﻿Three new species and one new record of Deimatidae (Echinodermata, Holothuroidea, Synallactida) discovered in the South China Sea and the Mariana fore-arc area using integrative taxonomic methods

﻿Abstract Deep-sea holothurian specimens were collected during five scientific expeditions (2018–2023) using the submersible vehicle ‘Shenhaiyongshi’. Our examination of specimens of Deimatidae from the South China Sea and the Mariana fore-arc area revealed three new species, which were described as Oneirophantaidsseicasp. nov., Oneirophantabrunneannulatasp. nov., and Oneirophantalucernasp. nov. These species were distinguished from each other and from congeners by the arrangement, and number of ventrolateral tube feet and ossicle types. We also reported Oneirophantamutabilismutabilis Théel, 1879 for the first time from the Mariana fore-arc area, and we recorded Deimavalidumvalidum for the second time from the South China Sea. The taxonomy of these new species and new records is discussed, and a phylogenetic analysis based on a concatenated dataset of 16S and COI genes was conducted. Additionally, the inter- and intraspecific genetic divergences we calculated among deimatid species. The results support the assignment of these new species to the genus Oneirophanta and their separation from congeners. A description of the main morphological characters of Oneirophanta species is also provided. The data were collected from geographically diverse areas and suggest that species of Deimatidae were abundant in the Pacific Ocean and occupied a wide range of depths.


Introduction
Echinoderms are abundant in Chinese seas, and the South China Sea has more species than the Yellow Sea and the East China Sea (Liao and Xiao 2012).The South China Sea covers ~ 3.5 million km 2 , and it is studded with 200 islands and islets.It is in the center of the Indo-Western Pacific Biogeographic Province, and it is one of the most biodiverse seas in the world.The maximum recorded depth for echinoderms was 5377 m, with considerable habitat and species diversity (Morton and Blackmore 2001;Teh et al. 2019 China began with the Jiaolong manned submersible in 2010, which has been used widely to uncover the deep-sea biodiversity in China seas and the Mariana Trench (Li 2017).Taxonomic research has revealed many new species and new geographical records of animal species in the deep waters of the South China Sea (Li et al. 2019).
Holothurians are the dominant epibenthic invertebrate taxon in many areas of the deep sea, and they account for 90% of that ecosystem's biomass (Hendler et al. 1995).However, our present knowledge of deep-sea holothurians is still limited due to the difficulty in collecting and depositing good specimens.Even the specimens collected by the submersible carefully and carried from the seabed to the sea surface in seawater-filled containers have arrived aboard the ship in very poor condition (Pawson 1982a).The epidermis and dermis of the whole holothurians begin to slough off, and the entire external body wall is often completely autotomized.The body wall contains a high proportion of water, even the most carefully preserved specimens will shrink greatly, and the shrinkage rate is frequently greater than 90% (Billett 1991).When preserved in absolute ethanol, the holothurians shrink, so all the external characters are easily distorted, which undoubtedly impacts their morphological identification negatively after collection.In recent years, few studies on the order Elasipodida have been conducted in the South China Sea (Li et al. 2018;Xiao et al. 2018Xiao et al. , 2023)), and studies on other taxa in the South China Sea have also been reported rarely.More morphological information and molecular data should be obtained for a comprehensive taxonomic evaluation of deep-sea holothurians in the South China Sea (Li et al. 2019).
The family Deimatidae formerly belonged in the order Elasipodida Théel, 1882, but was later transferred to the order Aspidochirotida by Smirnov (2012).Miller et al. (2017) subdivided paraphyletic ordo Aspidochirotida into three separate orders and placed family Deimatidae to the order Synallactida Miller, Kerr, Paulay, Reich, Wilson, Carvajal & Rouse, 2017.The order Synallactida includes the families Deimatidae Théel, 1882, Stichopodidae Haeckel, 1896, and Synallactidae Ludwig, 1894.Deimatidae is the smallest family in the order, and it contains 13 accepted species within three genera (Oneirophanta Théel, 1879, Orphnurgus Théel, 1879, and Deima Théel, 1879) (WoRMS 2023).Only two of the recognized species in the family Deimatidae, Orphnurgus protectus (Sluiter 1901b) and Deima validum Théel, 1879, have been recorded from the South China Sea (Liao 1997).The genus Oneirophanta was established with Oneirophanta mutabilis Théel, 1879 as the type species (Théel 1879).Currently, there are only three species of Oneirophanta, and none of them have been found in China.Eleven deimatid specimens were collected from the South China Sea and the Mariana fore-arc area from 2018 to 2023.Morphological observations of these specimens suggested that they represented three new species and one new record from the Mariana fore-arc area, and they all belonged to the genus Oneirophanta.
In addition, we present a morphological description of Deima validum validum Théel, 1879, which was recorded for the second time in the South China Sea; the present specimens show some variations compared with specimens that were recorded previously.Our study provides comprehensive a description of morphological characters, an assessment of intraspecific divergence between the new species and all other known species, and more molecular details that may be useful for further studies of the phylogeny and diversity of the family Deimatidae.

Sampling and preservation
Specimens were collected from the South China Sea and the Mariana forearc area (Fig. 1) using the manned submersible vehicle 'Shenhaiyongshi' from 2018 to 2023, at depths of 1340-3806 m.Samples were frozen or preserved in absolute ethanol, and then stored at the Institute of Deep-sea Sciences and Engineering (IDSSE), Chinese Academy of Sciences (CAS), Sanya, China.

Morphological observations
The specimens of each species were identified using a variety of original descriptions and literature (Théel 1879;Koehler and Vaney 1905;Hansen 1967Hansen , 1975;;Pawson 2002).External morphological features were examined under a dissecting stereomicroscope (OLYMPUS SZX7), and identification was based on in situ images or pictures photographed in our lab using a Canon EOS 6DII camera.To prepare the deposits, small pieces of body tissue (dorsal and ventral body wall, tentacles, papillae, and tube feet) were digested in a 15% sodium hypochlorite solution.The deposits were then washed three times in distilled water and dried in absolute ethanol before examination with a scanning electron microscope (Phenom ProX).

DNA extraction, PCR amplification, and DNA sequencing
Total genomic DNA was extracted from small pieces of 20-30 mg holothurian muscle tissue using a TIANamp Marine Animals DNA Kit (TianGen, Beijing), according to the manufacturer's instructions.Mitochondrial cytochrome c oxidase I (COI) and 16S rRNA were generated for various specimens using the primers and methods outlined in Miller et al. (2017).The PCR amplification program was as follows: initial denaturation at 98 °C for 3 min, followed by 40 cycles at 98 °C for 10 s, 52 °C for 10s, 72 °C for 10s, and a final extension at 72 °C for 5 min.Total reaction volume was 50 μL: 25 μL Premix Taq with 1.25U Taq, 0.4 mM of each dNTP, 4 mMMg2+ (Ex Taq version, Takara, Dalian, China), 0.5 μM each of the primers and ~ 100 ng template DNA.The sequence chromatograms were then checked using CHROMAS 2.23 (Technelysium Pty Ltd.).The forward and reverse sequences were assembled using CONTIG EXPRESS, which is a component of Vector NTI Suite 6.0 (Life Technologies, Carlsbad, California).

Phylogenetic analyses
Two partial sequences (COI and 16S) were obtained from specimens and were deposited in GenBank (Table 1), and some relevant sequences (from all available deimatid species) that were downloaded from BOLD (https://www.boldsystems.org/)and NCBI (https://www.ncbi.nlm.nih.gov/)databases were used for phylogenetic analyses.Apostichopus californicus and A. parvimensis in the family Stichopodidae (order Synallactida) were used to root the tree.
Sequence alignments were generated using MAFFT7 (Katoh and Standley 2013) with default parameters.Gblocks (Talavera and Castresana 2007) were used to remove batches of fragments from two alignments that were aligned ambiguously.The best partitioning scheme and evolutionary models for two pre-defined partitions were selected using PartitionFinder2 (Lanfear et al. 2017), with all algorithms and AICc criteria.Maximum likelihood phylogenies (ML) were inferred using the Shimodaira-Hasegawa-like approximation likelihood-ratio test (Gascuel 2010) and IQ-TREE (Lam-Tung et al. 2015) models with 20,000 ultrafast bootstraps (Minh et al. 2013).Bayesian Inference phylogenies (BI) were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under the partition model (two parallel runs, 5,000,000 generations).The initial 25% of sampled data were discarded as burn-in, and the remaining trees were summarized in a 50% majority rule consensus tree.The results were visualized using FigTree v. 1.4.4.The Kimura two-parameter (K2P) genetic distances of COI among deimatid species were calculated using model MEGA X (Kumar et al. 2018).Description of holotype.External morphology.Body elongated and cylindrical, ventrum flattened.29 cm long and 9 cm wide before fixation (Fig. 2A,  B).Color in vivo yellowish-white, tentacles, papillae, and tube feet often darker (Fig. 2C, D).Mouth anteroventral, anus posteroventral.Tentacle discs with rounded knobs at the edges, and the tentacles partially retracted into the mouth, making the number of tentacles difficult to calculate.Ventrolateral tube feet 40 pairs, arranged in alternating two or three rows on each ventrolateral ambulacrum.Midventral tube feet two and rudimentary, scattered along the mid-ventral ambulacrum, one positioned in the front third of the body, the other is positioned in the back third of the body, the anus surrounded by few small tube feet.Dorsal papillae roughly 18-20 on each body side, evenly distributed, measuring 4-10 cm in length, and placed in single rows along the dorsal radii.Ventrolateral papillae ~ 9-12 on each side, generally shorter than dorsal papillae, measuring 2.5-7 cm in length, arranged in single rows along the ventrolateral radii.All papillae slender and tapering from base to end.
Etymology.Consists of IDSSE and the Latin suffix icus (belonging to), to honor IDSSE's contributions and efforts to the field of deep-sea exploration.
Distribution.A seamount in APEI 4, Clarion Clipperton Zone; Xisha Trough, the northern slope of the South China Sea, at depths of 2985-3806 m.
Remarks.Oneirophanta idsseica sp.nov. is characterized by the arrangement of ventrolateral tube feet in two or three rows that number up to 40-50 pairs with distinctive cross-types in dorsal deposits.O. idsseica sp.nov. is distinct from Oneirophanta setigera (Ludwig 1893) due to the presence of small, perforated plates and crosses with open ramifications that are usually bifurcated.O. idsseica sp.nov.differs from Oneirophanta conservata Koehler & Vaney, 1905 and Oneirophanta mutabilis Théel, 1879 by the arrangement and high number of ventrolateral tube feet, and the absence of large, perforated plates on dorsum.
The phylogenetic trees showed that O. idsseica sp.nov., together with an unnamed species (Oneirophanta stet.CCZ_100, see below), formed a sister group that included Oneirophanta cf.mutabilis and O. mutabilis.From a morphological point of view, O. idsseica sp.nov.mostly resembled O. stet.CCZ_100 with ventrolateral tube feet arranged in two or three rows, two rudimentary midventral tube feet, spatulated crosses and small, irregular perforated plates on dorsum, and crosses with open ramifications in different stages of development on the ventrum.From a molecular point of view, the COI pairwise distance between O. idsseica sp.nov.and O. stet.CCZ_100 was 0.6% (Suppl.material 1).Compared with the description of O. stet.CCZ_100, our study provides more details of ossicle morphology of tentacles, tube feet, and papillae.Description of holotype.External morphology.Body elongated, dorsum convex, ventrum flattened.20 cm long, and 5 cm wide before fixation (Fig. 4A, B).Skin reddish brown, with darker coloration on tentacles and tube feet, a brown ring at the end of each tube foot.Tentacle 20, never with ramified processes, circum-oral papillae absent.Ventrolateral tube feet ~ 37 pairs, placed in alternating three rows, bilateral symmetry.Midventral tube feet two and rudimentary, one placed on half the body, the other placed on a rear quarter of the body, and several small tube feet surrounding anus.Dorsal papillae 23-26 on each body side, arranged in single rows along dorsal radii, measuring 5-6 cm.
Etymology.The species was named after the Latin word lucerna to commemorate the traditional Chinese Lantern Festival, which was relatively close to the time these specimens were collected.
Distribution.Northern slope of the South China Sea, depths of 1389-1393 m.
Remarks.The new species conformed to the genus Oneirophanta characterized by uncontracted the tentacles, the absence of oral papillae and tentacle discs without ramified processes.Oneirophanta lucerna sp.nov.differed from other species of Oneirophanta in possessing highly variable shaped spinous rods and ventrolateral tube feet that are only arranged in single rows, whereas, in other species (Table 2), they are arranged in two or three rows along ventrolateral ambulacrum: O. conservata, O. setigera, and O. mutabilis in double rows, O. idsseica sp.nov. in alternating two or three rows, and O. brunneannulata sp.nov. in three rows.The three accepted species of Oneirophanta and the other two new species in this study all have perforated plates that are absent in Oneirophanta lucerna sp.nov.This is the first record of a species in the genus Oneirophanta with mainly irregular spinous rods.Perrier, 1900: 117-118;R. Perrier 1902: 380-386, pls XIV: 3, 4, XVIII: 16-22.Oneirophanta alternata var.talismani R. Perrier, 1902: 386-388, fig.Description.External morphology.Body cylindrical, nearly equal in width throughout the whole length and tapering anteriorly.15 cm long and 4.5 cm wide after fixation with 95% alcohol for several days (Fig. 8A, B).Skin white, calcified and brittle.Tentacles 20, unretractile, 0.6-1.6 cm in length, with 7-9 marginal digits.Mouth and anus ventral.Ventrolateral ambulacra with 16 tube feet on each side, arranged in two irregular rows.Dorsal papillae 6 pairs, the maximum length up to 10 cm, placed in single rows along dorsal radii.Ventrolateral papillae 13 on each side, the maximum length up to 6 cm.Midventral tube feet not found due to damage to the mid-abdomen of the specimen, and only approximately six small tube feet observed near the anus.Ossicle morphology.The body wall and papillae with perforated plates (Fig. 9A1-C3), 0.9-1.5 mm in diameter.The entire periphery of fully developed plates usually surrounded by closed holes, with large central holes (especially in ventrum) and small peripheral holes.Some plates bearing 8-14 small, vertical spines (Fig. 9B1, C3), but no formation of a secondary layer of meshwork.Tube feet with rods up to 0.8 mm long (Fig. 9D1, D2, D4) and incompletely developed plates with open ramifications (Fig. 9D3, D5).The irregular rods in tentacles with variable bending angles (Fig. 9E1-E5).
Remarks.Oneirophanta mutabilis was first described west of the Crozet Islands (H.M.S. Challenger station 146: 46°46'S, 45°31'E) at depths of 2514 m (Théel 1879).It was divided into two subspecies, Oneirophanta mutabilis mutabilis Théel, 1879 and Oneirophanta mutabilis affinis Ludwig, 1893, based on differences in tentacle shape, the type of deposit, and the number of dorsal papillae (Hansen 1967).There are four main distinctions between them (see Table 2): (1) O. m. affinis was restricted to a relatively small area in the eastern Pacific where it replaced the otherwise cosmopolitan O. m. mutabilis.(2) tentacles with marginal digits were found in all specimens examined of O. m. mutabilis, but tentacle discs of O. m. affinis were smooth or had incised edges, and they lacked marginal digits.(3) the perforated plates of O. m. affinis were almost completely devoid of vertical spines, whereas those of O. m. mutabilis had vertical spines.(4) numerous rods of O. m. affinis were usually present in the tentacle discs, which was an additional difference from O. m. mutabilis.The morphological characteristics of our specimens were in accordance with the description of O. m. mutabilis (Hansen 1975).This was the first record of O. m. mutabilis from the Mariana fore-arc area.
Remarks.Deima validum was first described by Théel (1879) in the preliminary report of the exploring voyage of H. M. S. Challenger under Sir C. Wyville Thomson., with a detailed description of another novel species, Deima fastosum.Four new species were reported subsequently by different taxonomists: D. atlanticum Hérouard, 1898;D. blakei Théel, 1886;D. mosaicum Ohshima, 1915 andD. pacificum Ludwig, 1894.The family Deimatidae underwent a thorough revision by Hansen (1975), who regarded D. fastosum, D. atlanticum, D. blakei, D. mosaicum andD. pacificum as junior synonyms of D. validum. Hansen (1967) separated the species into two subspecies, cosmopolitan Deima validum validum and the eastern Pacific Ocean Deima validum pacificum Ludwig, 1894, based on differences in the number of dorsal papillae and the type of deposit in which they were found.
The two specimens examined here are consistent with the diagnosis of subspecies D. v. validum as described in detail by Hansen (1975).Perforated plates were like those of D. fastosum, with a large, reticulated, conical knob, which hardly ever approached in other known specimens that were used to investigate D. v. validum.This unique feature represents an extreme case of plate variation, where the spinous appearance of the skin is due to these very high and often vertically rising knobs on the plates.Hansen (1975) hypothesized that additional mesh structures on perforated plates varied with depth into two types: one-layered plates were characteristic of bathyal specimens, and many-layered plates were typical of abyssal specimens.However, it also exhibited a transitional type of plate in the abyssal Tasman Sea specimens and the bathyal Japanese specimens (perforated plates that were completely or almost completely devoid of additional meshwork), which prevented a clear distinction between a bathyal and an abyssal type of deposit.
Only in the Bay of Bengal (depth 1224-3365 m) did the development of additional layers of meshwork increase progressively with depth.In this research, high-knobbed plates were present in the abyssal South China Sea specimens (depth > 3000 m), but they were absent from the South China Sea specimens first reported (depth 1100 m) by Liao (1997).This was the second time that Deima validum has been recorded in the South China Sea.The proposition that the development of additional meshwork on perforated plates increased with depths needs to be investigated on more specimens at different depths in the South China Sea.

Genetic distance and phylogenetic analyses
The inter-and intraspecific genetic divergences of the COI gene were calculated to calculate the genetic distances in Deimatidae (Suppl.material 1).For the COI alignment, the interspecific distances in Oneirophanta ranged from 8.2-15.3%, the intraspecific distances in Oneirophanta were in the range of 0-0.6%, and the range of genetic distances among three genera was 15.1-21.9%.
In total, 11 COI sequences and 10 16S sequences were deposited into Gen-Bank (Table 1).To check the intrageneric relationships of species in Deimatidae, a Bayesian phylogenetic tree and a Maximum likelihood (ML) tree were reconstructed using concatenated 16S-COI sequences of length 1191 bp.The result of the phylogenetic analyses showed quite similar topologies in BI and ML trees (Fig. 12), except for one area of the BI tree, where Oneirophanta setigera formed an independent clade within Oneirophanta (BI 0.99).
The phylogenetic relationships of Deimatidae clustered into three portions and were consistent with the traditional classification system (Fig. 12).Portion 1: Deima validum was clustered with Deima validum validum (ML 99%, BI 1), which formed a monophyletic sister group (ML 89%, BI 1) with Oneirophanta.Portion 2: The three new species and the new record fell into Oneirophanta, which was divided into three clades.Clade 1: O. setigera was shown to be distinct from the other congeners in the BI tree (BI 0.99

Species delineation and generic assignment
Both the morphology and molecular phylogenetic analyses supported the assignment of the three new species to the genus Oneirophanta.The external morphological characteristics in Oneirophanta species were quite similar to those in Orphnurgus, but Oneirophanta never has tentacle discs with ramified processes, and they usually have rounded knobs on the margin.The three new species described in this study conformed to this feature.
Oneirophanta brunneannulata sp.nov., Oneirophanta idsseica sp.nov.and Oneirophanta lucerna sp.nov.can be separated from other congeners by ossicle types, the arrangement and the number of dorsal papillae and tube feet.The separations were confirmed by the p-distance analyses, which showed that the uncorrected p-distance for the COI among O. brunneannulata sp.nov.and other congeners was 8.2-13.1%;among O. idsseica sp.nov.and other congeners was 8.2-14.1%,and among O. lucerna sp.nov.and other congeners was 12.1-14.8%.These divergences were much higher than the known intraspecific variation in Oneirophanta.spp.(0-0.6%)(Suppl.material 1) and, thus, this warranted separation of O. brunneannulata sp.nov., O. idsseica sp.nov., and O. lucerna sp.nov.from other congeners.
The phylogenetic trees (Fig. 12) showed that O. idsseica sp.nov.clustered together with Oneirophanta stet.CCZ_100 from Clarion-Clipperton Zone, which was deposited in the Natural History Museum, London (voucher number: CCZ_100).Generally, taxonomic units with sequence differences of < 2% are likely to be the same species, and differences > 5% were confidently used to separate different species (Ward et al. 2008).Because the COI p-distance between the two species was 0.6% (Suppl.material 1), the divergences fell within the range of general intraspecific variation; both morphological characters and molecular data suggested that O. idsseica sp.nov.and O. stet.CCZ_100 are the same species.

Geographic distribution of deimatid species
There are a total of three genera and 16 species in the family Deimatidae, which include the three species that we described here.Ocean (New Zealand), Orphnurgus glaber Walsh, 1891 from the central and western Pacific Ocean, Orphnurgus protectus (Sluiter 1901) and Orphnurgus bacillus Cherbonnier & Féral, 1981 from the western Pacific Ocean (Celebes Strait and Philippines), and Orphnurgus vitreus (Fisher 1907) from the North Pacific Ocean (off Hawaiian Islands).On a vertical scale, all species in this genus inhabited the water from relatively shallow depths to the bathyal zone (depth 174-1301 m).Among the six species, which included the three new species of Oneirophanta, Oneirophanta mutabilis, the type species of the genus, is a cosmopolitan species (Hansen 1975).O. conservata was the only species recorded from the Indian Ocean (Koehler and Vaney 1905).All the other species were found mainly in the Pacific Ocean: O. setigera from the southern and eastern Pacific (Kermadec Trench and Gulf of Panama), O. mutabilis mutabilis is a cosmopolitan species with multiple records in the Pacific Ocean (Hansen 1975), and O. mutabilis affinis is restricted to a relatively small area in the eastern Pacific Ocean.The three new species were from the western Pacific Ocean (South China Sea).In addition, O. conservata has the shallowest record of the genus (depth 1315 m), and O. mutabilis has the deepest record (depth 6000 m).Oneirophanta was distinguished from the other two genera by its inhabitance in the bathyal-abyssal zone.
Based on their distribution, deimatid holothurians are abundant in the Pacific Ocean and inhabit a wide range of depths (174-6000 m).Future expeditions to the Pacific zone may discover even more species, and more research is needed to evaluate the species diversity and geographic distribution of these deep-sea holothurians.

Figure 1 .
Figure 1.Sampling sites for examined species of Deimatidae in the South China Sea and the Mariana fore-arc area.

Figure 12 .
Figure 12.Maximum likelihood (ML) and Bayesian inference (BI) trees based on concatenated 16S-COI sequences showing phylogenetic relationships among deimatid species.The new sequences provided in this study are in bold A ML tree, with bootstrap replications labeled B BI tree, with posterior probability labeled.

Table 1 .
List of GenBank accession numbers for all specimens used in this study.