﻿Rhynchospioaciliata sp. nov., a new spionid species (Annelida, Spionidae) from the Korea Strait

﻿Abstract A new spionid polychaete, Rhynchospioaciliatasp. nov., was discovered in the fine sandy sediments of an intertidal habitat from Korean waters. The new species is considered a simultaneous hermaphrodite, but no brooding embryos were found in any of the specimens collected in this study. This species is unique in the absence of ciliation in the anteriormost chaetigers. Rhynchospioaciliatasp. nov. is morphologically most similar to Rhynchospiofoliosa Imajima, 1991 from Japan in having an elevation on the prostomium, conspicuously large and foliaceous branchiae, and intersegmental lateral pouches. However, the new species differs from the latter by the following characteristics: (1) large and lanceolate notopodial postchaetal lamellae of chaetiger 1, (2) transverse ciliated bands and ciliation on the inner branchiae absent in anteriormost chaetigers, and (3) pygidium with one pair of ventral cirri and numerous elongated dorsolateral cirri. Detailed description and illustrations of the new species are provided with molecular information on mitochondrial cytochrome c oxidase subunit I (COI), 16S ribosomal DNA (rDNA), nuclear 18S rDNA, and 28S rDNA.


Introduction
Rhynchospio Hartman, 1936 is one of the less species-rich spionid genera mainly reported from the Pacific and adjacent waters and comprising 12 valid species (Radashevsky and Choi 2021). Some Rhynchospio species are known to be simultaneous hermaphrodites and brood their larvae on the dorsum (Radashevsky 2007;Radashevsky and Choi 2021). Adult members of the genus are characterized by a prostomium with frontolateral horns, branchiae appearing from chaetiger 2, notopodia with only capillary chaetae, and more than two pairs of pygidial cirri (Fauchald 1977;Blake and Kudenov 1978;Radashevsky et al. 2016). Some morphological characteristics, such as the first appearance of neuropodial hooded hooks and the number of dorsal pygidium cirri are highly variable and overlap between Rhynchospio species (Blake 1996;Radashevsky et al. 2014). For this reason, the status of some Rhynchospio species remains uncertain (e.g., R. cf. foliosa from USA, see Radashevsky et al. 2016). Identifying Rhynchospio species using only morphological characters has been considered very difficult, and a combination of detailed morphological and molecular analyses is required (Radashevsky et al. 2014;Simon et al. 2018). Additionally, information from the methyl green staining pattern (MGSP) can also be important for identifying Rhynchospio species (Simon et al. 2018).

Sampling and morphological observations
Adult samples were collected from sandflats in the intertidal zone of the Korea Strait (Fig. 1) using 500 μm mesh sieves. Morphological observations were performed on both live and formalin-fixed materials. The live specimens were relaxed using 10% MgCl2 solution in seawater, and characteristics were observed under a stereomicroscope (Leica MZ125, Microsystems Wetzlar GmbH, Wetzlar, Germany). After live observation, the individuals were fixed in 4% formaldehyde for morphological study and subsequently transferred to 70% ethanol. Some formalin-fixed specimens (intermittently transferred to distilled water) were stained with methyl green solution according to the method described by Meißner (2005). To observe the morphology of sperm and oocytes, a few formalin-fixed specimens were transferred to glycerol. Photographs were taken using a digital camera (Tucsen Dhyana 400DC, Fuzhou Fujian, China) with the capture program Tucsen Mosaic v. 15 (Fuzhou Fujian, China). Dissected appendages were mounted using the Eukitt Quick-hardening mounting medium (Sigma-Aldrich, St. Louis, MO, USA) for permanent slides. The specimens were fixed in 95% ethanol for molecular analyses. The specimens for scanning electron microscopy (SEM) were dehydrated using a t-BuOH freeze dryer (VFD-21S Vacuum Device; Ibaraki, Japan), covered with platinum, and observed using a Hitachi SEM model S-4300SE (Hitachi, Japan). All type and voucher specimens examined in this study were deposited at the National Institute of Biological Resources at Incheon, South Korea (NIBR). Non-type material of R. foliosa from Akkeshi, Hokkaido, northern Japan (NSMT-Pol 104819) deposited in National Museum of Nature and Science was also examined for morphological comparison.
Etymology. The specific name aciliata is a combination of the Latin prefix a-and the Latin word cilia, meaning "absence of cilia." This name refers to the absence of ciliation on the dorsum and inner margins of branchiae of the anteriormost chaetigers.
Habitat and ecology. The new species was found in fine sand in the intertidal zone. Distribution. Soan Island, Korea. Genetics. The partial mitochondrial COI, 16S rDNA, nuclear 18S rDNA, and 28S rDNA sequences from three specimens of R. aciliata sp. nov. were determined. The Gen-Bank accession numbers and sequence lengths of the species were as follows: ON206852-4 for COI (687 bp), ON206000-2 for 16S rDNA (517 bp), ON206003-5 for 18S rDNA (1,778 bp), and ON206006-8 for 356 bp (28S rDNA) ( Table 1). The intra-specific genet- ic distances were 0-0.4% in 16S rDNA, and no variation was detected in the other three gene regions. Based on the available molecular data of Rhynchospio from GenBank, the new species is genetically the closest to R. cf. foliosa from Oregon, USA. The genetic distance between the new species and R. cf. foliosa was 6.9% (22/294 bp, KR607514) in 16S rDNA, 0.4% (4/1,723 bp, KR607515) in 18S rDNA, and 0.7% (2/351 bp, KP986490) in 28S rDNA (Table 2). Phylogenetic analyses showed that R. aciliata sp. nov. formed a monophyletic clade with R. cf. foliosa from Oregon, USA (Fig. 6). This result implies that their close relationships share several morphological characteristics (see below). Unfortunately, the molecular information of R. foliosa from Japan (type locality) is still unknown. Further genetic studies on R. foliosa are needed to confirm their genetic relationships.

Discussion
Seven Rhynchospio species are known to be simultaneous hermaphrodites, three of which brood their larvae on the parent's dorsum (Radashevsky and Choi 2021). Rhynchospio aciliata sp. nov. examined in this study is a simultaneous hermaphrodite producing spermatozoa and oocytes in the anterior and posterior chaetigers respectively, but no brooding embryo was observed in any of the materials collected in May 2021. This finding is quite different from that of R. glandulosa specimens found with larvae on the dorsum collected in May 2013 and May to June 2016-2018. It seems likely that the brooding period is species-specific. The reproductive biology and sperm morphology of this new species is unknown.
The new species is unique in terms of the lack of ciliation in the anteriormost chaetigers. Among the known Rhynchospio species, R. foliosa from Japan has an elevation on the prostomium and conspicuously large, foliaceous anterior branchiae (Imajima 1991; see a key to the species by Radashevsky et al. 2014). We examined the nontype material of R. foliosa (NSMT-Pol 104819, collected by Imajima in 1991, from Hokkaido (43°00.9'N, 144°49.6'E) near the type locality) and it agreed well with the original description. Rhynchospio aciliata sp. nov. is morphologically very similar to R. foliosa in having large, foliaceous branchiae and the presence of intersegmental lateral pouches (described as "membranous ridge" by Imajima (1991)). However, the new species clearly differs from the Japanese species (characteristics present in parentheses) by large, lanceolate notopodial postchaetal lamellae of chaetiger 1 (small and similar to those of neuropodia), papilliform elevation in the posterior part (caruncle anteriorly elevated above the prostomium), ciliation absent in the anteriormost chaetigers (present), neuropodial hooks numbering 9 or 10 per fascicle (8 per fascicle), and pygidium with a pair of ventral cirri and elongated dorsolateral cirri (numerous foliaceous lobes) (Imajima 1991 (12-17), sperm present up to 32 segments (up to 4 segments), and pygidium with up to 15 pairs (up to 4 pairs). Simon et al. (2018) illustrated the MGSP of R. mzansi from South Africa for the first time in this genus. The new species and R. mzansi have similar staining patterns in having 2 transverse bands ventrally but differ in the dorsal side of the body: indistinctly stained in the new species and distinctly stained (especially anterior chaetigers) in R. mzansi (Simon et al. 2018). After at least 1 week after staining, the region of the chaetigers with sperm (male fertile chaetigers) remained conspicuously stained (in all examined specimens) but was not as prominent as that of the ventral transverse bands. These findings support the finding of Simon et al. (2018) that the MGSP is a reliable character for species identification in the genus Rhynchospio.
Phylogenetic analysis based on the sequences of three gene regions (16S rDNA, 18S rDNA, and 28S rDNA) showed that two monophyletic clades: R. aciliata sp. nov. and R. cf. foliosa in one clade, and all other known species in a second clade. The most conspicuous morphological features separating the two groups seem to be the branchial morphology (large and foliaceous vs elongated and normal in size) and the arrangement of male segments (much more than 12 segments vs not more than 12 segments).
The gene sequences obtained in this study along with morphological information, including MGSP and SEM observations, will be useful for further taxonomic or phylogenetic studies of genus Rhynchospio.