A new species of Notomastus (Annelida, Capitellidae) from southern China, with remarks on its morphology and distribution

Abstract The genus Notomastus is frequently encountered in Chinese waters. However, its species richness is poorly understood. In this study, a Notomastus species obtained from Xiamen Bay, southern China, was described and illustrated as a new species (N. sunaesp. nov.), based on morphological and molecular analyses. The new species is characterized by having uniramous chaetiger 1, the presence of palpode and eyespots on prostomium, chaetiger 11 with notopodial capillaries and neuropodial hooded hooks, and notopodial lobes with simple epithelial extensions on far posterior abdomen. With additional specimens collected from several localities along the southern coasts of China, the morphology and geographical distribution of the new species are discussed. A key is also provided for Notomastus species with neuropodial hooks in thoracic chaetiger 11.

prostomium and epithelial texture. The new species is distinguished from other closely related species by morphological characters and gene sequences. With specimens collected from the identical site in different months and from other localities along the southern coasts of China, the morphology and geographical distribution of the new species are also discussed. A identification key is provided for worldwide Notomastus species having the last thoracic chaetiger transitional.

Field sampling
A collection of over 90 specimens from eight localities along southern China ( Fig. 1) was examined in this study. Sediment samples were collected from intertidal or shallow subtidal coastal waters during surveys conducted from 2016 to 2019 using either a grab sampler (subtidal stations) or a sampling frame (intertidal). Sediment samples were washed through a 0.5 mm sieve in the field. Specimens retained were fixed with either 8% diluted formalin in seawater, and later transferred to 70% ethanol, or directly preserved in 95% ethanol.

Morphological analysis
Specimens were examined using a Leica MZ95 optical stereoscope. Light photographs were taken under a Leica M205A stereoscope equipped with DFC 550 digital camera. The structure of abdominal hooks was observed under Axio Imager Z2 (Carl Zeiss Inc., Oberkochen, Germany) using oil emersion. SEM observations were carried out on a scanning electron microscope (ZEISS SUPRA 55 SAPPHIRE) at Xiamen University, and methyl green staining pattern (MGSP) was used to identify the distribution of glandular areas, both as delineated by Lin et al. (2019). The type material and additional material examined in this study were deposited in the Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, China.

Molecular analysis
The total genomic DNA was extracted from organisms using Transgen Micro Genomic DNA EE 181 Kit (Transgen, Beijing, China) following the protocol provided by the manufacturer. The PCR reactions were conducted to amplify partial sequences of mitochondrial (COI) and nuclear (18S, H3) genes using primer sets as shown in Table 1. The PCRs (100 μL) contained 73.5 μL of deionized water, 10 μL of TakaRa 10× Ex Taq buffer, 8 μL of dNTP mixture (2.5 mM), 2 μL of each primer (10 μM), 0.5 μL of TakaRa Ex Taq (5 U/ μL) and 4 μL DNA template. The thermal cycling conditions were as follows: 95 °C for 240 s; 35 cycles of 95 °C for 45 s, 41 °C (COI) or 43 °C (18S1, 18S2, 18S3, H3) for 60 s, and 72 °C for 80 s; and 72 °C for 7 mins. 5 μL of the resulting PCR products were checked using 1% agarose gel electrophoresis, and the remaining products were purified using a Transgen Quick Gel Extraction EG 101 Kit (Transgen, Beijing, China) following the manufacturer's protocol. Sequencing of the purified DNA samples was performed at Biosune company (Xiamen, China) with an ABI 3730XL DNA analyzer (Applied Biosystems).
Obtained sequences were manually checked and assembled into a consensus sequence using the software DNAMAN 8 (Lynnon Biosoft, Quebec, Canada). Eventually, about 650 bp of COⅠ, 1637 bp of 18S, and 316 bp of H3 were successfully amplified in this study. The available sequences of related genera of Capitellidae in GenBank were used in phylogenetic analysis ( Table 2). Alignments of the sequences were performed using the MUSCLE algorithm (Edgar 2004) implemented in the software MEGA Ⅹ (Kumar et al. 2018) under default settings. The unaligned sequences and highly divergent regions were removed using Gblocks (Castresana 2000). A maximum likelihood (ML) analysis was conducted in RAxMLGUI 1.5 beta (Silvestro and Michalak 2012) on the concatenated sequence of 18S and H3 genes, using the model GTR+G+I and 1000 thorough bootstrap pseudoreplicates. The tree was edited using FigTree v. 1.4 (Rambaut 2012) and Adobe Photoshop CS5. The aligned and trimmed sequences were used as data sets to generate the interspecific genetic distance using the Kimura's two-parameter (K2P) model (Kimura 1980) implemented in MEGA X. with only capillaries in both rami or last thoracic chaetiger transitional with notopodial capillaries and neuropodial hooded hooks. Abdominal chaetigers with hooded hooks only. Branchiae present or absent. Genital pores present or absent. Lateral organs present on thorax and abdomen. Description. Holotype complete with over 100 chaetigers (Fig. 3A), measuring 33.74 mm long by 0.8 mm wide. Paratypes complete or incomplete, ranging from 6.81-43.02 mm long, 0.57-0.90 mm wide for 19-103 chaetigers. Color in alcohol tan (Fig. 3B). Thorax dorsally rounded, ventrally flattened, widest at chaetiger 3. Prostomium conical, with narrow palpode (Figs 2A-C, 3B, 4A, B). Everted proboscis globular, with numerous minute papillae ( Fig. 2A-C). Eyespots present on lateral sides of prostomium (Figs 2A, C,  3B). Peristomium achaetous, wider than long, as wide as first chaetiger, but longer ( Fig. 2A). Thorax slightly areolated in anterior 4-5 chaetigers, remaining chaetigers smooth.
Transition between thorax and abdomen marked by change in chaetal arrangement and methyl green staining pattern (Figs 2A, B, D, 3C, D, 4A). First abdominal segment as wide as last thoracic chaetiger, but slightly shorter ( Fig. 2A, B, 3C, D). Parapodial lobes reduced in anterior abdomen, well separated ( Fig. 2A, B). Notopodial lobes located dorsally (Fig. 2B), close together in anterior abdomen, becoming dorsolateral in posterior abdomen. Neuropodial lobes lateral, separated ventrally. Chaetal fascicles po-sitioned posterior to midsegment in anterior abdomen (Fig. 2B, D), and near posterior edge of segment toward the pygidium (Fig. 2E). In the far posterior, notopodial lobes with a simple epithelial extension (Figs 2E, 3E), broadly-based and rounded-tipped. In anterior abdomen, chaetal fascicles with approximately 10 hooks in notopodia and 16 hooks in neuropodia, decreasing to 6 hooks in notopodia and 10 hooks in neuropodia in posterior abdomen, and to 1-2 hooks in segments near pygidium. Notopodial and neuropodial abdominal hooded hooks of similar shape, with angled node, evident constriction, developed shoulder, posterior shaft longer than anterior one, attenuated to terminal end (Fig. 2F). Hood smooth, slightly longer than wide (Fig. 2F). Abdominal  hooded hooks (Fig. 4E-G) with multiple rows of teeth above main fang: 4-5 teeth in basal row, 6-8 teeth in second row, and at least 6 teeth in superior row.
No branchiae observed in abdomen. Regenerated pygidium simple, without anal cirri (Fig. 3E, F) Methyl green staining pattern (Figs 2B,D,3A,C,D). Thorax stained with blue with slightly different intensity whereas abdomen stained with very dark blue. From postchaetal area of chaetiger 12, abdominal segments dorsally stained with dark blue, extending ventrallaterally, interrupted by parapodial lobes and lateral organs. Toward posterior abdomen, blue stain on abdominal dorsum faded gradually. From chaetiger 13, abdominal segments with paired stripes of ventral stain with darker intensity, interrupted by intersegmental rings.
Distribution. The new species is widely distributed along the southern coasts of China, from Fujian Province westward to Guangxi Province, and southward to Hainan Province (Fig. 1).
Ecology. The examined specimens were collected from intertidal to shallow subtidal coastal waters (~23 m). Sediment was mainly characterized by mud or muddy sand. The new species is especially abundant in nearshore waters off eastern Xiamen Island, Fujian Province.
Etymology. The species is named after Professor Ruiping Sun, in recognition of her contribution to the study of polychaetes from China Seas.
Variation. Eyespots on prostomium were indistinct in several specimens due to preservation in alcohol. MGSP on chaetigers 11-12 may be different among individuals. Some specimens have darker stain on post-chaetal area of chaetiger 11.
Remarks. As the most species-rich genus of Capitellidae, Notomastus has more morphological variability, including variation in the structure of the last thoracic chaetigers. Although it is known that hooks may be replaced by capillaries in the middle-posterior thorax of capitellids during ontogeny (Blake 2000), such as the example of Heteromastus (Warren and Hutchings 1994), several authors have confirmed the presence of neuropodial hooks in posterior thorax of some Notomastus species even when in adulthood (Ewing 1982;Blake 2000;Green 2002, Magalhães andBlake 2017). For instance, among the 44 examined specimens of N. angelicae, Hernández-Alcántara and Solís-Weiss (1998) found that 43 specimens possessed only hooks in the neuropodium of chaetiger 11. Nevertheless, less efforts have been devoted to detecting whether this character change during the development of the specimens. In this study, Notomastus sunae sp. nov. specimens were collected from the identical site (sta. QPW1-4) in different months (January, April, July, September, and October). All the 61 specimens uniformly have the last thoracic chaetiger (chaetiger 11) transitional with notopodial capillaries and neuropodial hooded hooks, regardless of body size. Additional specimens from other localities also confirm the similar chaetal structure of chaetiger 11 to the type material. These indicate the stability of this character and that it could be used as an identification tool at the species level.
Notomastus sunae sp. nov. is readily distinguished from most congeners by the presence of neuropodial hooks in last thoracic chaetiger. Among the known Notomastus species with neuropodial hooks in chaetiger 11, N. sunae sp. nov. closely resembles N. mossambicus by the presence of uniramous chaetiger 1 and prostomial eyespots, but differs from the latter in that the new species has prostomial palpode and slightly areolated epithelium in anterior thorax, whereas N. mossambicus has prostomium without palpode and strongly areolated epithelium in anterior thorax as stated by Thomassin (1970) and Cinar (2005). The new species differs from the geographically close Korean species Notomastus koreanus described by Jeong et al. (2018) in that the new species bears eyespots on prostomium, reduced parapodial lobes in anterior abdomen, as well as neuropodial hooks in the last thoracic chaetiger. In terms of the MGSP, N. sunae sp. nov. has paired stripes of ventral stain, the feature shared by N. hemipodus and N. koreanus. However, N. sunae sp. nov. has very dark blue stain on abdominal dorsum and extending dorsolaterally, which is distinct from other Notomastus species.
Based on morphological description and illustration provided by Green (2002), a Notomastus species (labelled as N. near hemipodus) reported from Andaman Sea is closely similar to N. sunae sp. nov. in a number of characters: presence of palpode and eyespots on prostomium, uniramous chaetiger 1, slightly areolated epithelium on anterior 4-5 chaetigers, and the MGSP on abdomen which has very dark blue stain on dorsum and paired stripes of ventral stain. Green (2002) mentioned that some specimens had chaetiger 11 transitional with notopodial capillaries and neuropodial hooks, which also agreed with N. sunae sp. nov. As the specimens examined by Green (2002) only had anterior fragments (23-37 chaetigers) and lacked ultrastructure of hooded hooks and gene sequences, further comparison is hindered. According to the redescription of N. hemipodus by García-Garza et al. (2012), the specimens identified as N. near hemipodus could not belong to N. hemipodus in that: 1) they had slightly areolated epithelium on anterior thorax instead of strongly tessellated epithelium as in N. hemipodus; 2) they had reduced neuropodial lobes in the anterior abdomen instead of expanded neuropodial lobes as in N. hemipodus; 3) they had very dark blue stain on abdominal dorsum instead of moderate green stain as in N. hemipodus.
Notomastus sunae sp. nov. is commonly collected and abundant in Xiamen Bay, Fujian Province, widely distributed westward to Qinzhou Bay, Guangxi Province, and southward to western Hainan Island, based on the examined material obtained from several localities along southern China. Its specimens are found in great geographical ranges at latitude from 19.5N to 25.5N and at longitude from 108.8E to 119.5E. They prefer to inhabit soft sediments, like mud or muddy sand. So far, this species is found in shallow coastal waters less than 30 m deep.

Sequences analysis
No identical matches are found for mtCOI, 18S, or H3 of this new species when conducting a GenBank BLAST search. In this study, the maximum likelihood tree (Fig. 5) showed that the new species is sister to Barantolla lepte known from Australia with low support value (bootstrap value = 62). The situation might be due to the limited gene sequences included in the analysis, which need further verification. The K2P genetic distances between N. sunae sp. nov. and related Notomastus species ranged from 20.55% to 74.73% for mtCOI, from 4.107% to 4.109% for 18S rRNA, and from 3.29% to 9.87% for histone H3 (Table 3). For polychaete species, the K2P genetic distance was reported to be 12.3-23.7% among capitellid species (Jeong et al. 2017) and 19.4-26.5% among Timarete species (Magalhães et al. 2014) for mtCOI, and 2-9% among cryptic species of Nereis denhamensis (Glasby et al. 2013) for histone H3. Therefore, the genetic distance for mtCOI and histone H3 reported in this study, together with distinct morphological characters, indicates that N. sunae can be recognized as a new species. Key to Notomastus species with neuropodial hooks in thoracic chaetiger 11