A new eyeless species of Nicon (Annelida: Nereididae) from the deep Northwest Pacific Ocean

1. Introduction

Nereidids are among the most diverse annelid families, with more than 535 species classified into 43 genera (Bonyadi-Naeini et al., 2017). This family is one of the most diverse groups in the deep sea (Paterson et al., 2009). Comparison with shallow-water species, some morphological and genetic changes has been considered to be an evolutionary adaptation to extreme environment (Zhang et al., 2017). According to Gonzalez et al. (2018), the colonization of Aphroditiformia to deep sea and cave environments seems to be related with the loss of eyes. Many nereidid species belonging to subfamily Nereidinae from deep water are reported as being eyeless. Four Neanthes species (Neanthes abyssorum Hartman, 1967, N. kermadeca (Kirkegaard, 1995), N. typhla (Monro, 1930), N. shinkai Shimabukuro et al., 2017) are known as having no eyes (Shimabukuro et al., 2017). These species are found at depths of more than 2 000 m, with the exception of N. typhla. Neanthes abyssorum was described from Bransfield Strait, Antarctic Ocean; N. kermadeca was from Kermadec Trench, South Pacific Ocean; N. typhla was from off South Georgia Island; N. shinkai was from São Paulo Ridge, Southwest Atlantic Ocean. Nereis anoculopsis Fauchald, 1972 and N. anoculis Hartman, 1960 were from California deep water. They also lacked eyes (Fauchald, 1972; Hartman, 1960). Ceratocephale abyssorum (Hartman and Fauchald, 1971) and Nicon abyssalis at depth of more than 4 000 m are blind too. Nicon, one of the least species rich genera of Nereididae, was described by Kinberg (1866) and including nine species. Those species have a wide distribution from intertidal to deep sea. Subfamily Gymnonereidinae, including Ceratocephale Malmgren, 1867, Gymnonereis Horst, 1919, Micronereides Day, 1963 and Tambalagamia Pillai, 1961, erected by Banse (1977) and characterised by bifid ventral cirri and chaetae forming dense bundles in the anterior 10–15 chaetigers. Fitzhugh (1987) presented the first cladistic analysis of Nereididae and expanded Gymnonereidinae to include all taxa with fully biramous parapodia and with either a smooth or papillate proboscis. Therefore Nicon was included within Gymnonereidinae. Santos et al. (2005) analyzed phylogenetic relationship within Nereididae, and restricted Gymnonereidinae to Banse’s proposal.

In the present manuscript, a new species of eyeless Nicon (Nereididae) from the Northwest Pacific is described. The phylogenetic analysis was carried out to explore the relationship of Nicon inside Nereididae based on four molecular marker: mitochondrial 16S ribosomal RNA gene (16S rDNA) and cytochrome c oxidase subunit I (COI) gene; nuclear 18S ribosomal RNA gene (18S rDNA) and 28S ribosomal RNA gene (28S rDNA).

2. Materials and methods

The specimen was collected by Hailong III ROV (remotely-operated vehicle) during DY56 Cruise (the 56th China Ocean Scientific Research) in October 2019 and preserved in 95% ethanol solution then deposited in the Sample Repository of Second Institute of Oceanography (RSIO), Ministry of Natural Resources, Hangzhou, China. Specimen was examined under a stereomicroscope (Zeiss Discovery V20). To discern jaws and arrangement of the paragnaths, the withdrawn proboscis dissected. Details of the chaetae were observed using Scanning Electron Microscopy (SEM, HITACHI TM-1000).

The total genomic DNA was extracted from its muscle using DNeasy® Blood and Tissue Kit (QIAGEN, CA, USA) according to the manufacturer’s protocol. DNA concentration used a NanoDrop 2000 Spectrophotometer and a Qubit fluorometer. The qualified DNA was stored in sterilized Milli-Q® water at –80°C before sequencing. The genome sequence was obtained by high-throughput sequencing (PE150) on HiSeq^TM X-Ten platform (Illumina, CA, USA) at Zhejiang Tianke High Technology Development Co., Ltd. The de novo assembly was achieved by SPAdes genome assembler (Bankevich et al., 2012). The mitochondrial 16S rDNA and COI gene (cox1), and the nuclear 18S rDNA and 28S rDNA sequences were retrieved from the assembled contigs. The start and end position of the target sequences were checked by the reported universal PCR primer pairs (Norlinder et al., 2012). The 16S rDNA, COI gene, 18S rDNA and 28S rDNA sequences are deposited in GenBank with accession Nos MW525220, MW644966, MW525221 and MW525222, respectively.

The terminals consist of 28 nereidid species (Table 1). Vrijenhoekia balaenophila from family Hesionidae was chosen as the outgroup species. The four marker genes were aligned using the ClustalW algorithm with default settings (15/6.66 as gap/gap length penalties) in Geneious prime software (Biomatters Ltd., New Zealand). The alignments of 16S rDNA, COI gene, 18S rDNA and 28S rDNA sequences were performed separately, and then concatenated into one file after manually trimming unalignable regions. The phylogenetic tree was performed with Maximum-Likelihood (ML) analysis via RAxML GUI v.1.5 software (Stamatakis, 2014; Silvestro and Michalak, 2012) and IQ-TREE package (Nguyen et al., 2015, Chernomor et al., 2016). A partitioned ML analysis was performed in RAxML, with 1 000 bootstrap replicates and the GTRGAMMA model (Lanave et al., 1984; Yang, 1994). The auto-selected best substitution model (-m MFP option) was used in IQ-TREE, also with 1 000 bootstrap replicates. The tree file was visualized and edited in Figtree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree).

Table  1.  Information and GenBank accession No. of marker genes used in the phylogenetic analysis

Subfamily/family	Taxa	16S rDNA, length/bp	COI gene, length/bp	18S rDNA, length/bp	28S rDNA, length/bp
Nereidinae	Alitta succinea	MN823959, 352	MN823952, 1 515	AY210447, 1 891	AY210464, 3 504
	Ceratonereis longiceratophora	−	AY583701, 400	AB106251, 1 642	AF185189, 533
	Hediste japonica	LC323064, 416	LC323029, 570	−	LC380658, 804
	Hediste diadroma	KX499500, 1 196	KX499500, 1 535	LC323646, 1 698	LC380656, 804
	Nectoneanthes oxypoda	−	MN256616, 658	KX290701, 1 793	LC168841, 527
	Nereis heterocirrata	KC833492, 462	MN256591, 658	KC840697, 790	−
	Nereis pelagica	AY340470, 465	HQ024126, 660	AY340438, 1 795	−
	Nereis vexillosa	GU362677, 442	MF121661, 658	DQ790083, 1 829	DQ790043, 3 087
	Nereis sp.	MF960765, 1 182	MF960765, 1 534	−	−
	Paraleonnates uschakovi	KX462988, 1 191	KX462988, 1 533	−	−
	Perinereis wilsoni	LC482173, 422	MN256542, 658	KC840691, 779	−
	Perinereis aibuhitensis	KF611806, 1 197	KF611806, 1 534	−	−
	Perinereis cultrifera	MN812983, 1 016	MN812983, 1 534	−	−
	Perinereis nuntia	JX644015, 1 199	JX644015, 1 537	−	−
	Perinereis sp.	MN823971, 1 138	MN823962, 1 536	−	−
	Platynereis cf. australis	MN830369, 1 196	MN830369, 1 534	−	−
	Platynereis bicanaliculata	MN812984, 1170	MN812984, 1 534	−	−
	Platynereis dumerilii	AF178678, 1172	AF178678, 1 534	AY894303, 1 805	−
	Pseudonereis variegata	MN855213, 613	MN855134, 1308	KC840693, 779	−
Gymnonereidinae	Ceratocephale abyssorum	GQ426618, 493	GQ426683, 421	GQ426585, 1 709	−
	Gymnonereis sp.	KY704332, 472	KY805814, 633	−	−
	Laeonereis culveri	KU992689, 1 007	KU992689, 1 536	−	−
	Tylorrhynchus heterochaetus	KM111507, 1 236	KM111507, 1 534	−	−
	Nicon ablepsia sp. nov.	MW525220, 513	MW644966, 1 534	MW525221, 1 820	MW525222, 1 059
Namanereidinae	Namalycastis abiuma	KU351089, 1 019	KU351089, 1 534	−	−
	Namalycastis hawaiiensis	LC213728, 521	MN125542, 658	LC213729, 1 781	LC213727, 816
	Namalycastis indica	MF959005, 482	MF958995, 660	−	MF959019, 766
	Namalycastis jaya	JX483870, 462	JN790067, 699	JX483866, 1 744	−
Hesionidae	Vrijenhoekia balaenophila	JN571884, 512	JN571831, 658	JN571895, 1 777	JN571904, 781
Note: − means the sequence was not reported. The text in bold indicates the measurement results in this paper.

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3. Systematics

Family Nereididae Lamarck, 1818

Genus Nicon Kinberg, 1866

Nicon ablepsia sp. nov.

Material examined: Holotype, catalog number B6416500072, KIOST seamount (according to MarineRegions.org, https://www.marineregions.org) in the Northwest Pacific Ocean (Fig. 1), 13.391 5°N, 149.884 7°E, 2 763 m, 5 October 2019.

Figure  1.  Sampling site, KIOST seamount where the sample was collected.

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Description: The holotype specimen incomplete, 39 chaetigers, 17.0 mm in length, 1.5 mm wide at chaetiger 1 excluding parapodia (Fig. 2a). Prostomium slightly wider than long with one pair of frontal antennae and one pair of palps (Figs 2b–d). Antennae digitate, same length as palps. Palpostyles sub-conical (Fig. 2d). Eyes absent. Peristomium isometric to chaetiger 1, with four pairs of tentacular cirri; postero-dorsal pair the longest, extending back to posterior margin of 6th chaetiger. Jaws brown, dentate cutting edge with five teeth (Fig. 2e). Oral and maxillary rings without paragnaths and papillae.

Figure  2.  Nicon ablepsia sp. nov.: a. dorsal view of the holotype; b. lateral view of the holotype, white triangles mark tentacular cirri; c. dorsal view of head; d. ventral view of head; e. jaws; f. chaetigers 8–17, dorsal view; g. chaetiger 7, end view; and h. chaetiger 7, frontal view. Scale bars: 2.0 mm (a), 0.5 mm (e), 0.05 mm (b, d, f, h).

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First two chaetigers only with neuropodia. Biramous chaetigers present from chaetiger 3. Notopodia with dorsal cirri twice as long as notopodial ligule; dorsal cirri inserted in middle of ligule (Figs 2f–h; Fig. 3a). Notopodial ligule and notopodial lobe sub-conical; ligule slightly shorter than lobe. Neuropodial ligule conical, stouter than lobe. Ventral cirri inserted at base of ligule, slightly shorter than it. Notochaetae present from chaetiger 3, homogomph spinigers (Fig. 3d). Homogomph spinigers and heterogomph falcigers present in neurochaetae dorsal and ventral fascicle (Figs 3e and f). Acicular chaetae black in color. Notoaciculae visible on epidermis of notopodial lobe with curved distal ends on middorsal side of notopodial lobe (Figs 2f and 3b). Neoroaciculae straight with curved tips (Fig. 3e).

Figure  3.  Parapodia and chaetae (a−d, SEM; e−g, optical picture). a. Parapodium; b. notopodial lobe, arrow shows curved tip of notoacicula; c. neurochaetae, homogomph spinigers and heterogomph falcigers; d. notochaetae, homogomph spinigers; e. neuropodium with neurochaetae; f. heterogomph falcigers; and g. notochaetae, homogomph spinigers. Scale bars: 0.5 mm (a), 0.1 mm (b−d).

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Total segmental number and pygidium unknown as the specimen incomplete.

Etymology: The specific name ablepsia come from Latin and refers to the eyeless character of this new species.

Distribution: Known only for type locality.

Molecular analysis: The length of 16S rDNA, COI gene, 18S rDNA and 28S rDNA sequences were confirmed by respective sequence of PCR primers (Norlinder et al., 2012). The primer-sequence alignments of PCR primers located in relevant position of marker genes correctly. The partial 16S rDNA, the complete COI gene, the partial 18S rDNA and 28S rDNA sequences of N. ablepsia sp. nov. (voucher B6416500072) contained 1 820 bp, 1 059 bp, 513 bp and 1 534 bp length, respectively.

Genera Namalycastis and Tylorrhynchus formed a well-supported clade, which was sister to the group of Nereidinae + Nicon + Laeonereis clade. Neither the Gymnonereidinae sensu Fitzhugh (1987) nor Nereidinae were recovered as monophyleticis as Nicon and Laeonereis being clustered with genera from Nereidinae. The Nicon ablepsia sp. nov. and Alitta succinea showed different topological cluster in the RaxML tree (Fig. 4) and IQ-tree (Fig. A1 in Appendix) with very low support value.

Figure  4.  The Maximum-Likelihood phylogenetic tree of 29 species based on the concatenated dataset of 16S rDNA, COI gene, 18S rDNA and 28S rDNA sequences with 1 000 bootstrap replicates. Bootstrap support values (calculated by RAxML software) are indicated adjacent to each node. The red solid dot shows the inconsistent topology cluster between the tree constructed by RAxML and IQ-TREE. All the other nodes were identical.

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4. Discussion

The genus Nicon is characterized by having all notochaetae homogomph spinigers and neurochaetae falcigers, lacking paragnaths or papillae on oral and maxillary ring. de León-González and Trovant (2013) recognized eleven Nicon species. Later, N. polaris was transferred to Kainonereis since it bears elytriform structures (Conde-Vela et al., 2018). Nicon sinica Wu & Sun, 1979 was transferred to Sinonereis because it is a junior synonym of Sinonereis heteropoda based on affinities in chaetal and parapodial features (Conde-Vela and Wu, 2019). The new species lacks eyes, therefore, is easily distinguished from other species of Nicon. Nicon ablepsia sp. nov. is similar to N. abyssalis by absence of eyes but differs from the latter in the position of the antennae, length of tentacular cirri and relative length between peristomium and chaetiger 1. The antennae of N. abyssalis are inserted closely together in the frontal margin of the prostomium, and the bases of the antennae are in contact (Hartman, 1967). The antennae of N. ablepsia sp. nov. are separate from each other. Tentacular cirri are short in N. abyssalis (reaching chaetiger 2), but long in N. ablepsia sp. nov., reaching chaetiger 6. The peristomium of N. abyssalis is prolonged and nearly twice as long as chaetiger 1, while equal to the length of chaetiger 1 in N. ablepsia sp. nov.

The phylogeny of the Nereididae and the higher taxonomic relationships within it has been explored through molecular approaches recently (Alves et al., 2020). However, the molecular phylogenetic relationship between Nicon and other Nereidid genera has not been investigated yet. Santos et al. (2005) assessed relationships of nereidids in a morphology-based parsimony analysis of 41 terminal taxa. Gymnonereidinae was restricted to Ceratocephale, Gymnonereis, Tambalagamia and Micronereides. Systematic status of Nicon was not resolved in Santos’s results. In our tree, Nicon ablepsia sp. nov. is closely to Alitta succinea with low support. Alitta is characterized by conical paragnaths on maxillary and oral ring, and dorsal cirrus mid-dorsally to subterminally attached to dorsal notopodial ligule on posterior chaetigers (Bakken and Wilson, 2005). Nicon ablepsia sp. nov. has no paragnaths and dorsal cirrus attached to base of notopodium.

The explicit systematic status of Nicon is still undetermined due to the type species of Nicon, and some of its closely related genera are not included in the present analysis. Therefore, more extensive taxon coverage are required in further studies to elucidate the systematic status of Nicon.

The sequence data reported can provide base information for molecular phylogenetic research of Family Nereididae in the future, and can also supply the basic information for designing primers. A taxonomic key to species of Nicon is provided here.

Key to Nicon species (emended from de León-González and Trovant, 2013)

1. Superior notopodial lobe present.......................................... 2

– Superior notopodial lobe absent........................................... 5

2. Tentacular cirri short, reaching chaetiger 2.......................... 3

– Tentacular cirri reaching chaetiger 5.......... N. aestuarensis

3. Heterogomph falcigers present on supra-and subacicular fas cicle, dorsal ligule subtriangular................................................ 4

– Heterogomph falcigers absent, with sesquigomph falcigers in infracicular position, dorsal ligule long and thin on median and posterior parapodia........................................ N. orensanzi

4. With homogomph falcigers in neuropodial subacicular posi tion............................................................................... N. rotunda

– Homogomph falcigers lacking.............................. N. japonica

5. Tentacular cirri short, reaching chaetiger 2.............................. 6

– Tentacular cirri reaching chaetiger 5........................................ 7

6. Dorsal ligule cirriform, reduced in posterior chaetigers; falci gers with prolonged blade................................... N. abysssalis

– Dorsasl ligule subtriangular, similar in size throughout; falci gers with long, anteriorly blunt blade distinctly serrated along inner margin............................................................. N. yaquinae

7. Eyes present; tentacular cirri reaching chaetiger 5; supra and infracicular sesquigomph falcigers present......... N. pettibonae

– Eyes absent; tentacular cirri reaching chaetiger 6; supra and infracicular sesquigomph falcigers absent.. N. ablepsia sp. nov.

– Tentacular cirri reaching chaetiger 9–10................................. 8

8. Longest pair of tentacular cirri partially annulated on distal end; falcigers with long blade, denticulate along inner margin .................................................................................... N. maculate

– All tentacular cirri annulated, with cylindrical articles; falci gers with short blades, denticles on proximal inner margin ............................................................................. N. moniloceras

Acknowlegements

We thank the crew of R/V Dayang Yihao and technical staff of Hailong III ROV for their generous help during the investigation.

A1.  The Maximum-Likelihood tree constructed by IQ-TREE. Numbers near the branch nodes refer to SH-aLRT/ UFBoot support values (all based on 1 000 replicates). The red solid dot showed the inconsistent topology cluster between the tree constructed by RAxML and IQ-TREE. The partitioned scheme and best model information was auto selected by IQ-TREE as follows: Part 1 (1–1 158 nt, 16S rDNA), GTR+F+I+G4 model; Part 2 (1 159–2 697 nt, COI gene), TIM2+F+R4 model; Part 3 (2 698–4 600 nt, 18S rDNA), TIM2e+FQ+I+G4 model; and Part 4 (4 601–5 559 nt, 28S rDNA), TN+F model.

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