Neoamphitrite undevigintipes, a new terebellid species from South Korea (Annelida, Terebellida, Terebellidae)

Abstract A detailed description and illustrations of a new terebellid species are provided, and molecular information based on partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene are included. The new species, Neoamphitrite undevigintipessp. nov., is described from the deep sea off the eastern coast of South Korea. It is similar to Neoamphitrite groenlandica (Malmgren, 1866) in that the thorax has 19 notopodial chaetigers. However, Neoamphitrite undevigintipessp. nov. is clearly distinguishable from N. groenlandica in having the uncini of the first abdominal chaetiger arranged in a single row and in having 12 ventral shields. A taxonomic key to all known Neoamphitrite species is also included.


Introduction
The genus Neoamphitrite Hessle, 1917 is a terebellid polychaete assigned to the subfamily Terebellinae Johnston, 1846 (Fauchald 1977;Londoño-Mesa 2009). Members of this genus usually have distinct lateral lobes on anterior segments, three pairs of dichotomous branchiae, distally hirsute notochaetae, and the uncini beginning at segment 5 (Fauchald 1977;Reuscher et al. 2012). This genus has been confused with Amphitrite Müller, 1771 due to controversial morphological differences between the two genera (Hessel 1917;Fauvel 1927;Hutchings and Glasby 1988;Reuscher et al. 2012). Hessle (1917) considered that Neoamphitrite is distinguishable from Amphitrite by the branchial stem and nephridial papillae. Neoamphitrite has dichotomous branchiae with a well-developed stem and nephridial papillae with a free tube distinctly projecting from the body, while Amphitrite has filiform branchiae with a reduced stem and nephridial papillae with a fused tube retracted into the body. However, Fauvel (1927) and Hutchings and Glasby (1988) regarded that these differences were vague and not particularly useful in distinguishing Neoamphitrite from Amphitrite, and they considered Neoamphitrite to be a junior synonym of Amphitrite. Hessle's (1917) classification was re-accepted and currently the genus Neoamphitrite is recognized in recent taxonomic works (Hilbig 2000;Londoño-Mesa and Carrera-Parra 2005;Reuscher et al. 2012).
Here, the definition of Neoamphitrite, as described by Hessel (1917), is followed.
To date, 12 species of Neoamphitrite are known (Hessel 1917;Fauvel 1927;Caullery 1944;Hutchings and Glasby 1988;Hilbig 2000;Londoño-Mesa and Carrera-Parra 2005;Reuscher et al. 2012). Among the described species, N. edwardsi (Quatrefages, 1865), N. ramosissima (Marenzeller, 1884), andN. vigintipes (Grube, 1870), have been recorded from East Asia (Hessel 1917;Imajima and Hartman 1964;Paik 1989). While studying polychaetes from Korean waters, a new species belonging to the genus Neoamphitrite was found in the deep sea off the eastern coast of South Korea. In this study, a detailed description and illustrations of the new species are provided, and molecular information pertaining to the barcoded regions of mitochondrial cytochrome c oxidase subunit I (COI) gene are included. This study also includes a taxonomic key to all Neoamphitrite species and is based on the literature (Hessel 1917;Fauvel 1927;Caullery 1944;Hutchings and Glasby 1988;Hilbig 2000;Londoño-Mesa and Carrera-Parra 2005;Reuscher et al. 2012).

Sampling and morphological observation
Samples were collected from the benthos of the deep sea (500-1000 m depth). Specimens were sorted using sieves with a mesh size of 0.5 mm, initially fixed with 5% formaldehydeseawater solution, and transferred to 85% ethyl alcohol. Characteristics of the whole body were observed with appendages dissected in a petri dish using dissection forceps or surgical knives and needles under a stereomicroscope (SMZ1500; Olympus, Tokyo, Japan). Dissected specimens were mounted onto temporary slides using glycerol or permanent slides using polyvinyl lactophenol solution. Drawings were made under the stereomicroscope and light microscope (LABOPHOT-2; Nikon, Tokyo, Japan) with the aid of drawing tubes. Photographs were taken of appendages mounted on a permanent slide. Images of appendages were captured using an imaging system (LAS V4.7, Leica Microsystems, Heerbrugg, Switzerland). Specimens for scanning electron microscopy (SEM) were dehydrated using a t-BuOH freeze dryer (VFD-21S; Vacuum Device, Ibaraki, Japan). They were mounted onto stubs and coated with gold-palladium. SEM observations were conducted using a scanning electron microscope (SU3500; Hitachi, Tokyo, Japan). Type material and additional material examined were deposited at the National Institute of Biological Resources (NIBR) in Incheon, Korea and the National Marine Biodiversity Institute of Korea (MABIK) in Seocheon, Chungcheongnam-do, Korea, respectively.

Molecular analysis
Genomic DNA was extracted from posterior segments of three specimens selected among additional materials using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. Amplifications of partial sequences of mitochondrial cytochrome c oxidase subunit I (COI) from gDNA were conducted by polymerase chain reaction (PCR) method using a set of primers: LCO 1490 5'-GGT-CAACAAATCATAAAGATATTGG-3' and HCO 2198 5'-TAAACTTCAGGGT-GACCAAAAAATCA-3' in COI amplification (Folmer et al. 1994). PCR amplification was conducted in a total volume of 20 µL: 10 µL of 2× DyeMIX-Tenuto (Enzynomics), 0.5 µL of each primer, 1 µL of gDNA, and 8 µL of sterile water. Touchdown-PCR was conducted according to the following cycling program: 94 °C for 5 min, 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, followed by 20 cycles at decreasing annealing temperatures in decrements of 0.5 °C per cycle, followed by 1 min. at 94 °C, 15 cycles of 1 min. at 40 °C, 1 min. at 72 °C, and final extension at 72 °C for 7 min. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA, USA). Sequences for the new species were obtained with an Applied Biosystems 3730 DNA sequencer, and deposited in the GenBank under accession numbers MN306311 to MN306313. Sequences were aligned with those of other terebellid species and outgroup taxa using Geneious Pro v.9.1.8 (Biomatters, Auckland, New Zealand). Genetic distances between the new species and other species and the phylogenetic tree were produced by MEGA v.6.06 (Tamura et al. 2013).
Description. Holotype: complete, 11.0 cm long, 1.5 cm wide at segments 10, and with approximately 74 segments. Body uniformly light beige in alcohol, without pigmentation pattern, and consist of thorax with 19 chaetigers and abdomen; anterior thoracic segments compact until about 13 and then segments slightly narrower and longer than 13 anterior segments (Figs 1, 3A, B, E). Tentacular lobe short and collarlike. Peristomium with fleshy ridge on ventral side, separated anteriorly from lower lip by groove. Upper lip distinct and undulate with free margin. Lower lip well-developed, projecting forward. Buccal tentacles filiform with ventral groove. Lateral lappets paired on segments 2-4, distinct thickness flaps, protruding forwards, and with weakly developed glandular margin; first and second lappets well-developed, but third lappets reduced in length, located on nearby base of notopodia. Branchiae paired on segments 2-4, dichotomous, with 3 tiers of branches and weakly annulated stalk distinct. Nephridial papillae small, oval, present on segments 3-15, and located between noto-and neuropodia; those with fused tube retracted into body on segments 6-8 and other with free tube distinctly projecting from the body. Ventral shields trapezoidal, broader than longer, present on segments 3-14; first shield on segment 3 with glandular margin and others with smooth margin; thereafter shields replaced by mid-ventral groove extending to pygidium (Figs 2A, 3A, B, E). Notopodia short, rectangular, present on seg-ments 4-22 (chaetigers 1-19); last 2 or 3 pairs becoming much shorter. Notochaetae slightly curved, medially winged and distally serrated, types of 2 lengths; chaetae on anterior row at least half as long as those on posterior row (Figs 2C, 3E-H). Neuropodia beginning from segment 5 as low rectangular ridges, and with uncini arranged in single rows on segments 5-10 (chaetigers 2-7), uncini in double rows beak to beak arrangement on segments 11-22 (chaetigers 8-19), and in single row on all abdominal segments. Uncini avicular, short-handled with short triangular heel, distally pointed prow, minute dorsal button, and 5 rows of secondary teeth on main fang with subrostral guard. Dental formula MF: 4-5: 5-6: 7-8 (Figs 2B, 3C, D). Pygidium reduced with encircling 10 papillae.
Etymology. A combination of the Latin undeviginti and pes. This name means 'nineteen feet', referring to the 19 pairs of notopodia on the thoracic segments.
Habitat. This species is found on the soft bottom of deep waters (500-1000 m depth) and lives in a mud tube.
Remarks. In Neoamphitrite taxonomy, the number of notopodia is a key character for the identification of species (Hutchings and Glasby 1988;Hilbig 2000;Londoño-Mesa and Carrera-Parra 2005;Reuscher et al. 2012). Neoamphitrite undevigintipes sp. nov. has 19 pairs of notopodia on the thoracic segments regardless of body size and number of segments. In this respect, the new species is most similar to Neoamphitrite groenlandica (Malmgren, 1866), which was originally described from the Atlantic Ocean and also has 19 pairs of notopodia (Malmgren 1866;Hessle 1917;Fauvel 1927). However, the new species is clearly differentiated from N. groenlandica by two characteristic features. The uncini in the first abdominal chaetiger are arranged in a single row in N. undevigintipes sp. nov., but in double rows in N. groenlandica, and the new species has 12 ventral shields, but N. groenlandica has 14 (Malmgren 1866 ;Hessle 1917;Fauvel 1927).
In East Asia, three Neoamphitrite species, N. edwardsi (Quatrefages, 1865), N. ramosissima (Marenzeller, 1884), and N. vigintipes (Grube, 1870) from Japan, have been recorded (Hessle 1917;Imajima and Hartman 1964;Paik 1989). Neoamphitrite undevigintipes sp. nov. shows several differences from these species as follows: notopodia of N. undevigintipes sp. nov. are present on 19 chaetigers, compared to 17 in N. ramosissima and N. edwardsi. Neoamphitrite undevigintipes sp. nov. has uncini arranged in a single row in all abdominal chaetigers, while N. vigintipes has the uncini arranged in double rows in abdominal chaetigers except for some final chaetigers. The new species has 13 pairs of nephridial papillae, whereas six, nine, and 12 pairs are present in N. ramosissima, N. edwardsi, and N. vigintipes, respectively (Hessle 1917;Imajima and Hartman 1964;Paik 1989). Hessle (1917) suggested that Neoamphitrite species are distinguished from Amphitrite species by having nephridial papillae with free tubes distinctly projecting from the body rather than fused tubes retracted into the body. However, in several specimens of N. undevigintipes sp. nov., the nephridial papillae have fused tubes in two or three of all nephridial papillae pairs. Hutchings and Glasby (1988) mentioned that the form of nephridial papillae is difficult to use as a generic diagnostic feature because it can be variable according to the state of specimens. This character was overlooked in diagnoses of the terebellid genera (Fauvel 1927;Caullery 1944;Fauchald 1977;Hilbig 2000;Hutchings Reuscher et al. 2012). In this respect, we think that the form of nephridial papillae is not yet a useful diagnostic character and that its taxonomic value should be re-examined in detail using as many species as possible. We provide a key to the species presently regarded as members of Neoamphitrite.
Genetic information. In this study, partial COI sequences, each measuring 658 bp, were obtained from three specimens for genetic analysis of Neoamphitrite undevigintipes sp. nov. They are deposited in the GenBank under accession numbers MN306311 to MN306313. All COI sequences obtained were identical. Using data available from the GenBank (Carr et al. 2011;Siddall et al. 2011;Telfer and Dewaard 2017), we genetically compared the new species with two Neoamphitrite species, N. figulus (Dalyell, 1853) and N. robusta (Johnson, 1901), as well as six species belonging to the other terebelline genera: Amphitrite cirrata Müller, 1776, Nicolea zostericola Örsted, 1844, Pista maculata (Dalyell, 1853), Pista wui Safronova, 1988, Loimia arborea Moore, 1903, and Loimia medusa (Savigny, 1822. Thelepus cincinnatus (Fabricius, 1780) was used as the outgroup. GenBank accession numbers are represented in Table  1. Inter-specific genetic distances between the new species and two Neoamphitrite species, as measured by Kimura-2-parameter model, were distinct and ranged from 9.2 to 13.7%. The genetic distances between the new species and the six species in other genera ranged from 21.8 to 29.9%. In the maximum likelihood (ML) tree based on these genetic data (Fig. 4), all terebellid species showed specific validity. The new species was contained in a clade with N. figulus and N. robusta. At the generic level, the Neoamphitrite clade, including the new species, was closely related to A. cirrata, agreeing with the taxonomic view that Neoamphitrite and Amphitrite share many morphological features except for differences in the morphology of branchiae (Hessel 1917;Fauvel 1927;Hutchings and Glasby 1988;Reuscher et al. 2012). However, Neoamphitrite was monophyletic and clearly distinguishable from A. cirrata in the ML tree, supporting the known morphological differences between two genera (Hessel 1917;Fauvel 1927;Hutchings and Glasby 1988;Reuscher et al. 2012). Despite our results, further genetic studies with additional data and including more species of Neoamphitrite and Amphitrite are needed to confirm the phylogenetic relationship between the two genera.