﻿A new species of the genus Lindaspio Blake & Maciolek, 1992 (Annelida, Spionidae) from a cold seep near Hainan Island, China

﻿Abstract A new species of the spionid genus Lindaspio Blake & Maciolek, 1992 was collected from a cold seep near the Hainan Island at a depth of 1758 m. Morphologically, Lindaspiopolybranchiatasp. nov. differs from the congeners in having a narrow, folded caruncle and more neuropodial branchiae (from chaetiger 20). The 18S, COI, and 16S sequences of the new species have been submitted to GenBank. It is the first record of the genus Lindaspio from Chinese waters. A key to all species of Lindaspio is given.


Introduction
Spionidae Grube, 1850 is a group of polychaetes which have a worldwide marine distribution from the intertidal zone to the deep sea, including hydrothermal vent and cold seep environments (Blake and Maciolek 1992;Blake and Ramey-Balci 2020). They are very common and frequently dominant within polychaete communities (Meißner et al. 2014). The genus Lindaspio was established by Blake and Maciolek (1992) for Lindaspio dibranchiata, which was collected from a hydrothermal mound in the Guaymas Basin (27°01'N, 111°24'W, 2008 m depth). At present, Lindaspio comprises three valid species: L. dibranchiate Blake & Maciolek, 1992 and L. southwardorum Blake & Maciolek, 1992  In May 2021, a biodiversity survey in the area of a cold-seep (named "Lingshui") near Hainan Island was conducted by the Institute of Oceanology, Chinese Academy of Sciences (IOCAS) using the R/V Kexue. During the cruise, some specimens of an undescribed species of polychaetes were collected by the ROV Faxian at a depth of 1758 m (Fig. 1).

Materials and methods
All the specimens were fixed and preserved in 75% ethanol solution and are deposited in the Marine Biological Museum of the Chinese Academy of Sciences (MBMCAS) in the Institute of Oceanology, Chinese Academy of Sciences (IOCAS). The specimens were observed, measured, and photographed with a Nikon AZ100 stereomicroscope. The fine morphology of anterior end, notochaetae, and neurochaetae was observed using a Hitachi S-3400N scanning electron microscope (SEM).
Total DNA was extracted with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and stored at −20 °C. Partial fragments of COI (~700 bp), 16S (~450 bp), and 18S (~1800 bp) genes were amplified by the polymerase chain reaction (PCR) using primers polyLCO/polyHCO (Carr et al. 2011), 16S-AnnF/AnnR (Sjölin et al. 2005), and 18S-A/B (Medlin et al. 1988), respectively. Amplifications were carried out in a reaction mixture containing 2 μl of template DNA, 12.5 μl of Premix TaqTM (Takara, Otsu, Shiga, Japan), 0.5 μl of each primer (10 mM), and sterile distilled H 2 O to a total volume of 25 μl with cycling conditions as follows: initial denaturation at 94 °C for 10 min, followed by 35-45 cycles of denaturation at 94 °C for 30 sec, annealing at 45 °C for 40 sec, and extension at 72 °C for 90 sec. A final extension at 72 °C for 5 min was included. PCR products with distinct bands after electrophoresis on 1.5% agarose gels were sent to Qingke Laboratory (Qingdao, China) for sequencing using the same set of primers that were used for PCR amplifications. Fragments with overlapping sequences (forward and reverse) were merged into consensus sequences using CONTIG EXPRESS (a component of Vector NTI Suite 6.0, Life Technologies, Carlsbad, CA, USA). The assembled sequences were checked by BLASTing in GenBank to ensure that the DNA was not contaminated. Finally, all the new sequences were submitted to GenBank.
Anterior neurochaetae include row of heavy spines and ventral bundle of thin capillaries (Fig. 4D); neuropodial spines each with smooth shaft that tapers gradually, then continues as fine, pointed tip with fine bristles sometimes visible along edge (Fig. 4E); spines present until about chaetiger 40, then accompanied with thin capillaries; these capillaries accompanied by neuropodial hooded hooks from about chaetiger 65; each hook smaller, more delicate than notopodial hooks, without minute teeth (Fig. 4F). Pygidium simple, conical, lacking cirri (Fig. 3D, E).
GenBank Accession Number: COI OQ582086, 16S OQ592054, 18S OQ592055. Remarks. As mentioned above, three species have been reported in the genus Lindaspio. The new species is easily distinguished from L. dibranchiate and L. sebastiena in having a narrow, undulating caruncle, while L. sebastiena has no caruncle and L. dibranchiate has a short, mounded caruncle. The new species resembles L. southwardorum in having similar caruncle, ventral branchiae and dorsal clusters of spines in anterior notopodia, while the latter differs in having first neuro-branchiae from chaetiger 55 (vs. chaetiger 20 in the new species) and having more modified spines on chaetigers 2-4 (20 vs. 15).
The BLAST percent identity of the 18S sequence between the new species and Lindaspio dibranchiata is 99% (1758/1762 bp), suggesting that they are congeneric. Additionally, the COI and 16S sequences of our specimen are identical to those of Lindaspio sp. (GenBank accession number OK032597.1), which confirms that the unverified species of Lindaspio in GenBank is a Lindaspio polybranchiata sp. nov.
Etymology. The species is so named because it has more neuropodial branchiae than the congeners.
Distribution. Currently only known from the type locality, near Hainan Island, China, at a depth of 1758 m (Fig. 1).