayesian posterior probabilities. Some clades have been collapsed to triangles to facilitate readability. and Ofwegen 2004;Ofwegen 2005;Wood and Dipper 2008;Hoeksema 2009), an area also known for its scleractinian diversity (Baird et al. 2009;Veron et al. 2009). Ovabunda is mainly found in the Red Sea, where the highest number of species (11) occurs (Fig. 8). Benayahu et al. (2002)  hamsina. This is likely due to extensive collection efforts in Sudan (Reinicke 1995(Reinicke , 1997 and Saudi Arabia (Haverkort-Yeh et al. 2013). Similarly, nine species have been identified from the northern Red Sea in the Gulf of Suez and the Gulf of Aqaba. Extensive octocoral research in Eilat, Israel (Benayahu and Loya 1977, 1981, 1985Benayahu 1990) accounts for the numerous records at the northern most end of the Gulf of Aqaba. Janes (2008a) recorded three species from the Seychelles Islands (Fig. 9). There are additional reports of specimens collected from Madagascar (Reinicke 1997;Halász et al. 2013) and Reunion Island (Benayahu and Ofwegen 2012) in the Western Indian Ocean.
This new record from Thailand increases the know distribution of Ovabunda in the Indian Ocean approximately 5000 km eastward. Both the holotype and one para- type of O. andamanensis sp. n. were collected from Koh Doc Mai (Fig. 6) located along the eastern coast of Phuket Island. An additional paratype was collected from Koh Phi Phi, Hin Bida located at the southern end of the Gulf of Thailand. Xeniids have been recorded previously from the Andaman Sea (Chanmethakul et al. 2010) so it is likely that this or other species of Ovabunda will be found elsewhere in the Andaman Sea or eastern Indian Ocean if a thorough octocoral survey is carried out. Specimens of O. andamanensis sp. n. were all collected from horizontal shelves on vertical walls, and it is perhaps on horizontal surfaces such as this where Ovabunda species might occur.
The gross morphology, sclerite dimensions, and molecular results all support the description of O. andamanensis sp. n. as a new species. The range of intraspecific variability among xeniid species is poorly known. Living colonies of O. andamanensis sp. n. were observed to be smaller and less numerous than Ovabunda colonies found on the coral reefs in the northern Gulf of Aqaba (M. Janes, personal observation) and the largest sclerites found in O. andamanensis sp. n. had a maximal diameter of 0.020 mm, notably smaller than the 0.035-0.040 mm maximal diameter recorded by Halász et al. (2013: 36) in Red Sea species; whether this is a result of environmental factors such as light or water flow (Reinicke 1995) or is typical for colonies of O. andamanensis sp. n. remains unknown. The solid sclerite form (Fig. 4b) coated with microscleres that was rare in the Thai material is similar to sclerites recorded from Ovabunda impulsatilla collected in the Seychelles  (Janes 2008a: 612). While the smaller maximal diameter of sclerites from O. andamanensis sp. n. are considered a taxonomic character of this species, the occasional hollow cavities in the microscleres are not; primarily due to limited sampling of the microscleres.
The results of our study suggest both the holotype and two paratypes of O. andamanensis sp. n. are the same species which can exhibit one or two rows of pinnules. Previous authors have suggested the number of rows of pinnules can be variable within a species and is therefore not a highly reliable taxonomic character. Early on both Thomson and Henderson (1906: 410) and Kükenthal (1913: 6) recognized the variability in pinnule size and their arrangement. Later, Gohar (1940: 79) noted that "The number of rows increases with age…" and cited Heteroxenia fuscescens juveniles with two rows of pinnules where the adult colonies had up to five. Further, in their revision of Ovabunda Halász et al. (2013) mentioned the difficulty in identifying irregular rows of pinnules in the species O. crenata and O. hamsina. All three Thailand samples were nearly identical with regards to gross morphology, sclerite shape and size, pinnule spacing, lack of pulsation, and molecular genotype. O. andamanensis sp. n. differs from other Ovabunda species by having the following unique combination of characters: sclerites measuring from 0.010 to 0.018 mm in diameter on average, small colonies with slender tentacles up to 2.0 cm long in life, exhibits one or two rows of pinnules, and distinguished from all other Ovabunda species by a genetic distance of 0.5%.