New species of the genus Spio (Annelida, Spionidae) from the southern and western coasts of Korea

Abstract A new spionid polychaete, Spiopigmentatasp. nov., is described from the southern and western coasts of Korea. This new species differs from its congeners by the combination of the following morphological characteristics: the presence of orange-brown pigmentation on the anterior part of the prostomium, black pigmentation on the peristomium and along the body, U-shaped nuchal organs, a comparatively long extension of metameric dorsal ciliated organs, three pairs of white dots per chaetiger, two to three posterior abranchiate chaetigers, and the presence of tridentate neuropodial hooded hooks. The partial 16S ribosomal DNA (rDNA) and nuclear 18S rDNA sequences of the new species and Spio sp. 2 reported by Abe and Sato-Okoshi (2021) from Japan showed high similarity, indicating that these two specimens belong to the same species. A detailed description and illustrations of the new species, together with molecular information, are provided.


Introduction
Spio Fabricius, 1785 is one of the most speciose genera of Spionidae Grube, 1850. It currently comprises 37 species occurring all over the world (Read and Fauchald 2021). Spio filicornis (O. F. Müller, 1776), the type species of the genus, has been repeatedly misidentified and eventually regarded as a cosmopolitan species with worldwide distribution (Okuda 1937;Imajima and Hartman 1964;Paik 1975Paik , 1982Paik , 1989. The confusion regarding the taxonomy of this species has been considerable, and it was evident that the identity of S. filicornis needed to be stabilized. Against this background, a neotype of the species from the type locality in Greenland was designated by Meißner et al. (2011), with a detailed redescription based on traditional characteristics and additional diagnostic characteristics that had been rarely or only briefly described in previous publications. They examined traditional morphological characteristics such as the shape of the anterior margin of the prostomium, length of the first branchiae, and the beginning, shape, and number of hooded hooks. They also examined additional diagnostic characteristics including the pigmentation of the body, shape of nuchal organs, extension of dorsal ciliated organs, the number and arrangement of white dots (pores of ventral epidermal glands), shape of notopodial postchaetal lamellae in the posteriormost region of the body, and the number of posterior abranchiate chaetigers (see Meißner et al. 2011). Further, molecular information regarding the three gene regions, mitochondrial cytochrome c oxidase subunit 1 (COI), 16S ribosomal DNA (16S rDNA), and the nuclear 18S ribosomal DNA (18S rDNA), also has previously been provided . Thus, the re-examination of Spio specimens that have been identified as S. filicornis is now possible by additional morphological and molecular analyses, and the species can no longer be regarded as a species of worldwide distribution without scientific proof .
In this study, Spio specimens newly collected from the southern and western coasts of Korea were examined in detail to determine the species to which they belong. An illustrated description of the new species is provided together with the partial DNA sequences of three gene regions (COI, 16S rDNA, and 18S rDNA).

Sampling and morphological observations
Adult specimens examined in the present study were collected from the intertidal zones of the southern and western coasts of Korean waters (Fig. 1) using 500 μm-mesh sieves.
The observations were performed for both live and fixed specimens. The live specimens were relaxed in 10% MgCl 2 solution, and morphological characteristics were observed under a stereomicroscope (Leica MZ125; Germany). Photographs were taken using a digital camera (Tucsen Dhyana 400DC; Fuzhou Fujian, China) with a capture program (Tucsen Mosaic version 15; Fuzhou Fujian, China). After observation, the specimens were fixed in 4% formaldehyde for morphological analysis, washed, and subsequently transferred to 70% ethanol. For the molecular studies, the specimens were fixed with 95% ethanol. Some formalin-fixed specimens (briefly transferred to distilled water) were stained with methylene green solution to observe the pores of ventral epidermal glands, according to the method of Meißner (2005). The specimens for scanning electron microscopic examination were dehydrated using a t-BuOH freeze dryer (VFD-21S Vacuum Device; Ibaraki, Japan). The specimens were mounted on stubs and coated with gold-palladium and observations were performed using a scanning electron microscope (SU3500; Hitachi, Tokyo, Japan). Type and voucher specimens examined in this study were deposited at the National Institute of Biological Resources, South Korea (NIBR), the Senckenberg Research Institute in Frankfurt, Germany (SMF), and the Zoological Museum Hamburg, Germany (ZMH).

Molecular analysis
Genomic DNA was extracted from the tissues of the palps of five specimens (NI-BRIV0000829700-4) using a LaboPass Tissue Mini (Cosmo GENETECH, Seoul, South Korea) according to the manufacturer's protocol. Polymerase chain reaction amplification of the partial DNA sequences of three gene regions (COI, 16S rDNA, and 18S rDNA) was performed using the following primer sets: LCO1490 and HCO709 for COI (Blank et al. 2007), 16Sar and 16Sbr for 16S rDNA (Kessing et al. 1989), and 18E and 18B for 18S rDNA (Mincks et al. 2009). Molecular analyses were performed using the partial sequences aligned using Geneious 8.1.9 (Biomatters, Auckland, New Zealand). The maximum-likelihood tree was constructed based on the concatenated partial sequences of the COI, 16S rRNA, and 18S rRNA gene regions using IQ-TREE with the GTR+F+R3 model with 1000 replicates (Kalyaanamoorthy et al. 2017;Hoang et al. 2018). The obtained DNA sequences were registered in GenBank.
Diagnosis. Prostomium broadly rounded, slightly expanded at anterolateral margin, extending to chaetiger 1; nuchal organs with short median and long lateral ciliary bands, lateral bands extending up to transverse ciliated band (tcb) of chaetiger 3. Metameric dorsal ciliated organs double-paired, present from chaetiger 3. Branchiae from chaetiger 1 to almost end of body, length of first pair slightly shorter than that of second pair; branchiae mostly free from notopodial lamellae. White dots present from about chaetiger 3 to the end of the middle body region; three pairs of white dots per chaetiger. Neuropodial hooded hooks tridentate, present from chaetiger 11, uppermost tooth very inconspicuous. Pygidium with thin dorsolateral pair and stout but slightly longer ventral pair of anal cirri.
Pygidium with two pairs of anal cirri; dorsolateral pair shorter and thinner, comparatively widely spaced, and ventral pair longer, very stout, conical with rounded tip and closely spaced (Fig. 3C).
Pigmentation. Highly variable but conspicuous in live or well-preserved specimens (some specimens without pigmentation). Palps in live specimens with vari- able pigmentation, about 6-15 light to dark brown spots or black ringed appearance (Fig. 5A, B); pigmentation fades in formalin-and ethanol-fixed specimens, but light brown pigmentation along the food groove remains. Well-preserved specimens with orange-brown and black pigmentation as follows: medial part of prostomium with orange-brown pigmentation, often faded in formalin-and ethanol-fixed specimens; prostomium with black pigmentation on the anterior to transverse depression margin of the prostomium, dorsal side of the peristomium next to the prostomium; black pigmented patches in front of, and in particular, behind tcb dorsolaterally in about the first six chaetigers in holotype (Fig. 6A), and some specimens with distinct patches (Fig. 5B). If black pigmented patches are distinct on the ventral side, white dots (pores of ventral glands) are clearly visible (Figs 5D, 6B). Methyl green staining pattern (MGSP).The anterior part of the prostomium and peristomium, margins of branchiae and postchaetal lamellae, and anal cirri were intensively stained. Transfer of stained specimens to distilled water for approximately 10 min, resulted in white dots being visible against the bluish background on the ventral side (Fig. 6C). Three pairs of dots were visible on chaetiger 3 to end of middle body region; lateral two pairs closely spaced, easily confused as one pair.
Biology. In the present study, the specimens were found mostly in intertidal zones of fine sand, rarely muddy sand, and sometimes a mixture of gravel and macrophytes (Zostera marina). According to Abe and Sato-Okoshi (2021), planktonic larvae of Spio sp. 2 with two rows of black melanophore spots on each side of the dorsum from chaetiger 1 onward, linked by band-shaped medial black pigmentation from chaetiger 4 or 5 are found in Sasuhama and Onagawa Bay between April and August (see cited publication for further details). Adult specimens were collected from muddy sand sediments of shallow waters in Sasuhama, Japan (Abe and Sato-Okoshi 2021).
Etymology. The specific name, pigmentata, originates from the Latin word pigmentum, meaning "pigment" This name refers to the new species having conspicuous black pigmentation on the body.
Distribution. Along the southern and western coasts of Korea; Sasuhama and Onagawa Bay, north-eastern Japan.
The new species was present in a clade with S. blakei and S. symphyta.

Discussion
Morphological examination of Spio specimens from the southern and western coasts of Korea, combined with the molecular analysis of three gene regions from newly collected materials, revealed the presence of a previously undescribed species of Spio, S. pigmentata sp. nov. The new species agrees well with Paik's (1975Paik's ( , 1982Paik's ( , 1989 description of S. filicornis with respect to most diagnostic features and only differs in the dentation of hooded hooks (see above). The morphological examination in this study showed that the uppermost tooth in the tridentate hooks is very inconspicuous (e.g., new species in the present study and S. symphyta in Meißner et al. 2011). This can easily lead to erroneous conclusions. We suggest that the undescribed species newly collected during this study and the species previously known as S. filicornis by Paik's (1975Paik's ( , 1982Paik's ( , 1989 from Korean waters are the same. The new species is morphologically very similar to S. blakei Maciolek, 1990 from Australia in having the following characteristics: the length of first branchiae, the shape of nuchal organs, extension of dorsal ciliated organs, shape of hooded hooks, the number of abranchiate chaetigers, and the shape of the anal cirri (Meißner and Gotting 2015). However, the new species can be distinguished from S. blakei by the presence of three pairs of white dots per chaetiger instead of two pairs and having 7-9 tridentate hooded hooks instead of 4-5 (Meißner and Götting 2015). In the Far East of the temperate region, the new species and S. picta from the Kuril Islands share tridentate hooded hooks. The new species, however, differs from S. picta by the presence of orange-brown and black pigmentation instead of only light to dark brown, the shape of its nuchal organs (U-shaped vs straight), the number of hooded hooks (7-9 vs 8-13), and the fusion of notopodial postchaetal lamellae (mostly separated vs completely fused in the anterior and middle regions) .
According to the results of the molecular studies, the species was already recorded in Japan and published as unidentified Spio sp. 2 by Abe and Sato-Okoshi (2021). The known distribution of S. pigmentata sp. nov. ranges from the Korea Strait and the Yellow Sea of Korea to northeastern Japan.
The phylogenetic tree resulting from the analysis of molecular data revealed that S. pigmentata sp. nov. formed a clade with S. blakei and S. symphyta (Fig. 8). The morphological characteristics also imply a close relationship of these species. These three Spio species share U-shaped nuchal organs, and the two species that form a second clade (S. arndti and S. filicornis) share almost straight nuchal organs. Despite the high species diversity of the genus, the available DNA data are very poor. Further studies based on detailed morphological and molecular information of Spio species are needed to reveal additional information on their genetic relationships.
The identity of Spio species described in Northeast Asia should be verified based on both morphological and genetic studies (see Meißner et al. 2011). For example, the description of the specimens from China identified as S. martinensis showed morphological differences in the shape of the apical tooth of neuropodial hooded hooks and the number of posterior abranchiate chaetigers with the specimen from the type locality in France (Wu et al. 1965;Lavesque et al. 2015), and hence might be doubtful. Unfortunately, the DNA information of specimens from China and France is still unknown. Further studies are needed to resolve these and related problems.