The stoloniferous octocoral, Hanabira yukibana , gen. nov., sp. nov., of the southern Ryukyus has morphological and symbiont variation

Stoloniferan octocorals (Cnidaria: Anthozoa: Octocorallia: Alcyonacea) are a relatively unexplored fauna in the Ryukyus (southern Japan), known to be a tropical marine region of high biodiversity and endemism of species. Specimens of stoloniferous octocorals were collected during fieldwork along the coasts of two islands (Okinawa and Iriomote) in the Okinawa Prefecture. Despite their phenotypic polyp variation, this study shows their morphological and molecular uniqueness, leading to the description of a new genus with a single species: Hanabira yukibana, gen. nov., sp. nov. They are placed within the Clavulariidae and form a sister clade basally to the genus Knopia Alderslade & McFadden, tentacles. Moreover, the zooxanthellae hosted by our specimens form a clear, small-scale biogeographic pattern; all H. yukibana specimens from Okinawa Island contained zooxanthellae of the genus Cladocopium Lajeunesse & H.J. Jeong, 2018 (= former Symbiodinium ‘Clade C’) and all specimens from Iriomote Island hosted zooxanthellae of the genus Durusdinium LaJeunesse, 2018 (= former Symbiodinium ‘Clade D’). These results show the potential for variation among the Symbiodiniaceae floras within octocorals, something that has not yet been investigated for the large majority of zooxanthellate octocoral species.


Introduction
The Ryukyu Islands are the southernmost region of Japan and are a tropical marine region of high biodiversity and species endemism (Hughes et al., 2002;Cowman et al., 2017;Reimer et al., 2019). It was estimated that a large part (70%) of the marine biodiversity in Japanese waters has remained undescribed (Fujikura et al., 2010). Coral reefs are the most biologically diverse of shallow-water marine ecosystems (Roberts et al., 2002) and contain cryptic, usually small, inconspicuous species that are easily overlooked and therefore poorly observed and known (Wolf et al., 1983;Reaka-Kudla, 1997;Hoeksema, 2017). Octocorals of the subordinal group Stolonifera are characterized by their growth form by having polyps that are connected through stolons (Fabricius & Alderslade, 2001;Daly et al., 2007;McFadden & Ofwegen, 2012). Many stoloniferans have small inconspicuous colonies, which is one reason that such species are poorly known. Stoloniferan octocorals are a relatively unexplored fauna in the Ryukyus. This is especially true concerning molecular and ecological research, such as the examination of octocoral-Symbiodiniaceae associations. Previous research has revealed biogeographical patterns in the presence of Symbiodiniaceae genera occurring in different octocoral hosts in different geographic regions (Oppen et al., 2005;Goulet et al., 2008), but this is much less explored within smaller geographic areas, such as across islands of the southern Ryukyus.
Recent observations and collections in the north-western Pacific, including those from around the island of Okinawa (or Okinawajima, hereafter Okinawa Island), have revealed a high abundance of stoloniferous octocoral species on coral reefs that are either unrecorded or possibly undescribed (Okinawa Churashima Foundation and Biological Institute on Kuroshio, 2016;Lau et al., 2018) and mostly these octocorals are not only small in colony size with few polyps, but some species also have polyps which are only ~2-3 mm in diameter (Lau et al., 2018). Here, we use a combined approach of morphological and molecular data to describe one new monotypic genus within the family Clavulariidae based on recent collections from shallow waters surrounding Okinawa and Iriomote Islands (or Iriomote-jima, hereafter Iriomote Island). We also investigate the identity of their endosymbiotic Symbiodiniaceae and their relations to morphological polyp variations and geographic locations.

Specimen collection
Clavulariid specimens (total n = 38) were collected from locations in Okinawa Prefecture, Japan, around Okinawa Island (n = 18) from March to October 2011, and May 2017to August 2018, 25-27 July 2017. A total of 18 sites were visited, at Okinawa (n = 11) and Iriomote (n = 7) Islands ( fig. 1, table 1). Specimens were collected in depth ranges of 10-35 m by means of scuba and all material was preserved in 99% ethanol. High-resolution in situ images were taken with an Olympus Tough tg-4 in an Olympus pt-056 underwater housing. Vouchers and type material have been deposited at the National Museum of Nature and Science, Tokyo, Japan.

Molecular phylogenetic analyses
Multiple sequence alignments were performed using mafft 7 (Katoh & Standley, 2013) under default parameters. To determine the phylogenetic position of clavulariid specimens (n = 6), consensus sequences for markers 28S rDNA, coi and mtMutS were aligned to a reference dataset of 120 octocoral genera (n = 128), including Parasphaerasclera rotifera and Eleutherobia grayi as outgroup (total n = 134). Alignments of 895 bp for 28S rDNA, 786 bp for coi and 950 bp for mtMutS were separately run in maximum likelihood (ml) analyses (supplementary figs. S1-S3). To investigate morphological and symbiont variability in the clavulariid specimens, another analysis was performed on a dataset with more clavulariid specimens. This dataset included consensus sequences for markers 28S, coi, mtMutS and ND6. The four markers were aligned to a reference dataset of four octocoral genera (n = 14), including Parasphaerasclera rotifera and Eleutherobia grayi as outgroup (total n = 37). Alignments of 895 bp for 28S rDNA, 786 bp for coi, 950 bp for mtMutS, 534 bp for ND6 (available for all octocoral specimens), and 677 bp for its-rDNA (Symbiodiniaceae only) were separately run in ml analyses. The its-rDNA dataset for Symbiodiniacea was aligned with reference taxa Cladocopium spp. and Durusdinium spp., using Gerakladium sp. as outgroup (total n = 25) (supplementary figs. S4-S8). All new sequences generated in this study were deposited in GenBank (table 1). ml and Bayesian inference analyses were performed on the Naturalis OpenStack computing cloud using PhylOstack (Doorenweerd, 2016). Alignments of the different octocoral markers were concatenated using SequenceMatrix 1.8 (Gaurav et al., 2011), resulting in a 2631 bp dataset (three markers) of 121 taxa (n = 134) and a 3165 bp dataset (four markers) of five taxa (n = 37). ml analyses were run with raxml 8 (Stamatakis, 2014), using the gtrcat model. The best ml tree was calculated using the -D parameter. A multi-parametric bootstrap search was performed, which automatically stopped based on the extended majority rule criterion. The Bayesian inference was performed with ExaBayes 1.5 (Aberer et al., 2014) using the gtr substitution model. Four independent runs with each four Monte Carlo Markov Chains were run for 1,000,000 generations during which convergence (with a standard deviation of split frequencies <2%) had been reached. Bootstrap supports and posterior probabilities were depicted on the branches of the best ml tree using P4 (Foster, 2004). The resulting tree was visualized in Fig-Tree 1.4.2 (Rambout, 2014). Additionally, average distance estimations within species and within genera were computed using mega7 (Kumar et al., 2016) by analysing pairwise measures of genetic distances (uncorrected P) among sequences.

Morphological study
The present investigation revealed the morphological distinction of a new genus and species Hanabira yukibana, gen. nov., sp. nov. (Appendix). Several morphological characters separate this monotypic genus from other genera, based on colony and sclerite morphology, which is the main means of identification in octocoral species delimitation. The main distinguishing characters of the polyps include the typical petal-shaped tentacles and tentacle pinnules, which are fused together (pseudopinnules). Other characteristics include the morphological variation seen in the polyps of H. yukibana in both tentacle shape and sheen ( fig. 2). We divided all specimens into the three main polyp variations, based mainly on the shape of the tentacles; variation A, petal shaped, variation B, tongue shaped and variation C, feather shaped (figs. 2, 5). The sclerites seen in H. yukibana are different from those in closest sister genera Clavularia and Knopia in composition, types and size; the most typical sclerites in H. yukibana are platelets with a distinct waist in combination with small, smooth rods (figs. 3, 5).

Molecular phylogenetic analyses
This study has added 90 Hanabira yukibana sequences to the public reference database, representing a species for which no barcodes have been sequenced before. An additional 32 sequences have been added for Clavularia sp., of which 13 belong to a possibly undescribed species. For the family Symbiodiniaceae, 13 Cladocopium spp. and nine Durusdinium spp. sequences were added. The phylogenies resulting from the ml analyses of the separate host markers (supplementary figs. S4-S8) were highly congruent with those from the combined markers (figs. 4, 5). ml and Bayesian analyses of the combined datasets yielded almost identical tree topologies. Paratypes 26-28 were not analysed molecularly, as no sub-samples were available.

Hanabira yukibana from Okinawa
Sequences of Hanabira yukibana, from Okinawa and Iriomote Islands grouped together in a well-supported clade (100%/1.00). The new species grouped basally to two members of Clavulariidae (100%/1.00), the closest sister genera Clavularia and Knopia ( fig. 4), supporting the morphological data that justify the description of a new genus and species.
When examining morphological and symbiont variability in H. yukibana, there are two groups with moderately high support values (60%/0.90 and 45%/0.99) within the main clade of its specimens ( fig. 5), which could suggest multiple species or subspecies. The genetic distances (uncorrected p, expressed as percentage) between these groups and the main clade of H. yukibana specimens were 0.01% and 0.17% for coi and 0.00% and 0.00% for mtMutS (supplementary table S1). These values are well below average values typical of differences among congeneric octocoral species, but comparable to distances among conspecific octocoral species (McFadden et al., 2011). This observation is supported by values of the genetic differences within all Hanabira yukibana specimens; 0.09% and 0.00% for coi and mtMutS, respectively (supplementary  table S2), values below average within-species comparisons (McFadden et al., 2011). Thus, the major ramification and distances indicate a separation of this species from Clavularia at the generic level and neither support the distinction of multiple species nor subspecies.
Morphological characteristics support this separation at the generic level, as there are clear differences in sclerite features between Knopia, Clavularia and Hanabira (figs. 3, 5). The main difference is the contrast in sclerite types between the three genera. Hanabira specimens lack the spindle sclerite types seen in Clavularia, while in comparison with Knopia the Hanabira specimens have rodlets as an additional sclerite type. Hence, in concurrence with the molecular data, polyp morphology also does not support multiple species nor subspecies: the three groups of H. yukibana specimens seen in the phylogenetic Downloaded from Brill.com02/13/2020 09:11:20PM via free access Figure 2 In situ photographs of the three main polyp variations seen in Hanabira yukibana, gen. nov., sp. nov.; variation A: a) nsmt Co 1626, holotype and b) nsmt Co 1625, paratype 1; variation B: c) nsmt Co 1637, paratype 12; variation C: d) nsmt Co 1633, paratype 8. Images of Hanabira yukibana, gen. nov., sp. nov., holotype, nsmt Co 1626: e) stained with methylene blue to accentuate pseudopinnules of the tentacles and f) colony preserved in ethanol. Scale bar: 1 mm.    tree did not coincide with the three main polyp morphologies (variation A-C) and the polyp morphologies were scattered randomly through all groups ( fig. 5).

Hanabira yukibana-Symbiodiniaceae associations
Hanabira yukibana specimens were analysed for the presence of Symbiodiniaceae zooxanthellae to examine possible relationships with polyp morphology. With the exception of four specimens from Iriomote Island, its sequences of Symbiodiniaceae were obtained for all examined specimens; paratypes 3, 6, 8 and 11 were zooxanthellate, but no sequences were obtained. There was a clear biogeographical distinction in the presence of Symbiodiniaceae in the hosting octocorals. All H. yukibana specimens examined from Okinawa Island hosted genus Cladocopium (n = 12), while all specimens from Iriomote Island hosted Durusdinium (n = 8). The Symbiodiniaceae genus present was unrelated to neither polyp morphology ( fig. 5) nor depth.

Discussion
Analyses of separate gene regions (28S rDNA, COI and mtMutS) resulted in different positions of Knopia in the obtained phylograms. Nonetheless, Hanabira was consistently a strongly supported lineage in all the separate gene region analyses (supplementary figs. S1-S7). Concatenation of all four gene regions (28S rDNA, COI, mtMutS and ND6), which gives the highest resolution, demonstrated that Knopia octocontacanalis and Clavularia spp. were positioned basally to Hanabira yukibana specimens, with Knopia being its closest sister taxon (figs. 4, 5). Exclusion of gene region ND6 did not result in any differences in branch topology. Therefore, we recommend utilising such four region analyses for future studies of octocorals (Reijnen et al., 2014;Reijnen, 2016;Lau et al., 2018). Sclerite morphology also demonstrated similarity to both genera Clavularia and Knopia. Clavularia has similar sclerite types: platelets and rods in the anthocodiae and spindles in the calyx. However, sclerites differ significantly in size of the different types; sclerites seen in Clavularia have a much larger size range. The platelets are almost twice as large, ~0.02 mm in Hanabira and from ~0.04 to 0.05 mm in Clavularia, the rodlets in Hanabira range from ~0.08-0.17 mm and 0.23-0.26 mm in Clavularia and spindles are ~0.16-0.32 mm in Hanabira and ~1 mm in Clavularia (Schenk, 1896;Fabricius & Alderslade, 2001).
In contrast, Knopia has only two sclerite types, platelets and small scale-like sclerites, which are scattered amongst the platelets, and neither type is seen in Hanabira or Clavularia. The sclerites seen in Knopia are of similar size as those seen in Hanabira; platelets are ~0.011-0.025 mm, but they have a more oval, circular or kidney-shaped outline when compared to platelets seen in H. yukibana (Alderslade & McFadden, 2007). No comparison can be made between the three genera concerning the sclerites in the stolon, as these have not been described for Knopia (Alderslade & Mc-Fadden, 2007), nonetheless, the fused clumps seen in Hanabira yukibana are similar to the fused clumps seen in Clavularia, and are also similar in size (both ~0.05 mm in width of the fused sclerites) (Fabricius & Alderslade, 2001).
Polyp morphology of Hanabira also shows resemblance to that of both Knopia and Clavularia, even though the polyps in H. yukibana are much smaller (diameter expanded polyps ~2-3 mm) than seen in both Knopia (tentacle length ~5 mm; Alderslade & McFadden, 2007) and Clavularia (tentacle length up to 20 mm; Fabricius & Alderslade, 2001). Overall shape of the polyps show affinity to Clavularia, although tentacle pinnules show a likeness with Knopia; both Knopia and Hanabira have pseudopinnules ( fig. 2e).
The molecular results (figs. 4, 5) show that the relationship between Hanabira yukibana and Clavularia spp. is consistent, however the location of the monotypic genus Knopia is not well-supported in either ml or Bayesian inferences; 42/0.59, respectively, for the 3-marker dataset (28S rDNA+coi+mtMutS) and 48/ -for the 4-marker dataset (28S rDNA+coi+mtMutS+ND6). Both in colony form and sclerites, Knopia is different from the genera Hanabira and Clavularia, but as a result of the unresolved phylogenetic location of Knopia, it remains unclear how Hanabira and Clavularia are related to Knopia.
It is noteworthy that there is a wellsupported division within Clavularia spp. (fig. 5), in which one lineage of clavulariids (88%/1.00) branches off from Clavularia inflata (86%/1.00). Therefore, these specimens were investigated morphologically (supplementary figs. S10-S11). The sem images of the sclerites of this group appear to be different from the sclerites described for Clavularia inflata Schenk, 1896. This difference is also supported by values of genetic distances between this group and Clavularia inflata specimens, which are above the average of conspecific octocorals (supplementary table S3). Therefore, we suggest that these Clavularia specimens may represent an undescribed species, here designated as Clavularia sp. It is clear that Clavularia is in need of a revision that investigates this genus both morphologically and molecularly.
Phenotypic variation is not uncommon in octocorals. Morphological variation in octocorals has been investigated in Caribbean gorgonians, specifically with regard to the branching structure and sclerite morphology (Brazeau & Lasker, 1988;Sánchez et al., 2007;Prada et al., 2008;Sánchez, 2016), and also polyp characters, e.g., for xeniids, differing in pinnules and rows of pinnules . However, phenotypic variation in polyp morphology in octocorals is yet to be thoroughly examined for most groups, and morphological variation has not been studied sufficiently in other related genera to determine if the degree of variation in polyp morphology observed in Hanabira is of outstanding significance or not. In this study the morphological variations seen in the polyps of H. yukibana could not be explained by any clear pattern of their algal symbionts. Previously, comparisons of Symbiodiniaceae within various cnidarian species have been used to clarify intra-specific physiological (Goulet et al., 2005(Goulet et al., , 2008 or distributional differences (Kamezaki et al., 2013). Earlier studies have examined biogeographic patterns for octocoralzooxanthellae associations and these studies generally have comprised large datasets with wide taxonomic coverage spanning wide geographical regions (e.g., the Great Barrier Reef (gbr), the far eastern Pacific and the Caribbean). These studies have demonstrated that Clavularia spp. in the Caribbean, Torres Strait, and central gbr host Durusdinium (Goulet et al., 2004;Oppen et al., 2005;Goulet et al., 2008), and these are the only data available on stoloniferan octocoral-Symbiodiniaceae relationships.
The current work represents the first in depth small-scale examination of stoloniferan octocoral and Symbiodiniaceae relationships for Okinawan waters. Hanabira yukibana specimens were found on both the east and west coasts of Okinawa Island, but were only found at two out of seven locations around Iriomote Island. There were no outstanding noteworthy conditions at these two locations, Takosaki and Iriomote N, which could explain the occurrence of H. yukibana. Durusdinium was found in all H. yukibana specimens collected at Iriomote Island. Durusdinium includes species that are known extremophiles, adapted to survive in regions with large fluctuations in temperature, and many Durusdinium spp. tend to be resistant to bleaching (LaJeunesse et al., 2018). Iriomote Island has a recent history of extreme bleaching events; from the 1980s to 2001, there were five bleaching events recorded (Research Institute for Subtropics, 1999; Ministry of the Environment and the Japanese Coral Reef Society, 2004), with more events in 2016-2017 (Kayanne et al., 2017). On the other hand, members of Cladocopium are more adapted to a comparatively wider range of temperatures and irradiances, similar to what would be observed in more northern Okinawan waters. Thus, the observed Hanabira yukibana-Symbiodiniaceae relationship patterns at both islands fit well with this generalization. It is recommended for future octocoral research to continue investigating Symbiodiniaceae associations with the prospect of understanding octocoral-algal symbioses as global climate change will continue to severely decimate coral reefs. To fully understand the current Hanabira-Symbodiniaceae relationship, it would be worthwhile to investigate the occurrence of Hanabira yukibana around Miyako Island, situated southwest of Okinawa Island and northeast of Iriomote Island.
Understanding octocoral-Symbiodiniaceae relationships is becoming increasingly important, as octocorals constitute a large portion of the biodiversity in coral reef ecosystems, and are commonly the dominant benthos (Daly et al., 2007;Parrin et al., 2016). Additionally, previous research has shown that octocorals are able to mitigate bleaching through intra-colony migration of symbionts deeper into the colony (Parrin et al., 2016;Parrin, 2016) or into the stolons, as seen in the stoloniferan Phenganax parrini (Netherton et al., 2014), suggesting some capability to resist or even survive bleaching at least in some species (Dias & Gondim, 2015).
Finally, this study is an addition to the existing literature on the character of pseudopinnules in stoloniferan octocoral polyps. As discussed by Alderslade & McFadden (2007), the possession of pinnated tentacles is one of the major diagnostic features of the subclass Octocorallia, and thus the true diagnostic utility of this feature needs further examination. In conclusion, the molecular, morphological and ecological information of this study supplements the few studies that emphasize the need for a thorough investigation of the polyphyletic family Clavulariidae. saki, Iriomote Island. In Okinawa we thank mise members for their help in fieldwork, Y. Kushida for providing a specimen and image nsmt-Co 1650 from Nakagusuku Bay, Okinawa Island. We would like to thank Dr. Diagnosis. Colony with anthocodiae that retract into cylindrical to barrel-shaped calyces, which do not retract into the stolons. Tentacles with pinnules arranged adjacent to one another along either side of the tentacle rachis and are fused together (pseudopinnules). Sclerites of anthocodiae are platelets with a distinct median waist and small smooth rods, with the tentacles having only the platelet type. Sclerites of calyces are small spindles, which are larger than the anthocodial rods and are prickly and warty. Sclerites of stolon are a tubular network of fused sclerites. Zooxanthellate.
Remarks. The main morphological differences with closest sister taxa, genera Clavularia and Knopia: the tentacle rachis of Clavularia contains smooth to warty rods and long, narrow spindles (~1.0 mm), which are prickly or complexly warted, and sometimes branched. The calyces of genus Clavularia contain similar spindles, but can be twice as long (Fabricius & Alderslade, 2001). The anthocodiae of Hanabira do not contain warty rods and large, prickly or warted spindles are completely lacking.
Hanabira yukibana, sp. nov. Figs. 2, 3 Clavulariidae gen. sp. Imahara et al., 2017. Description. The holotype colony is attached to a sponge, which was fragmented into three pieces during sampling, although, this species is not always an epibiont and can be attached to other hard substrates. Small groups of polyps are attached by stolons to the sponge tissue (total ~10 polyps). Polyps are approximately 2-3 mm in diameter expanded and are spaced apart irregularly (from ~0.5 mm up to ~2 cm) and connected through flat and ribbon-like stolons that are 1 mm at the widest and 0.2 mm at the narrowest point. Anthocodiae can retract fully into cylindrical to barrel-shaped calyces, which are 1.5-2.5 mm in height and up to 1 mm in width; calyces do not retract into the stolon. Tentacles have pseudopinnules arranged adjacent to one another along either side of the tentacle rachis (~18 pseudo-pairs); when stained with methylene blue, the outline of the tentacles can be observed. A structure that seems to be the pinnule axis can be seen, although notches, which can distinguish the adjacent pinnules, are not observed in the contour of the tentacle (fig. 2e). In life the tentacles are elliptical to petal-shaped and the polyps have a pale, green-gold and white sheen. This could be caused by refraction on the minute sclerites, as described in Alderslade & McFadden (2007) (fig. 2a). Sclerites of anthocodiae are platelets with a distinct median waist (0.01-0.02 mm) and small smooth rods (0.1-0.2 mm), with the tentacles only having the type of platelets as seen in the anthocodiae ( fig. 3b, c). Sclerites of calyces are rods, which are larger than the anthocodial rods, that are prickly and warty (0.2-0.3 mm) ( fig. 3a). Sclerites of the stolon are a tubular network of fused sclerites ( fig. 3d, e). The polyps are zooxanthellate.
Morphological variation. Polyp density in H. yukibana is variable, as is also the broadness of the stolons (0.1-1.1 mm). Tentacles are not all elliptical in life; some specimens have a more pointed shape at the distal part of the tentacle. The number of pseudopinnules 'paired' alongside the tentacle rachis varies within a range of 15-31 'pairs' . The colour of the polyps is also very variable; in exception of paratypes 15, 16 and 25, all specimens have a clear recognisable sheen. The sheen is thought to be caused by refraction from the platelet sclerites in the tentacles (Alderslade & McFadden, 2007); usually green-gold and/or white. Paratypes 15, 16 and 25 do not lack the minute platelet sclerites but instead have a lower platelet density in the tentacles and only have this typical sheen (white) at the far distal part of their tentacles. Live specimens were overall brown in colour.
Etymology. From the Japanese language 'yukibana' (雪花), meaning 'snow flower'; denoting the resemblance of the sheen of the polyps (including tentacles) to the shimmer of snowflakes or snow crystals.
Habitat. Colonies encrust hard substrates with their stolons. Common substrates are rock, sponges, coral rubble and shells.
Distribution. Southwestern Japan, southern Ryukyu Islands, around Okinawa Island and the north and west coasts of Iriomote Island in the East China Sea. Specimens were collected from depths of 10-35 m.