Stolonifera from shallow waters in the north-western Pacific: a description of a new genus and two new species within the Arulidae (Anthozoa, Octocorallia)

Abstract A new genus and two new species of stoloniferous octocorals (Alcyonacea) within the family Arulidae are described based on specimens collected from Okinawa (Japan), Palau and Dongsha Atoll (Taiwan). Hanagen. n. is erected within Arulidae. Hanahanagasasp. n. is characterised by large spindle-like table-radiates and Hanahanatabasp. n. is characterised by having ornamented rods. The distinction of these new taxa is also supported by molecular phylogenetic analyses. The support values resulting from maximum likelihood and Bayesian inference analyses for the genus Hana and new species H.hanagasa and H.hanataba are 82/1.0, 97/1.0 and 61/0.98, respectively. Hanahanagasasp. n. and Hanahanatabasp. n. are the first arulid records for Okinawa, Palau, and Dongsha Atoll, and represent species of the second genus within the family Arulidae.


Introduction
Stolonifera is a subordinal group within Octocorallia, consisting of octocoral families that have been grouped together based mainly on the character of having polyps that arise separately from an encrusting horizontal, branching, ribbon-like stolon, or with polyps arising from broad, encrusting membranes. Stoloniferans are therefore morphologically different from other octocorals, which have their polyps embedded within common coenenchymal tissue. Like soft corals, stoloniferan octocorals are found in various marine ecosystems, such as coral reefs in shallow tropical and temperate seas (Fabricius and Alderslade 2001;Daly et al. 2007, McFadden andOfwegen 2012). Relative to some other octocoral groups (soft corals and gorgonians) little is known about stoloniferan octocorals, and this is especially true concerning molecular studies. Most Stolonifera studies involve the formal description of new species based on historical alpha-taxonomy methodology (Ofwegen et al. 2006, Alderslade and McFadden 2007, Williams 2013, Churashima Foundation and Biological Institute on Kuroshio 2016). The most comprehensive study on stoloniferan phylogenetic relationships to date was conducted by McFadden and Ofwegen (2012). Their results demonstrated that there is still much work to be done for this taxonomic group and confirmed the polyphyletic distribution of Stolonifera within Alcyonacea.
Until 2012, there were six families of Alcyonacea considered to belong to the Stolonifera; Acrossotidae Bourne, 1914, Coelogorgiidae Bourne, 1900, Cornulariidae Dana, 1846, Clavulariidae Hickson, 1894, Pseudogorgiidae Utinomi & Harada, 1973, and Tubiporidae Ehrenberg, 1828. Of these, the family Clavulariidae is the most speciose and most studied, comprising 27 genera and over 60 species (Cordeiro et al. 2018). The other five families are all either monospecific or monogeneric; having no more than a few described species. A seventh monotypic family, Arulidae, was erected in 2012 (McFadden and Ofwegen 2012), describing the single genus Arula and the single species Arula petunia, collected from subtropical South African waters. Arulidae is characterised by having polyps with tentacles that are fused together proximally, forming an expanded and broad circular oral membrane. Arulidae also has 'table-radiate' sclerites that are altar-like shaped, which had never been recorded before in any other octocoral species. The known distribution of Arula petunia is from the east coast of South Africa from Tanskei to northern Natal, and there are additional photographic records of similar species or relatives from Bali, Indonesia (McFadden and Ofwegen 2012), Oman (Weinberg 2017) and Sabah, Malaysia (Lau pers. obs.).
Recent observations and collections in the north-western Pacific have revealed a similar abundance of stoloniferous octocoral species in coral reefs that are either unrecorded or even undescribed (Churashima Foundation and Biological Institute on Kuroshio 2016). Many stoloniferan octocorals have small and inconspicuous polyps that are usually only ~2-3 mm in diameter and are often overlooked. These octocorals could potentially fill up important knowledge gaps concerning phylogenetic relationships within the subordinal group Stolonifera, as this group is polyphyletic within the Octocorallia. Thus, there is a need to investigate and identify Stolonifera in this region.
Here, we describe a new genus and two new species within the family Arulidae from recent collections in Okinawa (Japan), Palau, and Dongsha Atoll (Taiwan), which are situated in the north-western Pacific.

Specimen collection
A total of 16 arulid specimens were collected, at Okinawa Island, Japan (n=12) from June to August 2017, at Palau (n=2) from December 2017 to January 2018, and at Dongsha Atoll, Taiwan (n=2) from April to May 2018. All specimens were collected at depths between 5-30 m by means of SCUBA. Material was preserved in 99% ethanol. In total eight localities were visited for sampling, Okinawa Island (n = 4), Palau (n = 2), and Dongsha Atoll (n = 2) ( Figure 1). An overview with collection data of the specimens is presented in Table 1. Vouchers and type material have been deposited at the National Museum of Nature and Science, Tokyo, Japan.

Morphological study
Sclerites were isolated by dissolving entire polyps and stolons in 4% hypochlorite (household bleach). Sclerites were rinsed at least seven times with de-ionised water, dried, and initially studied by embedding the sclerites in Euparal on glass slides. In addition, for more detailed morphological studies, sclerites were mounted on scanning electron microscope (SEM) stubs and coated with Pd/Au for imaging on a JEOL JSM6490LV SEM operated at high vacuum at 15kV.

Molecular phylogenetic analyses
Multiple sequence alignments were performed using MAFFT 7 (Katoh and Standley 2013) under default parameters. Consensus sequences for each marker were aligned to a reference dataset of four octocoral taxa (McFadden and Ofwegen 2012), including two stoloniferan specimens of the family Arulidae, Arula petunia. The nearest sister taxa, Paratelesto sp. and Rhodelinda sp., were used as outgroup taxa in the alignments. Subsequently, alignments of 909 bp for COI, 714 bp for mtMutS and 825 bp for 28S rDNA were obtained. Each dataset was separately run for maximum likelihood (ML) analyses, to check for contamination [Suppl. material 1]. All new sequences generated in this study were deposited in GenBank (Table 1). Maximum likelihood and Bayesian inference were performed on the Naturalis OpenStack computing cloud using PhylOstack (Doorenweerd 2016). Alignments of different markers were concatenated using SequenceMatrix 1.8 (Gaurav et al. 2011), resulting in a 2448 bp dataset of 17 taxa. Maximum likelihood analyses were run with RAxML 8 (Stamatakis 2014) using the GTRCAT model. The best maximum likelihood 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 resulting RaxML bootstrap tree was analysed with RogueN-aRok (Aberer et al. 2013). 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. The effective sample size was confirmed using Tracer 1.6.0 (Rambaut et al. 2014). Bootstrap supports and posterior probabilities were depicted on the branches of the best maximum likelihood tree using P4 (Foster 2004). The resulting tree was visualized in FigTree 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. Diagnosis. Colony with polyps connected through flat and thin ribbon-like stolons. Anthocodiae (retractile portion of polyp) retract into cylindrical to clavate calyces. Tentacles are fused proximally, forming a broad, circular oral membrane. The oral membrane has eight deep furrows, which run from the intertentacular margin to the mouth of the polyp, giving it a plump appearance. Sclerites of anthocodia are rods. Sclerites of calyx are 6-radiates and table-radiates. The main difference between Hana and Arula is in sclerites found in the type species Hana hanagasa sp. n. and Arula petunia in the stolon. Sclerites of the stolon are fused sheets that form a flattened network of tableradiates in H. hanagasa, while in A. petunia they are similar to the separate table-radiates found in the calyx. Additionally, there is a difference in sizes of the table-radiates, being longer in H. hanagasa than in A. petunia. Sclerites colourless. Zooxanthellate.
Etymology. From the Japanese language 'hana' (花), meaning flower; denoting the shape of the polyps, which resemble flowers. Gender: feminine. Description. The colony consists of numerous small polyps (~50) growing on hard coral rock. Polyps are spaced apart irregularly (0.3-2.5 mm), connected by stolons that are 0.5 mm in diameter and flat thin ribbon-like in cross-section. Polyps have anthocodia fully retracted into calyces of 2.5-3 mm tall and up to 1.0 mm diameter at the widest point; calyces are slightly club-shaped or barrel shaped, wider near the distal end than at the proximal point of attachment to the stolon.
The oral disk expands into a broad circular membrane by fusion of the proximal regions of the adjacent tentacles. The margin of the oral membrane has eight broad lobes, with eight deep furrows, which run from the intertentacular margin to the mouth of the polyp, giving a plump appearance (Figure 2a). The distal two-thirds of the tentacles extend from fused margins of the oral membrane. Tentacles are long and thin, with 10 pairs of widely spaced pinnules, which are arranged in a single row on either side of the rachis.
Anthocodial sclerites are small rods with simple tubercles around margins 0.10-0.18 mm long (Figure 3a). Calyx containing small 6-radiates 0.05-0.06 mm long ( Figure 3b  white and brown in H. hanagasa, respectively. This would suggest assignment to the same genus, however, the combination of differences in genetic data and sclerite morphology indicate that they should be separate from each other at the generic level. The possibility that there are similar species or previous descriptions and reports on arulid species has previously been discussed (McFadden and Ofwegen 2012) and so far, no reports have been made on possible congeners. Etymology. From the Japanese language 'hanagasa' (花笠), the traditional Okinawan ceremonial dance headpiece worn by female performers; denoting the shape of the polyps, which resembles the flower headpiece. Description. The colony consists of small polyps (~30) growing on rock. Polyps are spaced apart irregularly (0.5-2.5 mm), connected by stolons that are 0.5 mm in diameter and flat thin ribbon-like in cross-section. Polyps have anthocodia retracted into calyces of 2.5-3.0 mm tall and up to 1.0 mm diameter at the widest point; calyces are slightly club-shaped or barrel shaped, wider near the distal end than at the proximal point of attachment to the stolon.
The oral disk expands into a broad circular membrane by fusion of the proximal regions of the adjacent tentacles (Figure 2c). The margin of the oral membrane has eight broad lobes, with eight deep furrows, which run from the intertentacular margin to the mouth of the polyp, giving a plump appearance. The distal two-thirds of the tentacles extend from fused margins of the oral membrane. Tentacles are long and thin, with eight pairs of widely spaced pinnules, which are arranged in a single row on either side of the rachis.
Anthocodial sclerites are rods with sparse simple tubercles around margins 0.07-0.24 mm long and rods ornamented with clustered tubercles on one end, giving it a clubshaped appearance, size 0.10-0.18 mm (Figure 4a). Calyx containing small capstans 0.02-0.05 mm long (Figure 4b) and table-radiates ranging 0.03-0.09 mm (Figure 4d). Sclerites of stolon are fused table-radiates forming a flat network (Figure 4c).  The oral disk and tentacles are white in life with brown in the proximal part of tentacle (Figure 2c), yellowish-white in ethanol. Zooxanthellate.
Morphological variation. Paratypes consist of colonies with 30-100 polyps, growing on hard substrates. Colonies show variations in the tentacles, sometimes having ten pairs of pinnules.
Distribution. The south-east of Palau in the Philippine Sea and the north to north-east reef of Dongsha Atoll, Taiwan in the South China Sea.
Remarks. Hana hanagasa and Hana hanataba have very similar polyp morphology, with minor colour differences, which could be due to differing abundances of zooxanthellae. Genetic data and sclerite morphology indicate that H. hanagasa and H. hanataba should be separated from each other at the species level. Sclerites found in H. hanataba are different from those in H. hanagasa in the presence of ornamented rods, which are lacking in H. hanagasa. It is noteworthy that both H. hanagasa and H. hanataba were found in environments with the presence of a comparatively strong current.

Molecular phylogenetic analyses
This study has added 24 sequences to the reference database, representing two species for which no barcodes have been sequenced before. The phylograms resulting from the ML analyses of the separate markers were highly congruent with those from the analysis of the combined markers ( Figure 5). ML and Bayesian analyses of the combined dataset yielded almost identical tree topologies.

Hana hanagasa and Hana hanataba from the north-western Pacific
Sequences of H. hanagasa, gen. n., sp. n., from Okinawa Island and H. hanataba, gen. n., sp. n., from Palau and Dongsha Atoll, Taiwan, grouped together in a wellsupported clade within the Arulidae. The sequences formed a separate clade from sequences of Arula petunia ( Figure 5). Molecular phylogenetic analyses support the distinctiveness of the genera Hana and Arula. The genetic distances (uncorrected p, expressed as percentage) between Arula and Hana taxa were 3.54% and 5.36% for COI and mtMutS, respectively [Suppl. material 2]. This is on the far upper end of the range typical of differences among congeneric octocoral species . The major ramification and distances indicate a separation of H. hanagasa, gen. n., sp. n., and H. hanataba, gen. n., sp. n., from Arula petunia at the generic level. Morphological features support this division; comparing sclerite characteristics between Arula and Hana specimens, there are several differences that stand out. The main morphological difference are the table-radiates of the stolon in genus Hana, which are fused together into a flattened network. The table-radiates of Arula petunia are of a smaller size range and lack two shapes of table-radiates that are seen in Hana hanagasa specimens, somewhat spindle and club shaped table-radiates. Additionally, there are differences in anthocodial rods; differing in size range, larger in Hana specimens. Additionally Arula specimens lack ornamented rods that are seen in Hana hanataba specimens (Figs 3-5).
The separation of H. hanagasa and H. hanataba was confirmed by molecular analyses and through the investigation of the sclerites. The genetic distances (uncorrected p, expressed as percentage) within the genus Hana between H. hanagasa and H. hanataba were 0.8% and 0.67% for COI and mtMutS, respectively [Suppl. material 2]. These percentages are far above margins typical of differences among intraspecific octocorals . Specimens of the two species do not differ much in polyp morphology ( Figure 2); however, investigation of the sclerites demonstrated differences between characters of sclerite morphology (Figs 3-4). Next to anthocodial rods with sparse tubercles arranged on the margins, a second type of rod was seen in H. hanataba; rods that are ornamented with lumps of tubercles on one end, giving the rods a club-like shape. Additionally, the table-radiates of the calyx seen in H. hanataba are of a smaller size range than of those in H. hanagasa, being much more similar to the table-radiates seen in Arula petunia. These morphological characters unique to H. hanagasa and H. hanataba are projected on the phylogenetic tree ( Figure 5).

Discussion
The new species H. hanagasa and H. hanataba bring the total number of species within the Arulidae to three and represent the first confirmed records of arulids for the northwestern Pacific. Arulids so far have been recorded only in South Africa with informal reports of other possible congeners occurring in Sabah, Malaysia ( Figure 2g)   Even though all new arulid specimens were amplified for four markers, here we used only three of the gene regions, 28S rDNA, COI and mtMutS, in the analyses. ND6 was excluded from the analyses, as the outgroup sequences lacked available ND6 sequences. There were no differences in results when including or excluding this region when performing analyses with concatenated datasets. However, utilising four region sequences resulted in better resolution. Therefore, for future analyses, it is recommended to include ND6 to obtain better resolution.
It has been made clear in previous studies that the subordinal group Stolonifera is polyphyletic (McFadden et al. 2006, McFadden and Ofwegen 2012, Conti-Jerpe and Freshwater 2017. There is still much work needed, with families in need of formal description in order to reflect adequately the phylogenetic distribution of stoloniferous genera. The addition of the genus Hana and its two species to the recently erected family Arulidae is a small step in the process to fully interpret the morphological and molecular distinctions amongst clades of Stolonifera and ultimately the Octocorallia. It is clear that many new records of Stolonifera still await discovery and that this group still has morphological surprises, such as previously discovered pinnuleless tentacles, sclerite-free clavulariids, and new sclerite types (Bayer et al. 1983, Williams 2000, Alderslade and McFadden 2007, McFadden and Ofwegen 2012.