Among polychaetes, polynoids have the highest number of symbiotic species found living with a wide variety of marine invertebrates, including other polychaetes (Salazar-Vallejo et al., 2015). Alentiana aurantiaca, the first and only member in this genus, living among the tentacles of the sea anemone Bolocera tuediae, was first described by Verrill (1885) as a member of Polynoe. After reviewing the type specimens, Hartman (1942) erected the genus Alentiana and recombined this species. Alentiana differs remarkably from the other genera of the family in having reduced notopodia without chaetae, and elytra present in every third segment in medial and posterior regions, neuropodial chaetae of two kinds: fine supraacicular chaetae with few rows of spines and single or few stouter, spine-like subacicular ones. Alentiana aurantiaca is described from northeastern coast of America (Verrill, 1885; Pettibone, 1963), and reported in European waters (Bellan, 2001).

Norlinder et al. (2012) analyzed the phylogenetic relationship of Polynoidae and other Aphroditiformia scale worms via the morphology and molecular sequences of 18S rDNA, 28S rDNA, 16S rDNA and the cytochrome c oxidase subunit I (COI) gene. There were 30 species of the Polynoidae family used in their study, but only two species, Hyperhalosydna striata and Lepidasthenia elegans belonging to the subfamily Lepidastheniinae, had reported as having those four marker genes. This study is the first report of molecular marker genes: two nuclear genes (18S rDNA and 28S rDNA) and two mitochondrial genes (16S rDNA and the COI gene) in the genus Alentiana (Lepidastheniinae). The marker-gene data set was retrieved from next-generation sequencing (NGS) and whole genome survey sequences (GSS) and provided a method to obtain the gene information from the NGS and GSS data.

In this contribution, Alentiana palinpoda sp. nov., the second member of this genus living as a commensal among the tentacles of the large sea anemone (Actiniidae), is described based on specimens collected from a seamount in the Northwest Pacific Ocean. This is the first report on the four molecular marker genes in the Alentiana species which can supplement the gene information of family Polynoidae in the public gene database.

The specimen was collected by Hailong III ROV (remotely-operated vehicle) DY56 Cruise (the 56th China Ocean Scientific Research) in September 2019. When photographs were taken of the sea anemone sample, this living scale-worm suddenly slipped out from the socket among the tentacles of Actiniidae. The iridescence of its elytra surfaces was remarkable and impressive.

The specimen was preserved in a 95% ethanol solution and deposited in the Sample Repository of Second Institute of Oceanography (RSIO), Ministry of Natural Resources, Hangzhou, China. The characteristics of the whole body were observed with appendages dissected under a stereomicroscope (Zeiss Stemi 305). Parapodium and neurochaetae were dissected, and mounted onto temporary slides using glycerol or permanent slides using Canada Balsam. Methyl green was used for staining in order to distinguish papillae on the Pharynx. Photographs of the chaetae were captured using light microscopy (Zeiss Axio Imager A2).

The total genomic DNA of this specimen was extracted from its muscle appendage using QIAGEN DNeasy^® Blood and Tissue Kits (Qiagen, CA, USA), according to the manufacturer’s protocol. Attempts to extract DNA from the elytra were unsuccessful, with little or nothing when we detected in the DNA concentration using a NanoDrop 2000 Spectrophotometer and a Qubit fluorometer. The qualified DNA was stored in sterilized Milli-Q^® water at –80°C before sequencing. The entire genome was obtained by Illumina sequencing. One pair-end (PE150) library was constructed with insert size of approximately 350 bp, and sequenced on HiSeq^TM X-Ten platform (Illumina, CA, USA) at Zhejiang Tianke High Technology Development Co., Ltd. A total of 61 640 260 sequence clean reads and 8.86 Gbp clean bases (Q20 ≥ 98.96%) were obtained after quality trimming, and then de novo assembled by the SPAdes genome assembler (Bankevich et al., 2012). In order to pick out the nuclear 18S rDNA, 28S rDNA and mitochondrial 16S rDNA, and COI genes, the assembled contigs were aligned against the downloaded reference sequences of those four genes in the GenBank database via the NCBI local blastn program (Cock et al., 2015). The complete length of the COI gene sequence was checked, based on the result of invertebrate mitochondrial genome prediction by the MITOS server online (Bernt et al., 2013). The start and end of target sequences were checked by the reported universal primer pairs (Norlinder et al., 2012). The accession number of sequences used in phylogenic analysis is listed in Table 1.

Nuclear and mitochondrial sequences 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 nuclear 18S rDNA, 28S rDNA and mitochondrial 16S rDNA and COI genes were performed separately, and then concatenated into one file after manually trimming unalignable regions on both flanks. The phylogenetic tree was performed with Maximum Likelihood analysis (ML), using the IQ-TREE Web Server (Trifinopoulos et al., 2016) with auto-selected substitution model, default parameters and 1 000 bootstrap replicates (command line: path_to_iqtree -s concatenated_alignments.phy -spp concatenated_alignments. partition.txt -pre concatenated_alignments.phy -m TEST -bb 1000 -alrt 1000). The SH-aLRT test (Guindon et al., 2010) was performed with the manual’s recommendation of adding “-alrt 1000” into the IQ-TREE command line.

Family Polynoidae Kinberg, 1856

Subfamily Lepidastheniinae Pettibone, 1989

Genus Alentiana Hartman, 1942

Alentiana palinpoda sp. nov. (Fig. 1)

Material examined. Holotype, catalog number B6317500003, unnamed seamount in the Northwest Pacific Oecan, 15°31′N, 161°48′E, 1 156 m depth, commensally with a sea anemone (Actiniidae), September 20, 2019.

Diagnosis. Body short with 37 segments. Elytra large, completely covering dorsal surface, present in every third segment in posterior regions. Notopodia reduced, without notochaetae. Neuropodia truncated, with two types of neurochaetae; prechaetal lobe larger than postchaetal lobe, with ventral part rolling upward and backward.

Description. Holotype complete, ca. 32 mm long, 13 mm wide, including parapodia, with 37 segments (including tentacular segment), last two small. Body dorsoventrally flattened, not tapering posteriorly, with very long parapodia.

Prostomium bilobed, wider than long, without cephalic peaks. Two pairs of conspicuous dark eyes, anterior pair largest, situated laterally on widest part of prostomium, posterior pair dorsally, near hind margin of prostomium. Median and lateral antennae present; medium antenna with bulbous ceratophore in anterior notch of prostomium and a long style about two times the length of lateral ones; lateral antennae inserted terminally on prostomium, with ceratophores formed by anterior prolongations of prostomial lobes. Styles smooth, tapering. Palps stout, 1.5 times thicker than antennae, tapered, smooth, and slightly shorter than median antenna. Facial tubercle absent. Pharynx with two pairs of jaws without any teeth (Figs 1h and i) and encircled distally by nine pairs of bulbous papillae (Figs 1b, c, and g). Two pairs of lateral papillae on subterminal pharynx (Figs 1b, c, and g). Tentacular segment (Segment 1) with two pair tentaculophores directed anteriorly, lateral to prostomium, without chaetae (Fig. 1j); tentacular cirri longer than median antenna. Second segment with first pair of elytra, sub-biramous parapodia, and ventral cirri obviously longer than the following ones. Parapodia same from second segment to the end of the body.

Sixteen pairs of large, bulbous elytrophores present on Segments 2, 4, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 26, 29, 32, and 35. Elytra large, completely covering the dorsum, oval to subreniform in shape, without papillae or microtubercles on margin and surface (Figs 1p and q). Dorsal tubercles on cirrigerous segments, indistinct (Fig. 1k). Parapodia sub-biramous (Figs 1m and n) throughout body, with embedded noto- and neuroacicula (Fig. 1n). Dorsal cirri on cirrigerous segments elongate, about six to seven times longer than ventral cirri. Notopodia reduced to a small digitate lobe, without chaetae (Figs 1k and m). Neuropodia distally truncate, elongate, notched dorsally and ventrally (Fig. 1o), prechaetal lobe larger than postchaetal lobe, forming upward flanged structure in ventral side, and enveloping ventral side of postchaetal lobe (Figs 1l and o). Neurochaetae of two kinds, including six to ten supraacicular chaetae toothed on one side (Fig. 1s) with a slightly inflated end and a heavy, smooth, subacicular chaeta (rarely two in a parapodium), with falcate tip and smooth sides (Fig. 1r). All chaetae and acicula yellow. The ventral nephridial papillae small, beginning on Segment 4.

Color: body and pharynx red, elytra dark brown in live specimen; body pale yellow with dark pigments (Figs 1a–d), elytra white in ethanol-preserved specimen (Figs 1e–m).

Etymology. The specific name palinpoda is a combination of the Greek palin- (backward) and poda (foot), referring to the ventral side of neuropodia of the new species rolling upward and backward.

Distribution. Only known from the type locality.

Remarks. Alentiana aurantiaca (Verrill, 1885) was the only known member of the genus Alentiana prior to the report of this new species. The major characteristics of Alentiana palinpoda sp. nov. are mostly similar to those of A. aurantiaca, originally described from northeastern coast of America (Verrill, 1885; Hartman, 1942). Both species are commensal among the tentacles of Actiniidae (sea anemone). However, Alentiana palinpoda sp. nov. clearly differs from A. aurantiaca in the number of chaetigers, the arrangement of elytra, neuropodium, and subacicular chaeta as follows. (1) About 39 chaetigers are present in A. aurantiaca, whilst there are 37 in A. palinpoda sp. nov. (2) Large elytra completely cover the dorsum in A. palinpoda sp. nov., whereas those of A. aurantiaca leave the median dorsum broadly exposed. (3) The neuropodium of A.aurantiaca has triangular chaetal lobes, and the prechaetal lobe slightly exceeds the postchaetal lobe in length. However, that of A. palinpoda sp. nov. is truncated with pre- and postchaetal lobes equal; the prechaetal lobe of A. palinpoda sp. nov. is larger than the postchaetal lobe in width, and rolls upward and backward on the ventral side. And (4) the distal end of the subacicular chaeta is more curved in A. palinpoda sp. nov.

The information of selected taxa for phylogenetic analysis is presented in Table 1. The length of 18S rDNA, 28S rDNA and 16S rDNA was confirmed by the respective sequences of PCR primers (Norlinder et al., 2012). The primer-sequence alignments of PCR primers located in the position of related genes are shown in the schematic map (Fig. A1 in Appendix). This study can also provides base information to design or edit primers when we need to amplify the gene fragments of relevant species. The data set of 18S rDNA, 28S rDNA, 16S rDNA partial sequence and COI complete sequence of Alentiana palinpoda sp. nov. (voucher B6317500003) contained 1 013 bp (1 013 bp used in phylogenetic analysis, total length 1 481 bp), 1 108 bp, 527 bp and 1 524 bp, respectively.

The ML tree was constructed based on the concatenated alignment file of Alentiana palinpoda sp. nov. and 75 reference species (Fig. 2). Molecular phylogeny revealed the subfamily Lepidonotinae Willey, 1902 is a polyphyletic group. Lepidasthenia elegans and Hyperhalosydna striata are cluseterd with members of Lepidonotinae. Both of the two genera belong to the subfamily Lepidastheniinae (Pettibone, 1989). This topology suggests a closed relationship between Lepidonotinae and Lepidastheniinae and the monophyly of Lepidonotinae is doubtful. Lepidonotinae differs from Lepidastheniinae in the form of neuropodia and elytra (Wehe, 2006). Neuropodia are deeply incised dorsally and ventrally in Lepidastheniinae, whilst not deeply incised in Lepidonotinae. Elytra are usually small, not covering the mid-dorsum and smooth in Lepidastheniinae while they are usually large, covering the mid-dorsum and ornamented in Lepidonotinae. Elytra of Alentiana palinpoda sp. nov. are large, covering the mid-dorsum. This characteristic makes it easily distinguishable from other members of Lepidastheniinae. Alentiana palinpoda sp. nov. and Bathymoorea_lucasi (subfamily Eulagiscinae) form a well supported clade. Bathymoorea has one pair of large eyes, elongate acicular lobe and numerous notochaetae. Bonifácio and Menot (2019) synonymize all these subfamilies lacking lateral antennae (Bathyedithinae, Bathymacellinae, Branchinotogluminae, Branchiplicatinae, Branchipolynoinae, Lepidonotopodinae, Macellicephalinae, Macellicephaloidinae, Macelloidinae, Polaruschakovinae and Vampiropolynoinae) with Macellicephalinae sensu Hartmann-Schröder, 1971. Hatch et al. (2020) reinstated Lepidonotopodinae comprised of Branchipolynoe, Branchinotogluma, Bathykurila, Branchiplicatus, Lepidonotopodium, Levensteiniella, Thermopolynoe, and Peinaleopolynoe. In our tree, Lepidonotopodinae clustered with partial members of Macellicephalinae with low supported value. Our results support the phylogenetic proposal of Bonifácio and Menot (2019). The phylogenetic relationship between Lepidastheniinae and other polynoid subfamilies has not been fully investigated yet. Lepidastheniinae comprised of 10 genera (Alentiana, Benhamipolynoe, Hyperhalosydna, Lepidasthenia, Lepidastheniella, Parahalosydna, Perolepis, Pseudopolynoe, Showapolynoe, Telolepidasthenia), while only three of them included in our analysis. Further analyses would be required to assess the taxonomic relationship between Lepidastheniinae and Lepidonotinae.

We thank the crew of R/V Dayang Yihao and technical staff of Hailong III ROV for their great help during the investigation. We thank Yang Li for his assistance in sea anemone identification.