1 INTRODUCTION

Holothurians (Echinodermata, Holothuroidea) are one of the most common benthic fauna on the deep-sea seafloor, widely distributed among all oceans, and occur in various marine environments (Hansen, 1975; Billett, 1988; Gebruk, 1990; Tyler et al., 1996; Rogacheva et al., 2009; Gebruk et al., 2010). At present, there are more than 1 700 described species of holothurians in the world (Paulay and Rogacheva, 2022), and echinoderm species are most widely distributed in the Indo-West Pacific Ocean (Yang et al., 2016).

The order Elasipodida and the family Psychropotidae were established according to the reports of the Challenger Expedition (Théel, 1882). Despite a comprehensive and systematic revision of Psychropotidae by Hansen (1975), the family was still least-known by the taxonomists (Rogacheva et al., 2009). Currently, Psychropotidae includes three valid genera: Psychropotes, Benthodytes, and Psycheotrephes, all erected by Théel (1882).

Benthodytes (Echinodermata, Holothuroidea, Elasipodida) differs from the other two genera in a dorsal anus, soft and pliable tentacle discs and circum-oral papillae. Eight species of Benthodytes were described by Théel (1882). Among them, three were accepted after several revisions by taxonomists: Benthodytes abyssicola Théel, 1882, Benthodytes sanguinolenta Théel, 1882, and Benthodytes typica Théel, 1882, of which B. typica was used as the type species to describe this genus. Subsequently, Ludwig (1893), Perrier (1896), Sluiter (1901a), and Koehler and Vaney (1905) described four new species. Hansen (1975) described eight species, including two new species. After that, there were few reports on the taxonomy of Benthodytes. In recent years, more new species have been discovered (Gebruk, 2008; Rogacheva et al., 2009; Martinez et al., 2014; Li et al., 2018; Xiao et al., 2018; Yu et al., 2021, 2022). To date, a total of 15 valid species from Benthodytes have been recorded (WoRMS, 2022), including Benthodytes abyssicola Théel, 1882; B. typica Théel, 1882; B. sanguinolenta Théel, 1882; B. incerta Ludwig, 1894; B. lingua Perrier R., 1896; B. sibogae Sluiter, 1901; B. superba Koehler & Vaney, 1905; B. plana Hansen, 1975; B. valdiviae Hansen, 1975; B. gosarsi Gebruk, 2008; B. wolffi Rogacheva & Cross in Rogacheva et al., 2009; B. violeta Martinez, Solís-Marín & Penchaszadeh, 2014; B manusensis Xiao, Li & Sha, 2018; B. marianensis Li, Xiao, Zhang & Zhang, 2018, and B. jiaolongi Yu, Zhang, Zhang & Wang, 2022. Most of them are distributed around the tropical Western Pacific Ocean, South Indian Ocean and Atlantic Ocean, and are usually found at depths of 600–8 000 m (Théel, 1882; Ludwig, 1894; Perrier, 1896; Sluiter, 1901a, b; Koehler and Vaney, 1905; Hansen, 1975; Gebruk, 2008; Rogacheva et al., 2009; Li et al., 2018; Xiao et al., 2018).

There are about 30 000 seamounts in the world (Yesson et al., 2011). Seamounts are natural obstacles for currents and have great influence on the variable hydrological environment. The environment changes will further affect the biological community, then relatively independent marine ecosystems are formed around them (Dower and Mackas, 1996; Genin, 2004; Clark et al., 2010). Numerous seamounts are scattered in the Western Pacific Ocean, one of the most oligotrophic oceans on Earth. During the cruise of R/V Kexue in June 2019, some holothurian specimens were obtained. Based on morphological and molecular biology methods, a psychropotid species was identified as new to science: Benthodytes tetrapapillata sp. nov. This study and recent studies suggest a high probability that species of Benthodytes and its allies are around the Western Pacific Ocean deep-sea seamounts (Li et al., 2018; Xiao et al., 2018, 2020; Yu et al., 2021, 2022).

2 MATERIAL AND METHOD 2.1 Sampling and morphological observation

The specimen was collected at a depth of 2 289 m by the Remotely Operated Vehicle Faxian, during the cruise of R/V Kexue in the tropical Western Pacific in June 2019. It was fixed and preserved in 4% buffered formaldehyde for better preservation of morphological details, except that some tissues obtained from fresh material were stored at -80 ℃. The holotype is deposited in the Marine Biological Museum (MBM) of the Chinese Academy of Sciences (CAS) at the Institute of Oceanology, CAS, in Qingdao, China, photographed by Canon EOS 6D camera before fixation. The identification of the holotype is mainly based on typical characters of psychropotid holothurians (Hansen, 1975). Features of external morphology were examined on the basis of images in situ. Small pieces of body tissues (after formaldehyde fixation) were digested in 15% sodium hypochlorite solution to obtain the deposits which were then examined by scanning electron microscope (Hitachi S-3400N).

2.2 PCR amplification and data analysis

Total genomic DNA was extracted from a small pieces of 20–30-mg holothurian muscle tissues (stored at -80 ℃) using OMEGA Mollusc DNA Kit (Omega Biotek Inc., America) according to the manufacture' s guidance. Mitochondrial cytochrome c oxidase I (COI) and 16S rRNA were generated for Benthodytes tetrapapillata sp. nov. using primers and methods as outlined in Miller et al. (2017). PCR amplification program was as follows: initial denaturation for 3 min at 98 ℃, followed by 40 cycles of 10 s at 98 ℃, 10 s at 52 ℃, 10 s at 72 ℃, and a final extension for 5 min at 72 ℃, with a total reaction volume of 50 μL: 1–3-μL template DNA, 25-μL T5 PolStar DNA Polymerase (Tsingke, Beijing, China), 8–10-μL DNase free ddH2O, 2-μL each primer. Then the sequence chromatograms was checked using CHROMAS 2.23 (Technelysium Pty Ltd.). The forward and reverse sequences were assembled by CONTIG EXPRESS (a component of Vector NTI Suite 6.0, Life Technologies, Carlsbad, California).

Two partial sequences (COI and 16S) were obtained from holotype of the new species and were deposited in GenBank (for accession numbers see Table 1). In addition, some relevant sequences retrieved from GenBank were used for phylogenetic analyses (Table 1). Two species of genus Peniagone were used to root the tree. Sequences were independently aligned using the MAFFT (Katoh et al., 2019). The alignments were further manually quality checked and corrected based on visual observation. Two genes from the same individual were concatenated into one single alignment (1 255 bp) using software SequenceMatrix v1.8 (Vaidya et al., 2011) for combined analysis.

The maximum likelihood analysis was constructed using RAxMLGUI v1.5 with a rapid bootstrap likelihood analysis (Silvestro and Michalak, 2012), under the GTRGAMMAI substitution model. Bayesian inference (BI) was conducted using the software MrBayes v. 3.1 (Ronquist and Huelsenbeck, 2003) and the optimal DNA substitution model for each partitioned dataset in the BI trees was estimated by the jModelTest 2.1.4, calculated by the Akaike information criterion (AIC) (Darriba et al., 2012), and GTR+G+I model was the most appropriate model for all partitions in the analysis based on a concatenated dataset and single COI gene. The Markov Chain Monte Carlo iterations were run for 1 000 000 generations, with sampling every 100 generations. The first 25% of trees were discarded as burn-in and the remaining trees were summarized in 50% majority rule consensus tree.

3 RESULT 3.1 Systematics

Class Holothuroidea Blainville, 1834

Order Elasipodida Théel, 1882

Family Psychropotidae Théel, 1882

Genus Benthodytes Théel, 1882

Benthodytes tetrapapillata sp. nov. (Figs. 1–4)

Type material examined

Holotype: 1 adult specimen, catalog number MBM286931, GenBank accession numbers MZ889521, MZ891643, collected on June 9, 2019 from the station FX-Dive 221 (10°02′54″N, 140°08′55″E) on a seamount (named unofficially as "M5") located on the Caroline Ridge in tropical Western Pacific, undersea muddy sediment, 2 289 m.

Type locality

M5 seamount located on Caroline Ridge, tropical Western Pacific Ocean, depth 2 289 m.

Etymology

The Latin word tetrapapillata refers to four pairs of distinct papillae on dorsum.

Diagnosis

Body elongated, dorsum inflated, ventrum flattened, tapered slightly at both ends. Skin dark violet, thick and gelatinous, numerous warts scattered on the surface. Mouth ventral, circum-oral papillae not visible. Tentacle 12. Anus dorsal. Brim broad with clearly visible violet channels. Four pairs of well-developed dorsal papillae, large and conical, tapering to distal extremity, arranged in two single rows along the dorsal radii, evenly distributed. Midventral tube feet present throughout length of the ventral sole. Calcareous ring not visible. Dorsal deposits cross-shaped, arms 200–400 μm long, central apophysis undivided, bipartite, rudimentary, tripartite and tetrapartite. Rods present in ventrum and tentacles, up to 970 μm long.

Description of the holotype

Holotype in situ 32 cm long, 16.5 cm wide (Fig. 1a & b), and about 27 cm in length, 6.5 cm in width after preservation in 4% formaldehyde solution for several days (Fig. 1c–d). Color uniformly dark violet both dorsally and ventrally. Anus dorsal. Skin thick covered with warts, soft and gelatinous. Brim broad and flat, but slight shrinkage occurred after being fixed a few days. Four pairs of dorsal papillae rather big and obvious, arranged in two single rows, distributed regularly along the dorsum, distance between each pair of papillae is approximately the same (Figs. 1a, c, & 2a). The first and fourth pairs are close to the anterior and posterior body ends, respectively; the second and third pairs are larger than the first and fourth pairs, and each papillae in situ is about 10 cm long. Mouth ventral. Tentacles 12, retracted to form the disc which is soft and pliable (Fig. 2b). Midventral tube feet minute and arranged in alternating double row throughout whole length of ventrum (Figs. 1d & 2b). Calcareous ring not visible.

Dorsal deposits crosses with slender and arcuate arms, arms about 200–400 μm in length, bearing many small irregular spines close to tips. Central apophysis with numerous spines, including presence of five main types: (1) undivided and very long, about 170–295 μm (Fig. 4d–e); (2) bipartite, about 130–160 μm in length, usually bifurcating from half position (Figs. 3a–d & 4a–b); (3) rudimentary (Fig. 3e); (4) tripartite (Fig. 3f); (5) tetrapartite, about 174 μm long (Fig. 4c). Deposits of dorsal papillae crosses, central apophysis 156–183 μm in length, containing three types: undivided, bifurcated, and tripartite, bifurcating from a quarter position from the top (Fig. 4f & h). Both ventral and tentacles deposits rod-shaped, with a length of 230–970 μm, slightly curved into two types: (1) small spines at both ends, center smooth (Figs. 3g–i & 4k); (2) an extra bifurcation in the center (Figs. 3j & 4k). Tube feet deposits crosses, with bifurcated central apophysis about 165–217 μm in length (Fig. 4i–j), bearing fewer spines compared to that of dorsal papillae (Fig. 4f & h).

Relationship

Benthodytes tetrapapillata sp. nov. closely resembles Benthodytes gosarsi Gebruk, 2008 in presence of four pairs of evenly distributed dorsal papillae. For external features, the new species differs from B. gosarsi in body color, the former is dark violet (Fig. 1) while the latter is green which is unique among psychropotid holothurians (normally violet). Meanwhile, dorsal papillae of B. tetrapapillata sp. nov. are relatively larger and more developed than those of B. gosarsi. For internal deposits morphology, B. gosarsi has dorsal crosses with a bipartite central apophysis, while B. tetrapapillata sp. nov. has dorsal crosses with varying types of central apophysis (Figs. 3a–f & 4a–e). In the original description, deposits are entirely lacked on ventrum in B. gosarsi, while rods present ventrally in B. tetrapapillata sp. nov. (Fig. 3g–j).

Distribution

Only in the type locality.

3.2 Molecular phylogenetic analyses

The molecular phylogenetic trees (ML and BI) reconstructed based on a total of concatenated data were shown in Fig. 5, and trees based on COI gene were provided in Supplementary Fig.S1. All trees showed that the genus Benthodytes was not monophyletic, as some species of genera Psychropotes and Psycheotrephes were nested within the genus Benthodytes, suggesting the classification of family Psychropotidae need to be redefined. The two consensus trees using ML and BI criteria based on concatenated data were generally congruent (Fig. 5), which showed that Benthodytes tetrapapillata sp. nov. clustered together with Benthodytes manusensis forming a well-supported clade (BS=95, BPP=1), sister to Benthodytes sibogae and Benthodytes palauta (BS=99, BPP=1). The phylogenetic trees inferred using ML and BI criteria based on single gene COI (Supplementary Fig.S1) showed similar overall topology. These results support the separation of Benthodytes tetrapapillata sp. nov. from other congeners.

4 DISCUSSION

State of main morphological characters in valid species of Benthodytes is provided in Table 2. Benthodytes was divided into two distinct groups based on the details of external and deposits morphology (Hansen, 1975; Yu et al., 2022). Benthodytes tetrapapillata sp. nov. belongs to the group characterized by cross-shaped deposits and well-developed dorsal papillae. The morphological identification was supported by molecular evidence. The phylogenetic trees (Fig. 5; Supplementary Fig. S1) revealed a congruent topology of Benthodytes, which is divided into two clades, verifying that Benthodytes tetrapapillata sp. nov. is a valid species within the Benthodytes lineage.

Phylogenetic analyses indicated that the new species was closely related to Benthodytes manusensis, with high support (16S-COI tree: BS=95, BPP=1; COI tree: BS=94, BPP=1). From a morphological point of view, both B. manusensis and the new species have 12 tentacles, big and conspicuous papillae which arrange in two single rows and evenly distributed (Table 2). In deposits morphology, the two species both have primary crosses with four arms and a central apophysis on dorsum (Table 2). However, the new species differs from B. manusensis in the number and position of dorsal papillae. B. tetrapapillata sp. nov. has four pairs of papillae, while B. manusensis only has three pairs. The position of the first pair of large papillae of B. tetrapapillata sp. nov. is approximately in the front quarter of the body (Fig. 2b), while that of B. manusensis is very close to the anterior end which is really unique among psychropotid species. Moreover, for dorsal deposits, B. tetrapapillata sp. nov. has crosses with undivided, rudimentary, tripartite and tetrapartite apophysis (Figs. 3e, f, & 4c–e) besides the bipartite apophysis of B. manusensis. On ventrum, B. tetrapapillata sp. nov. has rods (Fig. 3g–j) while B. manusensis has smaller crosses.

The molecular phylogenetic trees (Fig. 5; Supplementary Fig.S1) of the family Psychropotidae appear to be topologically similar and well in agreement with previous studies (Li et al., 2018; Xiao et al., 2018; Yu et al., 2021). Benthodytes was divided into two clades: Benthodytes sanguinolenta and B. jiaolongi grouped in one clade; B. marianensis, B. sp. SB-1-11, B. manusensis, B. sibogae, B. palauta, and the new species grouped in the other one clade. Since two groups also show great differences in morphological characters: the species in former group are characterized by mostly reduced rod deposits and minute dorsal papillae, while the species in later group are characterized by the regular crosses and well-developed dorsal papillae, it suggests that the species in former group may form a new genus under the family Psychropotidae, but the hypothesis cannot be verified due to the limited number of molecular data and specimens, and more studies need to be supplemented for a more complete taxonomic system. What' s more, further sequence data of Psychropotidae is needed to resolve the placement of B. tetrapapillata sp. nov. relative to other Benthodytes species. Sequences obtained in this study will enrich the database and provide more data to clarify the position of the genus Benthodytes in the taxonomy.

5 CONCLUSION

Phylogenetic analyses revealed the separation of Benthodytes tetrapapillata sp. nov. from congeners of genus Benthodytes, and supported the morphological identification that Benthodytes tetrapapillata sp. nov. was a new psychropotid species. The new species is characterized by four pairs of rather large and evenly distributed papillae, and crosses with varying types of central apophysis. Benthodytes tetrapapillata sp. nov. is the first species of order Elasipodida reported on a seamount (M5) located on the Caroline Ridge. The information about this new species can be significantly useful in expanding the diversity of the psychropotid species in deep-sea seamounts.

6 DATA AVAILABILITY STATEMENT

The data generated and analyzed to support the research result can be provided by the corresponding author upon reasonable request.

7 ACKNOWLEDGMENT

The authors are extremely grateful to the assistance of the crew of ROV Faxian for sample collection and in-situ pictures shooting. Many thanks to the members of Department of Marine Organism Taxonomy & Phylogeny at the Institute of Oceanology, CAS (IOCAS) for their help in sample collection and analysis. We are grateful to three referees for their constructive comments.

Electronic supplementary material

Supplementary material (Supplementary Fig.S1) is available in the online version of this article at https://doi.org/10.1007/s00343-022-2220-0.