﻿A new hexactinellid-sponge-associated zoantharian (Porifera, Hexasterophora) from the northwestern Pacific Ocean

﻿Abstract Symbiotic associations between zoantharians and sponges can be divided into two groups: those that associate with Demospongiae and those that associate with Hexactinellida. Parachurabanashinseimaruae Kise, gen. nov. et sp. nov., a new genus and a new species of Hexactinellida-associated zoantharian from Japanese waters, is described. It is characterized by a combination of the following: i) its host hexactinellid sponge, ii) very flat polyps, iii) cteniform endodermal marginal muscles, and iv) characteristic mutations in three mitochondrial regions (including a unique 26-bp deletion in 16S ribosomal DNA) and three nuclear regions. Parachurabanashinseimaruae Kise, gen. nov. et sp. nov. is the third genus in the family Parazoanthidae that is reported to be associated with Hexasterophora sponges. Although specimens have so far only been collected on Takuyo-Daigo Seamount off Minami-Torishima Island in Japan, unidentified zoantharians of similar description have been reported from the waters around Australia, indicating that the species might be widespread across the Pacific.


Specimen collection
A single specimen was collected on 19 June 2020 by using a remotely operated submersible on Takuyo-Daigo Seamount off southwestern Minami-Torishima Island in the northwestern Pacific Ocean during a cruise aboard the RV Shinsei-maru. Photographs of the specimen were taken in situ for gross external morphological observation. The collected specimen was fixed in 99.5% EtOH and stored at -80 °C.
Phylogenetic analyses were performed on the concatenated dataset using maximum likelihood (ML) and Bayesian inference (BI). ModelTest-NG v. 0.1.6 ) and the Akaike information criterion were used to independently select the best-fitting model for each molecular marker for both ML and BI. The best models for ML and BI analyses were TrN+I+G (BI: HKY+I+G) for COI, TPM3uf+I+G (BI: HKY+G) for mt 12S-rDNA, GTR+G for mt 16S-rDNA, HKY+I+G for 18S-rDNA, TPM1uf+I+G (BI: GTR+I+G) for ITS-rDNA, and GTR+I+G for 28S-rDNA. Independent phylogenetic analyses were performed using models partitioned by region in RAxML-NG v. 0.9.0  for ML, and MrBayes v. 3.2.6 (Ronquist and Huelsenbeck 2003) for BI. RAxML-NG was configured to use 12,345 initial seeds, search for the best tree among 100 preliminary parsimony trees, scale and automatically optimize branch length for each partition, and optimize the model parameters, with 1000 bootstrap replicates. MrBayes was configured as indicated by ModelTest-NG: 4 Markov chain Monte Carlo heated chains were run for 5,000,000 generations with the temperature of the heated chain set to 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 was consistently below 0.01.
ITS-rDNA has been considered as a useful marker to delineate species in Zoantharia (Reimer et al. 2007). Therefore, additional ML phylogenetic analysis for ITS-rDNA was performed using PhyML v. 3.0 (Guindon et al. 2010) with the best model (GTR) inferred by Smart Model Selection (SMS) implemented in the PhyML, with 1000 bootstrap replicates.

Morphological observations
External morphological characters of the preserved specimen were examined using in-situ images and a dissecting microscope. Internal morphological characters were examined by using histological sections; 10-15 µm serial sections were made with a microtome (LEICA RM2145; Leica, Germany) and stained with haematoxylin and eosin after decalcification with Morse solution for 48 h (1:1 vol; 20% citric acid: 50% formic acid) and desilication with 20% hydrofluoric acid for 18-24 h. Classification of marginal muscle shapes followed the scheme described by Swain et al. (2015). Cnidae analysis was conducted using undischarged nematocysts from the tentacles, columns, actinopharynxes, and mesenterial filaments of two polyps of the holotype specimen under a Nikon Eclipse80i microscope (Nikon, Tokyo). Cnidae sizes were measured using ImageJ v. 1.45 (Rasband 2012). Cnidae classification followed England (1991) and Ryland and Lancaster (2004) except for the treatment of basitrichs and microbasic b-mastigophores as mentioned by Kise and Reimer (2019). Associated hexactinellid sponges were identified based on morphology (Reiswig and Wheeler 2002a, b).

Abbreviations
CMNH Coastal Branch of the Natural History Museum and Institute, Chiba, Japan; NSMT National Science Museum, Tsukuba, Ibaraki, Japan; RMNH Rijksmuseum van Natuurlijke Historie (now at the Naturalis Biodiversity Center), Leiden, the Netherlands; RUMF Ryukyu University Museum (Fujukan), University of the Ryukyus, Okinawa, Japan.

Taxonomic description
Order Remarks. Parachurabana gen. nov. is differentiated from other sponge-associated parazoanthids based on a combination of host-sponge identity and morphological features. Parachurabana gen. nov. is easily distinguished from the genera Bergia, Parazoanthus, and Umimayanthus by its association with hexactinellid sponges, as the three other genera are associated with Demospongiae sponges. In Hexactinellida-spongeassociated Parazoanthidae genera, the association with subclass Amphidiscophora differentiates Parachurabana gen. nov. from Isozoanthus. Marginal muscle morphology differentiates Parachurabana gen. nov. (cteniform endodermal marginal muscles) from Vitrumanthus (cyclically transitional marginal muscles). Parachurabana gen. nov. can be distinguished from Churabana by polyp size, as Parachurabana gen. nov. has very flat polyps when preserved (0.5-1.0 mm in height, 0.5-3.0 mm in diameter) in comparison to Churabana (3.0-4.0 mm in height, 2.8-4.0 mm in diameter). In the16S-rDNA region, Parachurabana gen. nov. is characterized by a unique deletion of 26 bp (positions 136-150 and 168-178 in our alignment) (Suppl. material 2).
Etymology. Parachurabana alludes to its morphological similarities to Churabana. The Prefix "para" is a Greek word meaning "resembling."    (Fig. 1a, b). Surface of column rough, and ectoderm continuous. Polyps attached to hexactinellid sponge surfaces with pedal-disk-like structure (Fig. 1c, d). In contracted polyp, tentacles poorly covered by capitulum and actinopharynx visible. Preserved column creamy white in color and heavily encrusted with sand and silica particles. Capitulary ridges discernible, 12-14 in number (Fig. 1c). Tentacles 24-28 in number, shorter than or equal to expanded oral disk diameter. Living expanded polyps to ca. 10.0 mm in height and 5.0 mm in diameter. Preserved contracted polyps to 0.5-1.0 mm in height and 0.5-3.0 mm in diameter. Living column white and/or yellowish; capitulum and tentacle transparent (Fig. 1a).
Etymology. The species is named after RV Shinsei-maru, as the type specimens were collected by this vessel.

Discussion
Parachurabana gen. nov. is the third Parazoanthidae genus known to associate with hexasterophoran sponges. Each of these three genera is associated with different hexasterophorans: Parachurabana gen. nov. is known to associate with Farrea (family Farreidae); Churabana with Pararete Ijima, 1927 (Euretidae); and Vitrumanthus with     Reid, 1969 (Euretidae), Cyrtaulon Schulze, 1886 (Sceptrulophora incertae sedis), Aphrocallistes Gray, 1858 (Aphrocallistidae), and Tretochone Reid, 1958 (Euretidae) (Kise et al. 2022). In addition, Vitrumanthus is also known to associate to Parahigginsia Dendy, 1924 within the Demospongiae (Kise et al. 2022). Although Parachurabana gen. nov. may be host specific to the genus Farrea, recent studies suggest that the association between zoantharians and host organisms can be more flexible than initially presumed (see Vaga et al. 2020). Further studies on more taxa are required to evaluate host specificity between zoantharians and hexasterophorans. Recent studies indicate that the deep sea harbors high levels of zoantharian diversity (e.g., Sinniger et al. 2013;Carreiro-Silva et al. 2017;Reimer et al. 2019). However, taxonomic studies on the deep-sea zoantharians are generally lacking and numerous undescribed species await formal description. This study contributes to filling this taxonomic gap.