Four new species of Ctenodrilus, Raphidrilus, and Raricirrus (Cirratuliformia, Annelida) in Japanese waters, with notes on their phylogenetic position

Four new species of annelids, Ctenodrilus japonicus sp. nov., Raphidrilus misakiensis sp. nov., Raphidrilus okinawaensis sp. nov., and Raricirrus anubis sp. nov., are described based on specimens collected from Japanese waters. Ctenodrilus japonicus sp. nov. inhabits the interstitial environment and can be distinguished from the other congeners by the following features: (i) total of 16 chaetigers, (ii) chaetigers 1–3 with stout hooks, (iii) minute body (approximately 1 mm in length), (iv) all parapodia with the same number of chaetae (two notochaetae; two neurochaetae), and (v) presence of dorsal and ventral papillae. Raphidrilus misakiensis sp. nov. lives under intertidal stones and can be distinguished from other congeners by having pectinate neurochaetae. Raphidrilus okinawaensis sp. nov. inhabits the interstitial environment and can be distinguished from other congeners by: (i) absence of annulation on the peristomium and achaetous segment and (ii) presence of a heart body in chaetigers 4–5. Raricirrus anubis sp. nov. inhabits whale bones and can be distinguished from other congeners by the following features: (i) presence of a heart body in chaetigers 9–14, (ii) presence of capillary neurochaetae on chaetiger 1, and (iii) presence of simple curved spines. A phylogenetic tree based on partial sequences of cytochrome c oxidase subunit I and 16S rRNA from the new species and other cirratulid worms showed that Raphidrilus is included in Cirratuliformia. This is the first record of Raphidrilus and Raricirrus from Japanese waters.

Several specimens of cirratuliform polychaetes lacking dorsal tentacles were collected during the survey of polychaetes in Japanese waters. In this study, we describe the specimens as four new species and provide the phylogenetic tree based on two gene sequences. This is the first report of Raphidrilus and Raricirrus from Japan.

MATERIALS AND METHODS
Specimens of Ctenodrilus were collected on 17 th July 2020 in intertidal to subtidal areas at Tengan-sanbashi (26 28′04″N, 127 49′32″E), Okinawa-jima Island, Japan. Specimens of Raphidrilus were collected on 17 th July 2020 in intertidal to subtidal areas, Akasaki beach (26 28′04″N, 127 49′32″E) (collected with Actaedrilus okinawaensis Jimi, Fujimoto & Imura, 2020), Okinawa-jima Island and on 26 th July 2019 under intertidal rocks, Misaki, Japan (35 09′36″N, 139 36′41″E). Specimens of Raricirrus were collected on 12 th October 2014 from a whale ulna kept in an aquarium. The ulna was collected from a humpback whale carcass off Atami (35 04′29″N, 139 07′34″E, at a depth of 399 m) during a deep-sea research cruise using the ROV Hyper-Dolphin (cruise number: NT13-06, dive number: HD#1501) on March 26, 2013. The carcass was stranded and deployed in the bay on December 3, 2011. For morphological observation, the specimens were fixed in 10% formalin-seawater solution and later washed and preserved in 70% ethanol or fixed and preserved in 70% ethanol. For DNA extraction, the specimens were fixed and preserved in 99.5% ethanol. Fresh specimens were photographed using a digital camera (Nikon D5200). Preserved specimens were examined under stereomicroscopes (Nikon SMZ18 and Nikon ECLIPSE 80i). Drawings are made by using Wacom Cintiq and Clip Studio Paint. Traits that are not visible at the magnification used in the drawing are omitted. Specimens of Raricirrus for scanning electron microscopy (SEM) were post-fixed in 2% OsO 4 for 2 h, dehydrated through a series of ethanol and acetone, critical point dried (BAL-TEC CPD-030), osmium coated (Filgen OPC40), and observed using JEOL JSM-7001F. Specimens of Ctenodrilus and Raphidrilus for SEM observations were washed in deionized water or PBS buffer and dehydrated in a graded ethanol series, dried in a critical-point dryer (HITACHI HCP-1) using liquid CO 2 , and coated with gold in an ion sputter (HITACHI E-1045). Observations were conducted using a scanning electron microscope (HITACHI S-3000N).
Genomic DNA were extracted from small pieces of the paratypes. DNA extraction, sequencing, alignment, calculating pairwise genetic distances, as well as maximum likelihood (ML) and Bayesian inference (BI) phylogenetic tree construction followed the procedures of Jimi et al. (2021).
Additional sequences of other Cirratuliformia were obtained from GenBank (Table 1). Newly obtained sequences have been deposited in the GenBank (Table 1).
Type specimens were deposited in the National Museum of Nature and Science, Tsukuba (NSMT). The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:88A729DD-232C-4241-AF17-8C603D86C231. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE and CLOCKSS.
Etymology. The name is derived from the distribution of this new species. The specific name is a noun in the genitive case.
Distribution. Interstitial sand of Okinawa-jima, Okinawa, at a depth of 0.5 m.
Remarks. Ctednodrilus japonicus sp. nov. can be distinguished from the other members of the genus by the following features: (i) heart body from chaetiger 1 to the beginning of chaetiger 3, (ii) body with dark black and yellow spots, (iii) esophagus present to end of chaetiger 2, (iv) stomach present to the middle of chaetiger 10, and (v) 1-4 hooks with 3-4 inner teeth present in noto-and neuropodia. This species most resembles C. pacificus Magalhães et al., 2016 in having dark black spots and 1-4 hooks with 3-5 inner teeth in noto-and neuropodia. The new species has a heart body extending to the beginning of chaetiger 3 and the stomach extending to the middle of chaetiger 10, while C. pacificus has a heart body extending to the middle of chaetiger 3 and a stomach extending to the chaetiger 6-8. Ctenodrilus japonicus sp. nov. is closest to C. pacificus in terms of 565 bp of COI sequences; it was 12.3 % in K2P. From Japanese waters, Sudzuki & Sekiguchi (1972) described Ctenodrilus serratus limulicolus from an aquarium in Shimoda Marine Research Center. However, Wilfert (1973) concluded that the subspecies from Japan is not valid because of the variations in traits of the subspecies fall within the range of variation of those traits within C. serratus (Schmidt, 1857). The new species can be distinguished from C. serratus limulicolus (Sudzuki & Sekiguchi, 1972), by having a ciliated stomach and 1-4 hooks with 3-5 inner teeth in the noto-and neuropodia.
Esophagus ciliated to end of chaetiger 5, stomach transparent and ciliated to end of chaetiger 10. Heart body present from middle of chaetiger 3 to end of chaetiger 5 (Figs. 6A-6C). Intestine ciliated to end of body.
Pygidium elongated segment with dorsal anus; anal cilia not seen.
Etymology. The name is derived from the distribution of this new species. The specific name is a noun in the genitive case.
Etymology. The name is derived from the distribution of this new species. The specific name is a noun in the genitive case. Distribution. Only known from the type locality, the Akasaki beach, Okinawa, Japan, subtidal sands.
Etymology. "Anubis" is a Greek name of an ancient Egyptian god that was a grave keeper. This worm lived around whale skeletons perhaps as a grave keeper. The specific name is a noun in the nominative case.
Distribution. Only known from whale skeletons of the type locality, at a depth of 399 m.

Phylogenetic analysis
The topologies (Fig. 13) recovered by ML and BI analyses were identical. Ctenodrilus japonicus sp. nov. and C. pacificus Magalhães et al., 2016 formed a single clade with 97% of bootstrap support in ML and 0.97 posterior probability in BI. Raphidrilus misakiensis sp. nov. and Raphi. okinawaensis sp. nov. formed a single clade with 100 % of bootstrap support in ML and 1.00 posterior probability in BI. Raphidrilus is sister to the Dodecaceria-Ctenodrilus-Raricirrus clade, although they comprise a poorly supported clade (50 % bootstrap support, 0.6 posterior probability). Raricirrus anubis sp. nov. and Rari. beryli formed a single clade with 88 % of bootstrap support in ML and 1.00 posterior probability in BI. taxonomic studies with molecular analyses (Magalhães et al., 2016;Magalhães, Linse & Wiklund, 2017;Weidhase, Bleidorn & Simon, 2016). This topology warrants the revision of Ctenodrillidae and Cirratulidae. However, the scope of this study is to describe the four new species from Japan and infer their phylogenetic positions and no broad morphological re-assessment spanning the two families were performed. For this reason, we treated these genera as belonging to Cirratuliformia. Our study also provided the first molecular data of Raphidrilus. The genus is sister to the Dodecaceria-Ctenodrilus-Raricirrus clade. However, the Raphidrilus-Dodecaceria-Ctenodrilus-Raricirrus clade is poorly supported by bootstrap supports (50 BS) and posterior probability (0.6 PP). It is unclear what the relationship is between the three clades (1. Raphidrilus, 2. Cirratulus-Cirriformia-Timarete, 3. Dodecaceria-Ctenodrilus-Raricirrus) at this level of support. A previous morphology-based study inferred that Raphidrilus is close to Dodecaceria (Magalhães, 2015). In order to understand the evolutionary history of Cirratuliformia especially adaptation to the interstitial lifestyle, further OTUs and gene regions are needed for constructing robust phylogenetic trees. Especially, broader OTUs covering the cirratulid genera will help solving this problem.

DISCUSSION
In Japan, Ctenodrilus serratus limulicolus (Sudzuki & Sekiguchi, 1972) has been known as the sole Ctenodrilus species. Our new species C. japonicus sp. nov. is clearly separated from the species by several characters. There is no doubt that two species of Ctenodrilus inhabit Japan. However, the taxonomic status of C. serratus limulicolus is still unclear. Topotype sampling and DNA sequences of the species are needed for confirmation of C. serratus limulicolus validity in the future studies. Magalhães et al. (2016) described C. pacificus from Hawaii, Pacific Ocean. Ctenodrilus japonicus sp. nov. is morphologically most similar to the Hawaiian species, as described in the Remarks. In the phylogenetic tree, C. japonicus and C. pacificus form a clade with high support (97 BS/0.97 BP). Besides asexual reproduction by stolons (Scharff, 1887;Schmidt, 1857;Kennel, 1882;Magalhães et al., 2016; this study), Ctenodrilus is known to reproduce sexually by viviparity (Halt et al., 2006). Both ways have low dispersal ability and do not allow gene exchange to occur over long distances, which may promote geographical isolation in both species. Although it is not known how the common ancestor arrived in Hawaii, the phylogenetic relationships of Ctenodrilus species in different regions may shed light on the process of dispersal and speciation.
This study represents the first record of Raricirrus and Raphidrilus from Japan. Raricirrus anubis sp. nov., like other species of the genus, has been observed in whalebones and is consistent with known habitats (Petersen et al., 2012;Magalhães, Linse & Wiklund, 2017). Raphidrilus okinawaensis sp. nov. was collected from interstitial environments of sandy beaches like other species of the genus (Magalhães, Bailey-Brock & Davenport, 2011), but Raphidrilus misakiensis sp. nov. was collected from unusual environments such as attached to rocks. Raphidrilus misakiensis sp. nov. may have a slightly different ecology from other species of the genus because of its unusual habitat and unusual chaetae. Further field observations are needed.
Discovering an unusual morphological character (presence of pectinate chaetae) in Raphidrilus misakiensis sp. nov. led us to modify the genus diagnosis. In Qian & Chia (1989), an undescribed species of Raphidrilus was found to have genital spines which Magalhães, Bailey-Brock & Davenport (2011) did not include in the definition of the genus regarding it as a species-specific trait. It is possible that the special trait of R. misakiensis is also species-specific, but we have included it for now. As the diversity of the genus Raphidrilus becomes better known, the definition of the genus will become clearer.

CONCLUSIONS
The genera Ctenodrilus, Raphidrilus, and Raricirrus are a poorly known group in Cirratuliformia. In Japan, there has been only one study on this group, in which almost nothing is known about. Molecular phylogenetic analyses have been carried out in a number of previous studies in which some genera were not included. In this study we describe four new species from Japan and construct a phylogenetic tree using two genes to clarify the phylogenetic relationships within this group. As a result the phylogenetic position of Raphidrilus was not clearly determined, therefore further analyses should be required including additional OTUs in the future. This is the first record of Raphidrilus and Raricirrus from Japan.