A new Antipathozoanthus species (Cnidaria, Hexacorallia, Zoantharia) from the northwest Pacific Ocean

Abstract A new species of zoantharian within the genus Antipathozoanthus is described based on specimens collected from the coast of mainland Japan, northwest Pacific Ocean. Antipathozoanthustubussp. nov. is characterized by its substrate (epibiotic on polychaete tube) and habitat (exposed rock). As well, the results of molecular phylogenetic analyses using concatenated multiple genetic markers also support the distinction between A.tubussp. nov. and its congenerics. Antipathozoanthustubussp. nov. is the first species of Antipathozoanthus species reported to be epibiotic on polychaete tubes, and is the second species in the genus that is not associated with antipatharians.


Introduction
The order Zoantharia Rafinesque, 1815 (Cnidaria: Anthozoa) consists of primarily colonial hexacorallians that are commonly found in most marine environments, including extreme environments such as intertidal zones and methane cold seeps in DNA extraction, PCR amplification, and sequencing We extracted genomic DNA from tissue of the holotype specimen preserved in 99.5% EtOH using a spin-column DNeasy Blood and Tissue Extraction Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. PCR amplification using Hot Star Taq Plus Master Mix Kit (Qiagen, Hilden, Germany) was performed for each of COI (mitochondrial cytochrome oxidase subunit I), mt 12S-rDNA (mitochondrial 12S ribosomal DNA), mt 16S-rDNA (mitochondrial 16S ribosomal DNA), 18S-rDNA (nuclear 18S ribosomal DNA), ITS-rDNA (nuclear internal transcribed spacer region of ribosomal DNA), and 28S-rDNA (nuclear 28S ribosomal DNA) using published primers (Medlin et al. 1988;Folmer et al. 1994;Apakupakul 1999;Chen et al. 2002;Sinniger et al. 2005;Swain 2009Swain , 2010Fujii and Reimer 2011). All PCR products were purified with 1 U of shrimp alkaline phosphatase (SAP) and 5 U of Exonuclease I (Takara Bio Inc., Shiga, Japan) at 37 °C for 40 min followed by 80 °C for 20 min. Cleaned PCR products were sequenced in both directions on an ABI 3730Xl Genetic Analyzer (Applied Biosystems, Thermofisher) at the Fasmac Co., Ltd., Kanagawa, Japan. Obtained sequences in this study were deposited in GenBank under accession numbers MW652759-MW652774.

Molecular phylogenetic analyses
Forward and reverse sequences were assembled and edited in Geneious v10.2.3 (Kearse et al. 2012). Multiple alignments for each marker were performed with previously published Parazoanthidae sequences obtained from GenBank (Suppl. material 1: Table S1) using MAFFT ver. 7.110 (Katoh and Standley 2013) with the auto algorithm under default parameters for all genetic markers. In this study, sequences of two selected taxa within the zoantharian genus Epizoanthus were used as outgroups. We obtained a dataset of 549 bp for 34 sequences of COI, 757 bp for 22 sequences of mt 12S-rDNA, 772 bp for 40 sequences of mt 16S-rDNA, 1756 bp for 23 sequences of 18S-rDNA, and 902 bp for 33 sequences of ITS-rDNA, 936 bp for 16 sequences of 28S-rDNA. These alignments were subsequently concatenated to obtain a final dataset of 5672 bp for 40 OTUs. All aligned datasets are available from the first author and at treebase.org (ID: S27965). Phylogenetic analyses were performed on the concatenated dataset using Maximum likelihood (ML) and Bayesian inference (BI). ModelTest-NG v0.1.6 ) under the Akaike information criterion was used to select the best fitting model for each molecular marker, independently for ML and BI. The best selected models for ML and BI analyses were HKY+G for COI, GTR+I+G for mt 12S-rD-NA, SYM+I+G for mt 16S-rDNA, HKY+I+G for 18S-rDNA, TPM3uf+I+G (BI: HKY+I+G) for ITS-rDNA, and GTR+I+G for 28S-rDNA. Independent phylogenetic analyses were performed using model partition per each region in RAxML-NG v0.9.0  for ML, and MrBayes v3.2.6 (Ronquist and Huelsenbeck 2003) for BI. RAxML-NG was configured to use 12345 initial seeds, search for the best tree among 100 preliminary parsimony trees, branch length was scaled and automatically optimized per partition, and model parameters were also optimized. MrBayes was configured following the models and parameters as indicated by ModelTest-NG, 4 MCMC heated chains were run for 5,000,000 generations with a temperature for the heated chain of 0.2. Chains were sampled every 200 generations. Burn-in was set to 1,250,000 generations at which point the average standard deviation of split frequency (ASDOSF) was steadily below 0.01.

Morphological observations
Morphological data were collected from whole, dissected, and serial sections of the preserved specimens. Histological sections of 10-15 mm thickness were made using a RM-2125 RTS microtome (Leica, Germany) and were stained with hematoxylin and eosin after decalcification with a Morse solution for 48 h (1:1 vol; 20% citric acid: 50% formic acid). Classification of marginal muscle shapes followed Swain et al. (2015). Cnidae analyses were conducted using undischarged nematocysts from tentacles, column, actinopharynx, and mesenterial filaments of two polyps of holotype specimen under a Nikon Eclipse80i stereomicroscope (Nikon, Tokyo). Cnidae sizes were measured using ImageJ ver. 1.45 (Rasband, 2012). Cnidae classification generally followed England (1991) and Ryland and Lancaster (2004).

Abbreviations
NSMT National Science Museum, Tsukuba, Ibaraki, Japan; CMNH Coastal Branch of Natural History Museum and Institute, Chiba, Japan.  Ocaña & Brito, 2003, by original designation. Remarks. We herein modify the diagnosis of Antipathozoanthus, as A. tubus sp. nov. is clearly located within the clade of Antipathozoanthus with very high support in our molecular phylogenetic analyses. Skeletal secretion as has been reported in A. macaronesicus (Ocaña & Brito, 2003) was not found in any other Antipathozoanthus species, including A. tubus sp. nov. Type locality. Iwa Beach, Sagami Bay, Kanagawa, Japan Description. External morphology. Colonial zoantharian, with cylindrical polyps connected by well-developed dark red colored coenenchyme (Fig. 1a). External branched tube of dead polychaete mostly covered by coenenchyme. Scapus of column dark red in situ, dark brown in preserved specimens. Capitulum of column orange in situ, dark violet in preserved specimens. Column and coenenchyme heavily encrusted with visible sand and silica particles in ectodermal tissue to outer mesoglea (Fig. 1c, d). Preserved, contracted polyps 2.0-6.0 mm in height, 1.0-3.0 mm in diameter. In situ, opened polyps approximately < 8.0 mm in height, < 10 mm in diameter. Oral disk 5.0-8.0 mm in diameter, orange to light orange in coloration. Number of oral furrows the same as the number of tentacles, and cream white circular protrusion in central oral disk bears slit-like mouth aligned with directives. Tentacles arranged in two rows (15-17 inner endocoelic tentacles and 15-17 outer exocoelic tentacles), as long as the expanded oral disk diameter. Number of tentacles 30-34, transparent in coloration. 15-17 marginal teeth present under inner endocoelic tentacles (Fig. 1b). Tips of tentacles usually cream in coloration. Capitular ridges indiscernible. Internal morphology. Azooxanthellete. Mesentery number 30-34, complete 15-17, incomplete 15-17. Mesenteries in macrocnemic arrangement (Fig. 2c). Mesoglea thickness 0.01-0.10 mm, and thicker than ectoderm. Developed siphonoglyph distinct and U-shaped. Mesenterial filaments present (Fig. 2a). Endodermal marginal muscle, short comb-like mesogleal pleats supporting the entire length of the marginal muscle (cteniform endodermal marginal muscle: Fig. 2b). Basal canals of mesenteries absent (Fig. 2d). Additionally, possible gametes observed in several longitudinal sections.
Habitat and distribution. Northwestern Pacific Ocean: Sagami Bay, Kanagawa, Japan at depths < 14 m.
Associated host. We could not identify host polychaete species as there were no polychaetes in the tubes. However, the tubes that Antipathozoanthus tubus sp. nov. was attached to may belong to species within the genus Eunice, as polychaete species that build parchment-like branched tubes have been reported from this genus (e.g., Díaz-Díaz et al. 2020).  Fig. 4. The genus Antipathozoanthus appeared as a monophyletic clade located within the family Parazoanthidae with strong nodal support (ML=100%, BI=1) and was close to a Parazoanthidae clade containing species associated with stalked hexactinellid sponges. Within Antipathozoanthus, two subclades were formed; one subclade consisted of the antipatharian-associated species A. macaronesicus, A. hickmani, A. remengesaui, and A. cavernus (ML = 100%, BI = 0.97), and the other subclade consisted of A. tubus sp. nov. and A. obscurus (ML = 82%, BI = 0.92). Genetic distances in COI, 16S-rDNA, and ITS-rDNA sequences between A. tubus sp. nov. and other Antipathozoanthus species were 0.000 to 0.009, 0.002 to 0.010, and  Table 2. Cnidae types and sizes observed in Antipathozoanthus tubus sp. nov. Frequency: relative abundance of cnidae type in decreasing order; numerous, common, occasional, rare (n = number of cnidae). 0.010 to 0.128, respectively. As well, A. tubus sp. nov. and other Antipathozoanthus species shared unique insertion/deletion patterns in 16S-rDNA sequences. Remarks. Antipathozoanthus tubus sp. nov. can be easily distinguished from A. remengesaui Kise et al., 2017, A. macaronesicus (Ocaña & Brito, 2003), and A. hickmani Reimer & Fujii, 2010 by the number of tentacles as well as different coloration; Antipathozoanthus remengesaui, A. macaronesicus, and A. hickmani have up to 42 tentacles (Ocaña and Brito 2003;Reimer and Fujii 2010;Kise et al. 2017), while A. tubus sp. nov. has fewer tentacles (30-34). The dark red colored polyps and coenenchyme of A. tubus sp. nov. are not found in these other three Antipathozoanthus species. In addition, A. tubus sp. nov. differs from A. cavernus Kise et al., 2017 with regards to polyp coloration (A. cavernus has orange or light orange polyps: Kise et al. 2017). Although A. tubus sp. nov. and A. obscurus Kise et al., 2017 are phylogenetically close, their COI, 16S-rDNA, and ITS-rDNA sequences are all unique (genetic distances in COI, 16S-rDNA and ITS-rDNA sequences between A. tubus sp. nov. and A. obscurus were 0.009, 0.03, and 0.12, respectively). As well, these two species can be separated by coloniality; polyps of A. obscurus are connected by a stolon forming a mesh network (Kise et al. 2017), while polyps of A. tubus sp. nov. are connected by a well-developed coenenchyme. Furthermore, A. macaronesicus, A. remengesaui, A. cavernus, A. hickmani, and A. obscurus have holotrichs in their column (Ocaña and Brito 2003;Reimer and Fujii 2010;Kise et al. 2017), while holotrichs were not observed in the column of A. tubus sp. nov.

Tissue Type of cnidae
Antipathozoanthus is a circumglobally distributed genus, as species have reported from the Indian, Pacific, and Atlantic Oceans (Ocaña and Brito 2003;Sinniger et al. 2010;Reimer and Fujii 2010;Bo et al. 2012;Reimer et al. 2014;Kise et al. 2017), with members living from shallow waters (A. obscurus at 3 m depth; Kise et al. 2017) to mesophotic depths (153-169 m for Antipathozoanthus sp. sensu Reimer et al. 2019). The most distinctive attributes of A. tubus sp. nov. are its substrate and habitat.
Antipathozoanthus macaronesicus, A. hickmani, A. remengesaui, and A. cavernus are found on antipatharians within the families Antipathidae and Myriopathidae (Ocaña and Brito 2003;Reimer and Fujii 2010;Sinniger et al. 2010;Bo et al. 2012;Kise et al. 2017), while A. obscurus is directly attached to coral reef carbonate (Kise et al. 2017). On the other hand, A. tubus sp. nov. is the only species of the genus found to date on tubes of polychaetes. Four Antipathozoanthus species are known from low light environments; A. macaronesicus, A. remengesaui, and A. cavernus have been found in cave entrances, and A. obscurus is found in crevasses and/or coral reef caves (Ocaña and Brito 2003;Kise et al. 2017). On the other hand, the habitat of A. tubus sp. nov. is not a low-light environment, but the specimens were instead found on a polychaete tube attached to exposed rock.
Within Parazoanthidae, until now, Isozoanthus altisulcatus Carlgren, 1939 is the only species described as living on the tubes of polychaetes. However, several morphological differences exist between A. tubus sp. nov. and I. altisulcatus. Capitular ridges are developed and conspicuous in I. altisulcatus, whereas they are indiscernible in A. tubus sp. nov. The marginal teeth on the capitulum found in A. tubus sp. nov. were not observed in I. altisulcatus. Although Carlgren (1939) did not describe the numbers of tentacles of I. altisulcatus, the numbers of mesenteries are 34-42 (Carlgren 1939). As numbers of tentacles are known to be equal to the number of mesenteries (Bourne 1900), the number of tentacles of I. altisulcatus is likely to be 34-42, which is greater than the number of tentacles of A. tubus sp. nov. (30-34).
Genetic distances of COI sequence between A. tubus sp. nov. and other Antipathozoanthus species can be considered as intra-generic differences based on previous comparisons of genetic distances . As well, A. tubus sp. nov. shared unique insertion/deletion patterns in 16S-rDNA sequences with other Antipathozoanthus species. Thus, we consider that A. tubus sp. nov. should belong to the genus Antipathozoanthus and does not warrant the erection of a new parazoanthid genus.
Etymology. Antipathozoanthus tubus sp. nov. is named from the Latin tuba, as this species is found on polychaete tubes. The Japanese name is 'Iwa-tsuno-sunaginchaku'.

Discussion
Japanese waters are composed of a wide variety of physical, geographical, and topographical environments due to the latitudinal extension of Japan spanning from the near-tropics of Okinawa to the near-subarctic Hokkaido, and also to the dynamic geology of the region, and thus, Japanese waters have high marine species diversity levels (Fujikura et al. 2010). At the same time, it is estimated that more than 70% of the marine taxa in this region remain undescribed (Fujikura et al. 2010). The order Zoantharia is one such taxon for which much work remains to be done. Although many zoantharian studies have been conducted in Japan, taxonomic studies are still biased by region; southern Japan including Kochi and the Ryukyu Archipelago have been focused on in comparison to other regions . As a result, 16 species have been described based on type specimens collected from southern Japan (mainly from the Ryukyu Archipelago) (e.g., Irei et al. 2015). In other regions, historical taxonomic works have been conducted in Sagami Bay by Lwowsky (1913), Tischbierek (1929), andCarlgren (1934), with the description of three macrocnemic species; Hydrozoanthus gracilis Lwowsky, 1913, Epizoanthus cnidosus Tischbierek, 1929 (junior synonym of Hydrozoanthus gracilis), and Epizoanthus ramosus, Carlgren 1934. As well, Hertwig (1882 reported the carcinoecium-forming Epizoanthus parasiticus (Verill, 1864) based on the specimens collected from the Sea of Enshu during the Challenger expedition. However, few taxonomic studies have been conducted in these regions since these past historical works, and for many other regions, almost no literature exists (e.g. the Sea of Japan). Thus, in order to understand species richness and the distribution patterns of zoantharians in Japan, further diversity studies with sampling efforts focused on understudied regions are required.
Natural History Museum and Institute) for permission to subsample specimens, and assistance in visiting the CMNH. Prof. Akihiro Takemura (University of the Ryukyus) is thanked for giving us technical support. The first author was supported by JSPS KAKENHI grant number 19J12174. We thank two reviewers and the editor, who all provided helpful comments on earlier versions of this manuscript.