A new species of holothuroid, Pseudothyone labradorensis sp. nov. (order Dendrochirotida and family Sclerodactylidae), was discovered off the coast of Labrador (eastern Canada) at a depth of 740–969 m. Two specimens were described based on morphological and genetic parameters. Distinctive characters included pinkish body colour, presence of tube feet on a ‘tail’, supporting rod-shaped ossicles in the tube feet, and rod-shaped ossicles in the tentacles. To investigate its phylogenetic relationships, partial sequences of COI were obtained for the new species as well as for the type species P. raphanus and another North Atlantic species P. serrifera. According to the phylogenetic analysis, P. labradorensis sp. nov. appeared in a well-supported clade with P. raphanus and P. serrifera. Molecular data also suggest polyphyly of the genus, showing the Northeast Pacific species Pseudothyone belli recovered outside of the clade containing the type species. Pseudothyone labradorensis sp. nov. is the first species of the genus from the Northwest Atlantic. A key to the North Atlantic Pseudothyone is provided.

Bathyal fauna, distribution, Northwest Atlantic fauna, sea cucumbers, taxonomy

The genus Pseudothyone was established by Panning (1949) for Thyone raphanus Düben & Koren, 1846 within the new subfamily Sclerodactylinae. Apart from the type species, Panning assigned five other species to this genus: P. belli (Ludwig 1886), P. mosaica (Koehler & Vaney, 1910), P. poucheti (Barrois, 1882), P. argillacea (Sluiter, 1910), P. buccalis (Stimpson, 1855) and P. trachyplaca (Clark, 1924). The latter two species were later assigned to other genera, whereas P. poucheti was synonymized with P. raphanus according to Théel (1886), and P. argillacea with P. belli according to Deichmann (1954).

Pseudothyone currently includes seven species characterized by a wide range of morphological characters. The original diagnosis by Panning (1949) included the following: ten tentacles, undivided pieces of calcareous ring and its radial pieces with two fork-shaped processes of medium length undivided or consisting of few large pieces, and body-wall ossicles composed of only plates. Later investigations of this genus added more variations to taxonomic characters (Lambert and Oliver 2001; Martins 2019): tentacles ten equal-sized (in P. levini Lambert & Oliver, 2001) or eight bigger and two ventral smaller in all other species; body-wall ossicles smooth plates and knobbed buttons (P. belli), or smooth plates only (all other species); tube feet ossicles tables and end plates (P. belli and P. mosaica), rods and end plates (P. sculponea Cherbonnier, 1958, P. serrifera (Östergren, 1898) and P. levini), or end plates only (P. raphanus and P. furnestini Cherbonnier, 1969); radial pieces of calcareous ring with short posterior processes (P. levini), or of medium length (all other species), with posterior processes undivided (P. raphanus), or divided into several pieces (P. belli, P. mosaica, P. sculponea).

Most species of Pseudothyone are distributed in the Atlantic Ocean (Fig. 1). Pseudothyone furnestini, P. raphanus, and P. serrifera occur in the Northeast Atlantic, and the latter two are also known from the Mediterranean. Pseudothyone sculponea is known only from the Mediterranean, and P. belli is from the Western Atlantic (Atlantic US coast, Caribbean and Brazil). Moreover, two species are known outside the Atlantic: P. mosaica from the Persian Gulf, and P. levini from the northeastern Pacific. Bathymetric distribution also differs between the species. The shallowest is P. belli occurring at sublittoral depths from the low-tide mark to 37 m (Pawson et al. 2010). Among shallow-water representatives are also P. levini occurring from the intertidal to 70 m depth and P. sculponea reported from 21 to 41 m (GBIF.org 2023). Pseudothyone raphanus and P. serrifera occur deeper, at 10–1200 m and 200–1200 m, respectively (Madsen and Hansen 1994; Fernández-Rodríguez et al. 2019). The deepest-dwelling species is P. furnestini reported from 440–1347 m (Fernández-Rodríguez et al. 2019). Pseudothyone mosaica is known from a single record at 97 m.

Figure 1. 

Distribution of Pseudothyone labradorensis sp. nov. and other species in the same genus, based on published records and GBIF (2023). Map was prepared using QGIS 3.16.5-Hannover.

Phylogenetic relationships of species within the genus Pseudothyone, as well as the latter’s position within Sclerodactylidae, remain unclear. The taxon was originally described by Panning (1949) as Sclerodactylinae, a subfamily of Cucumariidae. Later, Pawson and Fell (1965) upgraded its status to the family Sclerodactylidae with two subfamilies, Sclerodactylinae and Cladolabinae. Thandar (1989) restricted the diagnosis of Sclerodactylidae and described a new subfamily, Sclerothyoninae. Smirnov (2012) recognized the subfamilies Cladolabinae and Sclerothyoninae as separate families. Available molecular data (Miller et al. 2017) partly support the system of Smirnov, showing no sister relationships between sclerothyonins Afrocucumis africana (Semper, 1867) and Euthyonidiella huwi O’Loughlin in O’Loughlin et al., 2014 and sclerodactylins Pachythyone rubra (Clark, 1901) and Sclerodactyla briareus (Lesueur, 1824).

In this study we describe a new species, Pseudothyone labradorensis sp. nov., from the bathyal depths of the Labrador Sea (Northwest Atlantic Ocean) based on morphological and molecular data. Using molecular data on partial sequences of the mitochondrial gene cytochrome c oxidase subunit I (COI), we examined phylogenetic relationships of P. labradorensis sp. nov. with two Atlantic congeners, P. raphanus (type species of the genus) and P. serrifera, for which we obtained additional genetic data, as well as with the Northeast Pacific species P. levini. COI is commonly used for recovering relationships within the genera of Holothuroidea (O’Loughlin et al. 2014; Li et al. 2018; Ogawa et al. 2022; Ogawa et al. 2023). To test monophyly of Pseudothyone we included available data on the representatives of Sclerodactylidae sensu Smirnov (2012) as well as Pentamera calcigera (Stimpson, 1851), as close relationships of Pentamera to Sclerodactylidae has been shown previously (Arndt et al. 1996; Miller et al. 2017).

Two specimens of Pseudothyone labradorensis sp. nov. were collected together in the same location (North Atlantic Fisheries Organization NAFO, Zone 2J https://www.marineregions.org/gazetteer.php?p=details&id=23382) using a rock dredge deployed during the ISECOLD scientific expedition aboard the research icebreaker CCGS Amundsen on 30 August 2020 (Table 1). The rock dredge net had a 7 mm mesh. Due to the large volume of mud collected at this station, a fourth of the material was sieved through a 2 mm sieve and the rest was scanned for larger individuals through a 17 mm diameter mesh sieve. The specimens later identified as Pseudothyone were preserved in 100% ethanol for morphological and molecular analysis.

The specimens of P. raphanus and P. serrifera had previously been collected in 2006–2016 with a Van Veen grab, Triangular dredge and Agassiz trawl deployed from the research vessels R/V Gunnerus, R/V Håkon Mosby and R/V Hans Brattström (Table 1). They were preserved and subsequently stored in 96% ethanol.

Morphological examination, dissection and photographing were performed using a Leica M205C stereomicroscope equipped with a Leica FLEXACAM C1 digital camera. To extract ossicles, small fragments of the body wall, introvert, tube foot and tentacle skin were digested in a domestic bleach water solution followed by several rinses in distilled water. For light microscopy, ossicles were transferred onto a glass slide, dried using a heating stage and mounted in Canada Balsam. For scanning electronic microscopy (SEM), ossicles were dried with 96% ethanol, mounted on a stub and sputter coated with gold. Ossicles were examined and photographed under a light microscope (Olympus BX43) with a ToupCam U3CMOS08500KPA digital camera and SEM examination was performed using a TESCAN Vega 3.

To evaluate phylogenetic relationships of Pseudothyone labradorensis sp. nov. within the genus, partial sequences of cytochrome c oxidase, subunit I (COI) were obtained from two examined specimens. Additionally, COI data were obtained for three specimens of P. raphanus and seven specimens of P. serrifera (Table 1). Some of these specimens were collected close to their type localities (Bergen area and Trondheimsfjorden, respectively). Also, three sequences of Pseudothyone, publicly available in GenBank, were used in the analysis: P. raphanus (MG934913) and P. levini (MH242951 and MH242950). To test monophyly of Pseudothyone, published sequences of Sclerodactylidae sensu Smirnov (2012) were analysed (Table 2). A COI sequence of Pentamera calcigera (Phyllophoridae) was also included in the analysis as it was recovered in a sister clade to the sclerodactylid Pachythyone rubra (Miller et al. 2017). Stichopus horrens was set as an outgroup. GenBank accession numbers of the sequences used in the analysis are listed in the Table 2.

Molecular work was carried out in two laboratories applying two different protocols. Genetic data on Pseudothyone labradorensis sp. nov. (voucher AMLAB-02), P. raphanus and P. serrifera were obtained at the Canadian Centre for DNA Barcoding, University of Guelph following protocols by Ivanova et al. (2006), Ratnasingham and Hebert (2007), and deWaard et al. (2008). Data on another specimen of P. labradorensis sp. nov. (voucher PT1210) was generated at IORAS using the following methods: DNA was extracted using QuickExtract^TM DNA Extraction Solution (Lucigen) following the manufacturer protocol; PCR amplification was conducted using Encyclo Plus PCR kit (Evrogen, Moscow) according the manufacturer protocol with annealing temperature set at 48 °C; PCR products were purified from agarose gel using HiPure Gel DNA Mini Kit (Magen); the purified samples were sequenced using the Sanger method on Applied Biosystems ABI 3900 (ThermoFisher Scientific) by Evrogen (Moscow, Russia). All PCR amplifications and sequencing were carried out using the LCOech1aF1 (5′-TTTTTTCTACTAAACACAAGGATATTGG-3′; D. Eernisse unpublished) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′; Folmer et al. 1994) primers.

Contigs were assembled from forward and reverse chromatograms using the MUSCLE algorithm implemented in Geneious v.10.0.9 and then manually edited. Sequences were aligned in MEGA 7 (Kumar et al. 2016) also using the MUSCLE algorithm, and then checked for stop-codon presence. The final dataset included 840 aligned positions. Phylogenetic analysis was performed using maximum-likelihood (ML) and Bayesian inference (BI) approaches. PartitionFinder 2 (Lanfear et al. 2017) was used for selecting best-fit partitioning schemes and models of nucleotide evolution. The defined models were TRNEF+I+G for positions 1 and 2, and GTR+G for position 3. ML tree search and bootstrapping was conducted in RAxML-NG (Kozlov et al. 2019) using auto MRE option with cutoff=0.03; the analysis converged after 12950 replicates. BI analysis was performed using MrBayes v.3.2 (Ronquist et al. 2012). The analysis was conducted in two runs, four chains (one cold and three heated) with trees and parameters sampled every 500 generations. The traces were analysed in Tracer v.1.7.1, and then 10% of the trees were discarded as burn-in. Run convergence was evaluated by analysing sump output parameters in MrBayes log file and Tracer v.1.7.1. Genetic distances were calculated using Kimura 2-parameter model (K2P; Kimura 1980) implemented in MEGA 7.

Molecular data supported close relationships of Pseudothyone labradorensis sp. nov. to the type species of the genus, P. raphanus, and to the Northeast Atlantic species P. serrifera. According to body-wall ossicle morphology, P. labradorensis sp. nov. is most closely related to P. raphanus (Fig. 5A), from which it differs by its pinkish body colour, presence of tube feet on the ‘tail’, absence of fern-like ossicles on the tentacles, and by the presence of rod-shaped ossicles in the tube feet. From the genetically closest species, P. serrifera (Fig. 5B), the new species differs by the pinkish body colour, absence of S-shaped rods on tentacles, and by the smooth margins and perforations of body-wall ossicles. From another Northeast Atlantic species, P. furnestini, the new species differs by body colour and body-wall ossicles. Pseudothyone furnestini is characterized by whitish colouration and thick body-wall ossicles that are from rounded to oval in shape and often possess solid, unperforated extensions. Also Pseudothyone furnestini lacks rod-shaped ossicles in the tube feet.

Figure 5. 

Pseudothyone raphanus (A) and P. serrifera (B) collected from the type locality areas. A ZMBN 120547 B NTNU-VM-72187. Collection data is given in Table 1. Image courtesy of Katrine Kongshavn.

According to molecular data, Pseudothyone levini was not closely related to the species of the raphanus clade. This species has ten tentacles that are equal in size, whereas other species of Pseudothyone have two ventral tentacles smaller than others. From species of the raphanus clade, it also differs by a less prominent ‘tail’, less perforated body-wall plates and by more robust rod-shaped ossicles on tube feet.

Some other species of Pseudothyone also have remarkable morphological differences. Apart from plates, the body-wall ossicles of P. belli include knobbed buttons and plates with handles, which do not occur in P. raphanus and most other species of the genus. Also P. belli differs by the ossicles from the tentacles, which are plates and rosettes (not rods as in P. raphanus and other species), by introvert ossicles pillared tables and plates, and by tube feet ossicles arched pillared tables. Therefore, P. belli differs remarkably from P. raphanus in most ossicle types. Marked differences in ossicle types are also noted for P. mosaica. This species is characterized by large rounded plates on the body wall and arched pillared tables on its tube feet.

The present results suggest that the taxonomy of Pseudothyone requires further investigation. Particularly, the generic affiliation of P. levini, P. belli and P. mosaica may require additional evaluation. The taxonomic position of the genus also remains unclear. Based on COI data, the raphanus clade (P. raphanus + P. serrifera + P. labradorensis sp. nov.) does not form any well-supported clade with other examined representatives of Sclerodactylidae sensu Smirnov (2012). More data, both morphological and molecular, are needed to analyze the phylogenetic relationships of Pseudothyone.

Scientific collections of fauna in the Labrador Sea are extremely limited so the distribution of this species is yet to be resolved. Pseudothyone labradorensis sp. nov. is known from a single locality in the Labrador Sea, at a depth between 740–969 m. Metabarcoding surveys of the ISECOLD transects in the Labrador Sea (Côté et al. 2023) did not detect this species but several other holothuroids were detected over benthic habitats at a depth of 1026 m about 300 km to the north (Côté unpubl. data): Chiridota laevis (O. Fabricius, 1780), Benthogone sp., Enypniastes sp., Psolidae gen. sp., and two unresolved species from the orders Dendrochirotida and Apodida.

Apart from P. labradorensis sp. nov., three more species of Pseudothyone are known from bathyal depths. The type species P. raphanus and another Northeast Atlantic species, P. serrifera, were reported down to 1200 m (Fernández-Rodríguez et al. 2019), with most of their records obtained shallower than 250 m and 600 m, respectively. The deepest species of the genus, P. furnestini, occurred between 440 and 1347 m (ibid.). According to McKenzie (1991), the body colour in P. raphanus can be “sometimes a pale pink”. Combined with personal unpublished observations of pinkish specimens of P. cf. raphanus off south Iceland, it is possible that P. labradorensis sp. nov. may have a wider distribution in the North Atlantic.

We would like to thank Kaitlin Casey, as well as the researchers and crew aboard the CCGS Amundsen for their help during the collections. We would also like to thank Torkild Bakken at the NTNU University Museum for arranging the workshop at Sletvik Field Station and the cruise time on R/V Gunnerus from which our specimens of P. raphanus and P. serrifera from Trondheimsfjorden were collected in 2016. We are grateful to Gustav Paulay and Francisco Alonso Solís Marín whose valuable comments helped to improve the manuscript. We are also grateful to Alexander Filippov (IORAS) for technical assistance in SEM observation, and to Katrine Kongshavn for providing photographs of P. raphanus and P. serrifera.