A new Notomastus (Annelida, Capitellidae) species from Korean waters, with genetic comparison based on three gene markers

Abstract Notomastus koreanus sp. n., collected from the sublittoral muddy bottom of Korean waters, is described as a new species. The Korean new species closely resembles N. torquatus Hutchings & Rainer, 1979 in the chaetal arrangement and the details of abdominal segments, but differs in the position of genital pores and the absence of eyes. DNA sequences (mtCOI, 16S rRNA, and histone H3) of the new species were compared with all the available sequences of Notomastus species in the GenBank database. Three genes showed significant genetic differences between the new species and its congeners (COI: 51.2%, 16S: 38.1–47.3%, H3: 3.7–9.3%). This study also includes a comprehensive comparison of the new Korean Notomastus species with its most closely similar species, based on the morphological and genetic results.


Introduction
Capitellid polychaetes build spiral burrows or U-shape tubes in bottom sediments, which increase the subsurface penetration of water and oxygen, thus improving the recruitment and growth of small benthic organisms (Fauchald andJumars 1979, Scaps 2002). In particular, the genus Notomastus Sars, 1851 is one of the most common and species-rich genus in the Capitellidae Grube, 1862 and occurs from the intertidal to the deep sea in a variety of sediment types including fine, medium, and silty sand and mud (Dean 2001). It currently contains 43 valid species, which is the highest number of species among the capitellid genera (Gil and Bellan 2017). Despite their ecological success and high species diversity, the lack of good generic characters and the incorrect descriptions in several previous records have led to taxonomic confusion in the genus (Green 2002). For instance, the hooded hook dentition of N. latericeus Sars, 1851 has been described differently in the published records of the species, and the protruded lateral organs had been mistaken as the branchiae in the former records of Notomastus species from Japan and Vietnam (Day 1967, Fauvel 1927, Green 2002, Thomassin 1970. The taxonomic boundary of the genus Notomastus has been continually modified over the last century. The genus was designated by Sars (1851) with the description of the type species, N. latericeus. Eisig (1887) divided Notomastus into two subgenera, Tremomastus and Clistomastus, by the presence/absence of genital pores in the abdomen and the development of hooded hooks. Fauvel (1927) suggested that the subgeneric name of Notomastus (Notomastus) should replace Notomastus (Tremomastus) and Hartman (1947) accepted this view. However, Day (1967) and Fauchald (1977) did not agree with these subgeneric categories in their diagnoses of the genus. Ewing (1982) placed three genera, Dodecaseta McCammon & Stull, 1978, Paraleiocapitella Thomassin, 1970, and Rashgua Wesenberg-Lund, 1949, within Notomastus. Green (2002 clarified that Dodecaseta and Rashgua differed from Notomastus in the chaetal distribution, which was regarded as a good generic character. Green (2002) also suggested the need for a review of the taxonomic boundary of Notomastus and its species. In this study, Notomastus is defined based on the characteristics of its 12 thoracic segments, which comprise an achaetigerous peristomium and 11 chaetigers, including a uniramous or biramous first chaetiger, subsequent chaetigers usually with only capillaries, and posterior thoracic chaetigers with capillaries and sometimes neuropodial hooks; abdominal segments have only hooks. Although this study provides detailed descriptions of the Notomastus species from Korean waters, the comparison with closely related species was limited due to the insufficient morphological information of many records. To overcome this difficulty, studies using a combination of morphological analysis and DNA barcoding have been conducted to distinguish closely related capitellid species and to improve species recognition between them (Jeong et al. 2017b, Silva et al. 2016. The aim of the present study is to clarify the taxonomic status of the undescribed Notomastus species of Korea by morphological and genetic analysis using three different partial genes (mtCOI, 16S rRNA, and H3) and to compare Korean species with their closest congeners.

Materials and methods
Morphological analysis. Samples were collected from seven stations in sublittoral areas of Korea using a 0.05 m 2 Van Veen grab (Fig. 1). The sediment samples were elutriated over a 0.5 mm sieve in a 30 l seawater container, and the organisms were transferred to a 1 l collecting jar with 7% MgCl 2 solution for anesthesia. The relaxed samples were fixed in a buffered solution of 10% formalin within 2 hours and finally preserved in 90% ethanol. In the laboratory, Notomastus specimens were sorted under a stereomicroscope (SMZ745T, Nikon). Line drawings were generated using a differential interference contrast microscope (Eclipse Ci-L, Nikon) and a digital pen display (Cintiq 22HD, Wacom). Methyl green staining patterns (MGSP) and scanning electron microscopy (SEM) analyses were described and photographed, as delineated by Jeong et al. (2017b). The examined type materials were deposited in the collections of the Marine Biodiversity Institute of Korea (MABIK) in Seocheon, Korea (Table 1). Two additional specimens (voucher numbers: NIBRIV0000634919 and NIBRIV0000634920) were deposited at the National Institute of Biological Resources (NIBR) in Incheon, Korea.
Molecular analysis. Genomic DNA was extracted from tissue obtained from partial dissection of the middle part of the abdomen of the ethanol-preserved specimens. To extract the genomic DNA, 1.5 mL centrifuge tubes each containing 90 μL of 10% Chelex suspension (Bio-Rad Laboratories Inc.), 10 μL of Proteinase K (10 mg/ml, iNtRON Biotechnology, Inc.) and dissected tissues (ca. 1/2 segment) were incubated at 56 °C for 3-12 hours.
The forward and reverse sequences were compared and edited using Chromas software version 2.3 (Technelysium Pty. Ltd.). The partial sequences of the COI, 16S rRNA and H3 genes were aligned with the sequences of available Notomastus species obtained from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) using the Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 (Kumar et al. 2016). Table 1 summarizes information for all sequences used in the analyses. The aligned sequences were used as data sets to generate the genetic distance using Kimura's two-

BIOUG03550-A09
BOLD Systems (2017) parameter (K2P) model (Kimura 1980). Based on the K2P distances, we calculated the intraspecific genetic differences within the Korean specimens and the interspecific genetic differences among the closest taxa.

Systematics Family Capitellidae Grube, 1862
Genus Notomastus Sars, 1851 Type species. Notomastus latericeus Sars, 1851 Type locality. Komagfjord, Norway Generic diagnosis (modified after Green 2002). Thorax with 12 segments including an achaetous peristomium and 11 chaetigers with capillary chaetae. Last three thoracic chaetigers may have capillary chaetae in both rami or may be transitional with capillary chaetae in notopodia and hooded hooks in neuropodia. Remaining chaetigers with hooded hooks only. Hooded hooks with one or more rows of teeth above main fang; more than two teeth in basal row. Branchiae may be present or absent.
Transition between thorax and abdomen distinguished by changes in shape of chaeta and segment (Figs 2A-B, 3A-B); last thoracic chaetiger bi-annulated, with capillaries only, slightly thinner than first abdominal chaetiger; anterior abdominal segments multi-annulated, with better developed neuropodial lobes than thoracic ones, having hooded hooks only (Figs 2A-B, 3B). Parapodia in anterior to mid abdominal region not protruded, well separated (Figs 2A-B, 3A-B). Notopodial lobes not protruded in anterior abdomen, middorsal on anterior few segments, becoming dorsolateral in following abdominal region, with 6-15 hooded hooks only per fascicle, having posteriorly extended and semicircular lamella from chaetiger 160 to end of body ( Hooded hooks with main fang extending slightly beyond hoods; hood slightly flared. Main fang of hooded hooks with 3 rows of small teeth; 5 in basal row, 6-8 in second row, and at least 6 in superior row (Fig. 3F-G).
Etymology. The new species is named for its wide distribution in coastal waters of Korea. Distribution. The subtidal areas (10-20 m) near Korea (Fig. 1). The subtidal habitat (ca. 20 m) of Bohai Sea, China (see details in Discussion).
Ecology. Notomastus koreanus sp. n. was sampled from soft sediments throughout the year. Most well-developed individuals (having over 250 segments) were obtained between October and January. The sediment of the collecting stations was mainly composed of sandy mud with shell fragments. Leiochrides yokjidoensis Jeong, Wi & Suh, 2017 and an undescribed Heteromastus Eisig, 1887 species co-occurred in southern stations of this study. micrographs (using additional specimens from type locality) D anterior 6 thoracic segments in left lateral view E capillary chaetae of chaetiger 4 F-G abdominal hooded hooks in frontal view. Abbreviations: cc, capillary chaetae; Ch, chaetiger; genP, genital pore; hh, hooded hooks; lo, lateral organ; mf, main fang; neu, neuropodium; no, notopodium; per, peristomium; pro, prostomium; prob, proboscis; transition, transition between thorax and abdomen.
Remarks. Notomastus koreanus sp. n. is distinguished from other species of the genus by the morphological combination of absence of distinct eyes and first neuropodia, last thoracic chaetigers with only capillary chaetae, presence of genital pores between chaetigers 7-11, non-protruded lateral organs and neuropodial lobes in anterior abdomen, and posteriorly extended parapodial lobes in posterior abdomen. The new Korean Notomastus species closely resembles N. torquatus Hutchings & Rainer, 1979 in the chaetal arrangement, the absence of developed neuropodial lobes in anterior abdomen, and the presence of posteriorly extended parapodial lobes in the posterior abdomen (Table 2). However, they differ in the presence of eyes on posterior prostomium (eyespots vs. absence) and the location of genital pores (between chaetigers 3 or 5-10 vs. 7-11, Table 2). Additionally, N. torquatus is regarded as an endemic species of Australia and has a much wider thorax (4 mm vs. 1.3 mm) than comparable specimens of N. koreanus sp. n., which have 280 segments when fully developed (Doyle 1991, Hutchings andRainer 1979). Notomastus hemipodus Hartman, 1945 andN. tenuis Moore, 1909 are also similar to N. koreanus sp. n. in the chaetal arrangement and the absence of protruded neuropodial lobes in anterior abdomen, but clearly differ in the details of the eyes, the genital/lateral organs, and the MGSP (Table 2). Moreover, they have the unique features of the indistinct palpode and the bilobed notopodial lobes, respectively.
Genetic comparison with the published sequences of Notomastus species. To confirm the genetic distances among the new species and its closely related species, we used the partial sequences of mitochondrial (mtCOI and 16S rRNA) and nuclear (histone H3) genes. In all genetic comparisons, the intraspecific differences among the Korean specimens were negligible (0-0.1%, Table 3). The mean interspecific differences for mitochondrial COI (50.9%) and 16S rRNA (43.2%) genes were much higher than the mean interspecific difference for the nuclear histone H3 gene (7.6%). In the mtCOI gene comparison, the mean genetic difference between N. koreanus sp. n. and N. profondus (Eisig, 1887) of Portugal (KR916899) was substantial (51.2%, Table 3). In the interspecific comparison for the 16S rRNA gene, N. koreanus sp. n. was well distinguished from N. hemipodus (38.1%, HM746714) of Canada and N. latericeus (47.3%, AY340469) of Sweden (Table 3). In the histone H3 gene comparison, N. koreanus sp. n. genetically differed from N. torquatus (3.7%, AF185258) of Australia, N. latericeus (7.0%, DQ779747) of Sweden, and N. hemipodus (9.3%, HM746759) of Canada (Table 3). Previously known genetic difference of the mtCOI and the 16S rRNA genes among the capitellid species is generally about 18-20% (Jeong et al. 2017b, Silva et al. 2016. In contrast, the histone H3 gene difference between cryptic nereidid polychaetes is around 2-9% (Glasby et al. 2013). Thus, the genetic differences between N. koreanus sp. n. and its closely related species (COI: 51.2%, 16S: 38.1-47.3%, H3: 3.7-9.3%) is significant at the species level revealing the speciation among them. On the other hand, the mtCOI gene sequence of the Chinese specimen (BIOUG03550-A09, Table 1) is genetically matched with N. koreanus sp. n. (0.007 in K2P distance, Table 3), although it has been reported as N. latericeus on BOLD (www.barcodinglife.org) database (BOLD Systems 2017). Notomastus latericeus was originally described from Norwegian waters, and it is easily discriminated from our new species in terms of morphology ( Table 2). The published histone H3 and 16S rRNA sequences of N. latericeus from Swedish waters are clearly distinguished from the sequences of N. koreanus sp. n. by the significant genetic difference (Table 3). Thus, the mtCOI sequence of the Chinese specimen on the BOLD database is regarded as a misidentification at the species level and confirms the additional occurrence of our new species in the Bohai Sea of northeastern China.