Revisiting the deep-sea Atlantic gastropod species Scaphander gracilis Watson, 1883 (Gastropoda: Cephalaspidea: Scaphandridae): first data on its anatomy, systematics, and ecology

ABSTRACT

Scaphander gracilis Watson, 1883 is a deep-sea Cephalaspidea gastropod species endemic to the Azores in the mid-North Atlantic, and which was up to now only known from empty shells with a unique thick callus. Here, we describe for the first time the morphology of this snail species from anatomical dissections and provide a DNA barcode. A phylogeny based on the gene marker cytochrome c oxidase subunit I was inferred to test the position of S. gracilis among the genus Scaphander. In addition, the ABGD species delimitation method was employed and uncorrected p-distances were estimated. The sister relationship of S. gracilis was not resolved, but it clustered with the Atlantic species S. meridionalis and S. nobilis and the Pacific species S. grandis, S. mundus, and S. subglobosus. The smallest genetic distance was to the NE Pacific species S. grandis (4.29% in COI). Foraminifera were found in the digestive tract and the species is confirmed alive at depths between 2065−2066 m. The fact that S. gracilis was found on both sides of the Mid-Atlantic Ridge suggests that this significant geological phenomenon is not an insurmountable barrier for dispersal in this species.

KEYWORDS:

• Azores
• biogeography
• cephalaspidea
• mid-Atlantic ridge
• Mollusca
• phylogenetics

Introduction

The deep sea (waters and bottoms below 200 m depth) is the largest ecosystem on the planet and covers 65% of the Earth (Thistle 2003; Tyler 2003; Jobstvogt et al. 2014). It was long thought to host little or no life (Tyler 2003), but today we know that a high diversity is found in the deep oceans (Grassle and Maciolek 1992; Van Dover 2000). In fact, an increasing number of studies have investigated deep-sea biodiversity in several regions of the world, but often these studies focus on a reduced number of taxa and are typically characterised by a limited spatial scale of investigation (e.g.: regional species diversity off the eastern US coast (Etter and Grassle 1992; Grassle and Maciolek 1992); species diversity in the Mediterranean (Danovaro et al. 2010); Scaphander snails in the Atlantic (Eilertsen and Malaquias 2013a, 2015)), and overall deep-sea benthic diversity, particularly of abyssal plains, remains poorly known.

One deep-sea benthic faunal group that received considerable attention in recent years in the Atlantic Ocean was the gastropod genus Scaphander Montfort, 1810 (Eilertsen and Malaquias 2013a, 2013b, 2015; Siegwald et al., 2020). Scaphander includes 33 species worldwide (MolluscaBase 2021) that inhabit predominantly deep-sea soft bottoms where they feed preferentially upon foraminiferans, including agglutinating forms, but also bivalves, gastropods, scaphopods, and polychaetes with calcareous tubes (Eilertsen and Malaquias 2013b). Ten species are recognised in the Atlantic Ocean, seven of those have representative DNA barcodes. The species Scaphander clavus Dall, 1889 is only known from its shell and anatomy, Scaphander gracilis Watson, 1883 is only known from empty shells (Eilertsen and Malaquias 2013a; Siegwald et al. 2020) and S. imperceptus Bouchet, 1975 is only known from old material (1883) not suitable for regular DNA extraction methods.

Scaphander gracilis is a rare species endemic to the Archipelago of the Azores in the mid-North Atlantic Ocean, and was described from empty shells that have a distinctive parietal callus. Shells have been collected between 1299 and 2995 m depth, but no complete specimen including soft tissue, has ever been collected (Watson 1883; Dautzenberg and Fischer 1896; Locard 1897; Bouchet 1975; Eilertsen and Malaquias 2013a). The current work aims to contribute to a better understanding of Atlantic deep-sea molluscan biodiversity by providing the first data on the anatomy and ecology of S. gracilis. An inference of relationships for this species within its genus using molecular phylogenetics is discussed as is the biogeography of this species.

Material and methods

One specimen of S. gracilis was collected and preserved in 96% ethanol during the Meteor cruise M150, Biodiversity of the Azores (BIODIAZ) in 2018, an expedition led by the Senckenberg am Meer, German Center for Marine Biodiversity Research. The specimen was dissected for examination of the digestive and reproductive systems, which were drawn using a stereo microscope fitted with a camera lucida. The buccal bulb was dissolved in a Proteinase K solution composed of 20 μL Proteinase K and 180 μL buffer ATL obtained from the Qiagen DNeasy Blood and Tissue kit, and incubated at 56°C overnight (protocol modified from Holznagel 1998; Vogler 2013). The gut content was collected under a stereo microscope then visualised with SEM. The male reproductive system was dissected and the penial chamber was critical-point dried for SEM analysis. The shell was photographed using a DSLR camera equipped with macro lens. Details of the shell and gizzard plates were photographed using a Leica M205C stereo microscope equipped with a Leica DMC5400 camera, and images were stacked using Zerene Stacker 1.04 software (http://zerenesystems.com/cms/home). The radula, penial chamber section and gut content were coated with gold-palladium and examined with a ZEISS SUPRA 55VP electron scanning microscope (SEM) at the Department of Earth Science (University of Bergen).

DNA was extracted from foot tissue using the Qiagen DNeasy Blood and Tissue Kit following the manufacturer’s instructions. The barcoding gene marker cytochrome c oxidase subunit I (COI) was amplified and purified following the methods described by Eilertsen and Malaquias (2013a). Purified products were sequenced using an ABI 3730XL DNA Analyser (Applied Biosystems). The software programme Geneious (v. Prime 2021.1.1) was used to inspect, edit and assemble the chromatograms. The sequences were blasted in GenBank to check for potential contamination. An additional 23 COI sequences were pulled from GenBank (22 Scaphandridae and one Bulla vernicosa for outgroup). The final dataset (Table 1) was edited and aligned using MUSCLE (Edgar 2004) implemented in Geneious and the quality of the alignment was assessed by translating to proteins and checking for stop-codons. Saturation of codon positions was assessed by plotting uncorrected p-distances against total substitutions (transversions + transitions) using MEGA 10.0.1 (Kumar et al. 2018). The best-fitting model of evolution was selected using Akaike’s information criterion (Akaike 1974) implemented in jModelTest 2.1.10 (Darriba et al. 2012). A Bayesian Inference analysis was performed in MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001) with three parallel runs of five million generations and sampling every 100 generations. Convergence was assessed in Tracer 1.7.1 (Rambaut et al. 2018) for each run by plotting the likelihood against the number of generations, with a burn-in set to 10%. Tree annotation was done in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree), and adjustments were made in Adobe Illustrator CS6.

Table 1. Taxon set used for molecular phylogenetics with locality, voucher numbers, and GenBank accession numbers (* novel sequences generated for this study)

Family	Species	Seq nr.	Voucher nr.	Locality	COI
Scaphandridae	Scaphander bathymophilus	SC13	RMNH unnr.	Azores	KC351559
Scaphandridae	Scaphander bathymophilus	SC52	MZUSP 75708	Puerto Rico	KC731430
Scaphandridae	Scaphander darius	SC21	MZUSP 29016	Brazil	KC351560
Scaphandridae	Scaphander gracilis	SJ104	DBUA 1630	Between São Miguel and Santa Maria, Azores	MZ457932*
Scaphandridae	Scaphander grandis	CH207	MIMB 36540	Kurile-Kamchatka Trench, NW Pacific	MK952767
Scaphandridae	Scaphander lignarius	GB2	NHMUK 20060114	Algarve, Portugal	DQ974664
Scaphandridae	Scaphander lignarius	SC1	ZMBN 87998	Cadiz, Spain	KC731431
Scaphandridae	Scaphander lignarius	SC19	MCZ371884	Vilanova i la Geltrú, Spain	KC351561
Scaphandridae	Scaphander lignarius	SC37	ZMBN 88000	Bergen, Norway	KC351563
Scaphandridae	Scaphander meridionalis	st11	ZMBN 127881	Off Mar del Plata, Argentina	MN433681
Scaphandridae	Scaphander meridionalis	SJ36	ZMBN 127882.1	Off Mar del Plata, Argentina	MN433682
Scaphandridae	Scaphander mundus	SC29	MNHN-IM-2009-4319	Philippines	KC351565
Scaphandridae	Scaphander mundus	SC31	MNHN-IM-2009-4318	Philippines	KC731429
Scaphandridae	Scaphander nobilis	SJ67	ZMBN 127875	Azores	MN433679
Scaphandridae	Scaphander punctostriatus	SC4	MNHN-IM-2009-29695	Newfoundland	KC351566
Scaphandridae	Scaphander punctostriatus	SC36	ZMBN 88004	Skagerrak, Norway	KC351569
Scaphandridae	Scaphander sp. A	SC30	MNHN-IM-2009-4317	New Caledonia	KC351572
Scaphandridae	Scaphander sp. B	SC32	MNHN-IM-2009-4371	New Caledonia	KC351573
Scaphandridae	Scaphander subglobosus	SC33	MNHN-IM-2009-4339	Philippines	KC351574
Scaphandridae	Scaphander watsoni	SC15	USNM 1151226	Gulf of Mexico	KC351575
Scaphandridae	Scaphander watsoni	SC17	USNM 1151240	Gulf of Mexico	KC351576
Scaphandridae	Hamineobulla kawamurai	208	NHMUK 20070016	Maui, Hawaii	MH933093
Scaphandridae	Hamineobulla kawamurai	SJ103	ZMBN 88206.1	Kauai, Hawaii	MN433677
Outgroup
Bullidae	Bulla vernicosa	C21	MNHN-IM-2007-42245	Panglao, Philippines	DQ974661

The molecular species delimitation method Automatic Barcode Gap Discovery (ABGD; Puillandre et al. 2012) was used based on the alignment. The web version of the ABGD programme (available at https://bioinfo.mnhn.fr/abi/public/abgd/) was run for the three different models available (Jukes Cantor, Kimura, and Simple Distance) using default settings (Pmin = 0.001, Pmax = 0.1, 10 steps, 20 Nb bins) and the relative gap width (X) adjusted to 1.0.

Geographical distributions of known S. gracilis specimens were collected from the study of museum specimens, literature records that could confidently be attributed to the species, and newly collected material, and were plotted using R (R Core Team 2021). Shoreline data were taken from the Global Self-consistent, Hierarchical, High-resolution Geography Database (GSHHG v.2.3.7 https://www.soest.hawaii.edu/pwessel/gshhg; Wessel and Smith 1996). Mid-Atlantic Ridge data were taken from Bird (2003).

Abbreviations

DBUA: Department of Biology of the University of the Azores, Ponta Delgada, Azores, Portugal; MCZ: Museum of Comparative Zoology, Harvard University, Boston, MA, USA; MIMB: Museum of the National Scientific Center of Marine Biology, Vladivostok, Russia; MNHN: Muséum national d’Histoire naturelle, Paris, France; MZUSP, Museu de Zoologia da Universidade de São Paulo, Brazil; NHMUK: The Natural History Museum, London, UK; NMINH: National Museum of Ireland (Natural History Division), Dublin, Ireland; RMNH: National Museum of Natural History (Naturalis), Leiden, The Netherlands; USNM: National Museum of Natural History [United States National Museum], Smithsonian Institution, Washington DC, USA; ZMBN: Department of Natural History, University Museum of Bergen, Norway; H: shell height, PP: posterior probability.

Results

Taxonomy

Order Cephalaspidea P. Fischer, 1883

Family Scaphandridae G.O. Sars, 1878

Genus Scaphander de Montfort, 1810

Scaphander gracilis Watson, 1883

(Figures 1–3, Tables 1–2)

Table 2. COI uncorrected p-distances between and within Scaphander species

	S. gracilis	S. grandis	S. mundus	S. nobilis	S. meridionalis	S. subglobosus	S. punctostriatus	S. lignarius	S. watsoni	S. darius	S. bathymophilus	Scaphander sp. A	Scaphander sp. B
S. gracilis	–
S. grandis	4.29	–
S. mundus	4.47	5.55–5.90	0.36
S. nobilis	6.62	6.44	6.08	–
S. meridionalis	7.33–7.51	6.26–6.44	6.80–7.33	7.87–8.77	1.25
S. subglobosus	7.87	8.77	9.48	10.2	10.20–10.38	–
S. punctostriatus	10.73–10.91	11.09–11.27	10.73–10.91	12.34–12.52	12.34–13.06	12.70–12.88	0.18
S. lignarius	14.85–15.56	16.46	13.95–16.64	16.46–17.35	16.28–17.17	14.49–15.38	14.85–16.82	0.89–10.20
S. watsoni	12.52–13.06	14.85–15.03	13.95–14.13	14.31–14.49	15.56–16.10	12.70–12.88	14.85–15.56	12.70–15.56	0.89
S. darius	15.21	15.56	14.49	14.67	16.64–16.82	14.85	16.10–16.28	15.38–16.64	12.34–12.52	–
S. bathymophilus	14.49–15.38	16.28–16.82	15.03–15.56	16.46–17.17	16.46–16.82	14.49–15.03	13.77–14.85	14.85–16.10	14.85–15.92	14.49–16.10	5.72
Scaphander sp. A	19.14	18.78	19.68	19.5	19.50–20.04	18.6	18.60–18.78	17.53–20.04	18.25–18.43	16.28	17.17–17.35	–
Scaphander sp. B	15.38	17.53	15.74	18.25	17.35–18.07	15.92	15.74–15.92	15.56–17.35	15.38–15.56	18.25	16.28	16.28	–

Figure 1. Scaphander gracilis Watson, 1883. (a) apertural (left), adpertural (right), apical (bottom) views of shell from south off Flores Island, Azores, lectotype, NHMUK 1887.2.9.2183–6, H = 13.5 mm (images courtesy of the NHMUK photographic unit). (b) apertural (left), adpertural (right), apical (bottom) views of shell from south of São Miguel Island, Azores, paralectotype, NHMUK 1887.2.9.2187 − 8, H = 13.0 mm (images courtesy of the NHMUK photographic unit). (c) apertural (left), adpertural (right), apical (bottom) views of shell from between São Miguel and Santa Maria Islands, Azores, DBUA 1630, H = 20 mm. (d) stereo microscope image of the sculpture of the shell illustrated in C. Scale bar = 1 mm.

Figure 2. Anatomical details of Scaphander gracilis Watson, 1883 (DBUA 1630, H = 20 mm). (a) radula; (b) rachidian tooth; (c) denticulation of the inner edge of lateral teeth; (d) anterior part of digestive tract; (e) gizzard plates, inner side of the paired plates and lateral view of the unpaired plate; (f) male reproductive system; (g) lining of the penial chamber. bb, buccal bulb; c, crop; go, genital opening; m, mouth; o, oesophagus; p, prostate; pc, penial chamber; pd, prostatic duct; pgp, paired gizzard plates; sg, salivary gland; ugp, unpaired gizzard plate. Scale bars: A, G = 200 µm; B, C = 20 µm; D, F = 1 mm; E = 2 mm.

Figure 3. Scanning electron micrographs of foraminifera taken from the gut content of Scaphander gracilis (DBUA 1630, H = 20 mm). (a–b) agglutinating foraminifera; (c−j) calcareous foraminifera. Scale bar = 1 mm.

Scaphander gracilis Watson, 1883: 345−346; Watson, 1886: 645−646, pl. 47, figs 4a–c; Pilsbry, 1893: 247−248, pl. 31, figs 19−20; Dautzenberg & Fischer, 1896: 402; Locard, 1897: 47–49, pl. 1, figs 15–18; Bouchet, 1975: 338, pl. 3, fig. i; Eilertsen & Malaquias, 2013a: 404−405, figs 2.10; 14.

Figure 4. Bayesian phylogenetic tree based on partial sequences of the COI gene. Figures on nodes are posterior probabilities, scale bar refers to branch lengths. The tree was rooted using the species Bulla vernicosa.

Scaphander gracilis var. minor Locard, 1897: 47–49.

Scaphander gracilis var. major Locard, 1897: 47–49.

Type material

Western Azores, south of Flores Island (38°30ʹN, 31°14ʹW; 1829 m), 4 syntypes (images seen; https://data.nhm.ac.uk/object/80cd6292-170e-4636-9c32-859ad933b36e/1631750400000), lectotype and paralectotypes here designated (lectotype: H = 13.5 mm; paralectotypes: H = 8.8, 5.1, 11.3 mm), NHMUK 1887.2.9.2183–2186; Azores, south of São Miguel Island (37°26ʹN, 25°13ʹW; 1829 m), 2 syntypes (images seen; Natural History Museum 2014), specimens designated here as paralectotypes, NHMUK 1887.2.9.2187–2188 (H = 13.0, 14.4 mm) (Eilertsen and Malaquias 2013a); 1 syntype (image seen), here designated as paralectotype, NMINH 1899.114.182 (H = 14 mm).

Other material examined

Azores, between Santa Maria and São Miguel Islands (37°10.820ʹN, 025°11.750ʹW − 37°10.368ʹN, 025°12.957ʹW; 2065 − 2066 m), 1 spc. dissected and sequenced for the mitochondrial gene COI, DBUA 1630, H = 20 mm.

Revised diagnosis

Shell solid, white, pyriform. Parietal callus thick, white, smooth, can present an angular hump around aperture inflexion point. Spire concealed. Outer lip protruding posteriorly beyond apex. Spiral sculpture composed of medium and small punctuated striations. Prostate cylindrical, prostatic duct short, narrower than prostate. Penial chamber lined with soft longitudinal grooves. Penial papilla absent.

Shell (Figure 1)

Maximum H = 24 mm (Locard 1897). Shell solid, white, pyriform, covered by thin white to pale yellow periostracum. Parietal callus thick, white, smooth, can present an angular hump around aperture inflexion point. Spire concealed. Outer lip protruding posteriorly beyond apex in small wing. Spiral sculpture composed of regular lines of small or medium-sized punctuations.

Radula (Figure 2(a)−(c))

Radular formula 15 × 1.1.1. Lateral teeth hook-shaped, curved, with weak denticulation on inner edge. Small rachidian teeth tetragonal, with expanded outward corners, straight or curved.

Digestive system (Figure 2(d)−(e))

Salivary glands long with uneven surface. Paired gizzard plates subtriangular to kidney-shaped; unpaired gizzard plate thinner, elongated.

Reproductive system (Figure 2(f)−(g))

Prostate elongate, cylindrical, narrowing towards penial chamber. Prostatic duct short, narrower than prostate, separating prostate from penial chamber. Penial chamber cylindrical, lined with soft longitudinal ridges running from genital opening to prostatic duct. Penial papilla absent.

Ecology

Feeds on foraminifera, agglutinating and non-agglutinating, ingested along with sand and mud (Figure 3). Live specimen collected between 2065 − 2066 m (present study), empty shells records between 1299 − 2995 m (Watson 1883; Dautzenberg and Fischer 1896; Locard 1897; Bouchet 1975; Eilertsen and Malaquias 2013a).

Distribution

Only known from the Archipelago of the Azores, where it is reported west and southwards of Flores Island, on the western slope of the mid-Atlantic ridge (Watson 1883) and eastwards of the Islands of São Miguel and Santa Maria, on the eastern side of the mid-Atlantic ridge (Watson 1883; Dautzenberg and Fischer 1896; Locard 1897; Bouchet 1975; present study).

Molecular analysis and species delimitation

The COI alignment dataset included 658 bp, and the best-fitting model of evolution was TIM3 + I + G. The resulting COI tree clustered all Scaphander specimens together with maximum support (PP = 1). The S. gracilis specimen was part of a larger clade together with other Atlantic (S. meridionalis, S. nobilis) and Pacific species (S. grandis, S. mundus, S. subglobosus) (PP = 0.98; Figure 4). Sister relationships were generally poorly resolved and this was also the case for S. gracilis (Figure 4).

COI uncorrected p-distances ranged from 0.18% to 1.25% within Scaphander species and from 4.29% to 20.04% between species. Genetic distances between S. gracilis and other Scaphander species varied from 4.29% to 19.14%. The smallest genetic distance was to the NE Pacific species S. grandis (4.29%), followed by the western Pacific species S. mundus (4.47%) and to the amphi-Atlantic species S. nobilis (6.62%).

The three independent models of evolution implemented in the ABGD method recovered 15 Scaphander lineages, including S. gracilis as a candidate species.

Discussion

Watson (1883) described Scaphander gracilis based on shells found during the ‘Challenger’ expedition around the Azores Islands. He remarked on its similarities to the shells of S. punctostriatus (Mighels & C. B. Adams, 1842), but stressed the uniqueness of the striations and thick callus differentiating S. gracilis. Additional shells were subsequently found during various cruises around the Azores (e.g. ‘Hirondelle’ reported by Dautzenberg and Fischer (1896); ‘Travailleur’ and ‘Talisman’ reported by Locard (1897); ‘Charcot-Biaçores’ reported by Bouchet (1975)).

Locard (1897) described two varieties of S. gracilis: major, a larger and more pyriform variety; and minor, a smaller shell with a thicker callus, but Eilertsen and Malaquias (2013a) attributed these differences to intraspecific variability. The shell of S. gracilis has been likened to the elongate form of the Atlantic Scaphander punctostriatus (Locard 1897; Marcus 1974), however, it is easily distinguishable from all other known Scaphander species by its thick callus, which even in the form without an angular hump, is conspicuously more pronounced than in other pyriform species of Scaphander.

Marcus (1974) mentioned the occurrence of S. gracilis in the Caribbean, while at the same time expressed uncertainty about the identification of the specimens, stating that they could be S. punctostriatus, a known amphi-Atlantic species commonly found in the Caribbean. Eilertsen and Malaquias (2013a) did not find evidence for the presence of the species in the western Atlantic and considered S. gracilis to be restricted to the Azores. This is also our opinion after comparison of shells between Caribbean and Azorean specimens.

Eilertsen and Malaquias (2013a) recognised eight valid species of Scaphander in the Atlantic, and Chaban et al. (2019) reassigned Meloscaphander imperceptus Bouchet, 1975 to Scaphander, to which Siegwald et al. (2020) added S. meridionalis from offshore Argentina. Among these 10 species, S. gracilis was the only one known solely from shells. The present study describes for the first time the anatomy of S. gracilis and includes the first molecular sequence data from it; contributing to a preliminarily test of its phylogenetic position among Scaphander. Our illustration of the radula (Figure 2(a)) may lead to the false impression that only few rows possess a rachidian tooth, but this is only because they are fragile and fall off easily during the cleaning and preparation for SEM. Another noteworthy feature is the comparatively larger size of the salivary glands when compared with other Atlantic species. Only S. bathymophilus and S. nobilis have similar or larger size glands (Figure 2(d); see Eilertsen and Malaquias 2013a).

The COI uncorrected p-distances and the phylogenetic tree suggest a closer relationship to the other white sub-rectangular shelled Scaphander species, namely S. grandis, S. mundus, S. nobilis and S. meridionalis (Figure 4; Table 2), but the thickened smooth and often angular callus of S. gracilis makes it easy to distinguish from its congeners.

Interestingly, the known and restricted distribution of S. gracilis, which is only confirmed from deep waters in the Archipelago of the Azores, makes it the only Scaphander species with a geography limited to offshore Islands. The fact that S. gracilis was found on both sides of the Mid-Atlantic Ridge (Figure 5) suggests that this significant geological phenomenon is not an insurmountable barrier for dispersal in this species. The role of the Mid-Atlantic Ridge as a putative barrier for dispersal of bathyal and abyssal organisms across the western and eastern sides of the Atlantic realm remains poorly understood and heavily hampered by lack of molecular studies (Mullineaux et al. 2002; Zardus et al. 2006; Etter et al. 2011), and this work contributes to a greater understanding of the bathyal malacofauna of the Atlantic.

Figure 5. Distribution of Scaphander gracilis, based on reliable literature records (shells) and newly sampled material (complete specimen).

Acknowledgements

We are grateful to S. Ávila and R. Cordeiro (University of the Azores) for making specimens available for study, and to A. Salvador and K. Webb (NHMUK), as well as to A. Geraghty and P. Viscardi (NMINH) for providing type specimen images. We also thank I. Heggstad (Laboratory for Electron Microscopy, Department of Earth Science, University of Bergen) for help with critical-point drying and SEM work and L. Lindblom (DNA Lab, Department of Natural History, University of Bergen) for help with molecular sequencing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was funded through a PhD scholarship attributed to the first author by the Department of Natural History, University Museum of Bergen, University of Bergen, Norway.