INTRODUCTION
The deep sea is one of the largest habitats on the planet, but our knowledge of the habitat and the deep-sea fauna remain relatively poor. The expansion of commercial fishing activities into deeper waters has raised major concerns (e.g. UNGA Resolution 61/105) regarding the potential impact of fishing on benthic ecosystems and biodiversity, but has also provided an additional source of biological samples for researchers. There is clearly an urgent need to describe the components of deep-sea environments and understand how these systems will cope with disturbance from fisheries.
The cirrate octopods (Octopoda: Cirrata) are the most abundant cephalopods in deep-sea benthic and bentho-pelagic communities (Collins & Villanueva, Reference Collins and Villanueva2006). The paired fins, well developed webs, large internal shell and paired cirri interspersed in a single row of suckers distinguish them from the generally shallower incirrate forms. Cirrates are known from all oceans, typically at depths of 300–7000 m, but are found shallower in cold waters at high latitudes and include some of the largest invertebrates in the deep-sea (Collins & Villanueva, Reference Collins and Villanueva2006). They are usually caught in small numbers, are extremely fragile and easily damaged on capture, and often distort during preservation (Vecchione et al., Reference Vecchione, Collins and Sweeney2002).
The cirrates are divided into four families (Cirroteuthidae, Grimpoteuthidae, Opisthoteuthidae and Cirroctopodidae (Collins & Villanueva, Reference Collins and Villanueva2006)). The family Opisthoteuthidae includes some of the shallower species, characterized by a broad U-shaped shell. Nineteen species of Opisthoteuthis are considered valid (Collins & Villanueva, Reference Collins and Villanueva2006). The taxonomy of the Atlantic species of Opisthoteuthis was evaluated by Villanueva et al. (Reference Villanueva, Collins, Sanchez and Voss2002), with O. borealis added later (Collins, Reference Collins2005). Opisthoteuthis hardyi Villanueva et al. (Reference Villanueva, Collins, Sanchez and Voss2002) was described from a single, male specimen caught in a pelagic trawl near Shag Rocks to the north-west of South Georgia. Since the original description no further specimens have been reported. Recent deep-water trawling on the Patagonian slope targeting grenadiers (Macrourus holotrachys) caught a number of specimens of Opisthoteuthis and here we have used morphological measurements and molecular genetics to confirm that these specimens are O. hardyi and are thus able to provide a more extensive description of the species and basic data on the ecology of the species.
MATERIALS AND METHODS
Sample collection
Opisthoteuthis sp. were collected during deep-sea surveys by a midwater trawl (codend mesh size 40 mm) onboard RV ‘Dorada’ (16 and 19 July 2006) and by a bottom trawl (codend mesh size 110 mm) onboard FVs ‘Jose Antonio Nores’ (3 September 2006) and ‘Manuel Angel Nores’ (8 and 21 November 2007) on the Patagonian slope (Figure 1) between 630 and 1391 m depth (Table 1). Specimens were frozen whole shortly after capture for subsequent analysis in the laboratory. Upon thawing, specimens were investigated and then preserved in 10% buffered saline formalin.
Fig. 1. Map of the south-western Atlantic showing location of captures of Opisthoteuthis hardyi. The dotted line indicates the mean position of the Antarctic Polar Front. The 200 m and 1000 m isobaths are also illustrated.
Table 1. Opisthoteuthis hardyi. Details of capture locations in the south-western Atlantic Ocean, including the holotype.
NMSZ, National Museums of Scotland (Zoology); SBMNH, Santa Barbara Museum of Natural History; BMNH, British Museum (Natural History); FIFD, Falkland Islands Fisheries Department.
Morphological measurements
Definitions of counts and measurements used here generally follow Voss & Pearcy (Reference Voss and Pearcy1990) and the basic measurements are defined in Collins (Reference Collins2003). The dorsal mantle length (ML) was measured as for other octopods (Roper & Voss, Reference Roper and Voss1983). Fin length and fin width are measured in accordance with Voss & Pearcy (Reference Voss and Pearcy1990) where the length of the fin (in contrast to fin length in teuthoids) is measured from the midpoint of the base of the fin to the outer tip, and fin width is the greatest width across the fin measured perpendicular to the fin length. Finspan follows Guerra et al. (Reference Guerra, Villanueva, Nesis and Bedoya1998), and is the distance between the apices of the fins. The gill lamellae counts refer to the total number of lamellae on each gill; 7/6 means 7 lamellae on the left gill and 6 on the right. Specimens were considered sexually mature on the basis of the presence of spermatophores in the seminal vesicle and/or penis of the males, and presence of eggs in the oviducts and/or oviducal gland of the females.
Female reproductive systems were opened from three specimens after preservation and all oocytes were counted and measured.
Molecular analyses
DNA extraction and PCR amplification followed the methods outlined in Allcock et al. (Reference Allcock, Strugnell, Ruggiero and Collins2006). Primers for a fragment of the mitochondrial gene 16S rDNA are detailed in Allcock et al. (Reference Allcock, Strugnell and Johnson2008).
DNA sequences were aligned by eye in Se-Al v2.0a11 Carbon (Rambaut, Reference Rambaut2002). PAUP v4.0b10 (Swofford, Reference Swofford1993) was used to perform full heuristic searches. Starting trees were generated by the neighbour-joining (NJ) method (Saitou & Nei, Reference Saitou and Nei1987). A GTR+Γ+I likelihood model incorporating four rate categories was used. Branch swapping was performed using tree-bisection-reconnection (TBR). Parameters were then re-estimated and branch swapping was then performed using nearest-neighbour-interchange (NNI). Substitution model parameter values for each data set are included in the supplementary information. Maximum likelihood (ML) bootstrap values of clade support were generated using the above parameters, with starting trees obtained using NJ.
MrBayes 3.1.2 (Ronquist & Huelsenbeck, Reference Ronquist and Huelsenbeck2003) was used to calculate marginal posterior probabilities using the GTR+I+Γ model of nucleotide substitution for each partition. Model parameter values were treated as unknown and were estimated in each analysis. Random starting trees were used and analyses were run for 1,000,000 generations, sampling the Markov chain every 100 generations. The analysis was performed twice, in each case starting from a different random tree to ensure the analyses were not trapped in local optima. Stationarity was deemed to be reached when the average standard deviation of split frequencies, shown in MrBayes 3.1.2, was less than 0.01 (Ronquist & Huelsenbeck, Reference Ronquist and Huelsenbeck2003).
The program Tracer v 1.3 (Rambaut & Drummond, Reference Rambaut and Drummond2003) was used to determine the correct ‘burn-in’ for the analysis (i.e. the number of initial generations that must be discarded before stationarity is reached).
RESULTS
Morphological data
Thirty-three specimens of Opisthoteuthis were examined from the Patagonian slope (Table 1; Figure 1). General body shape, sucker counts and locations of enlarged suckers were consistent with those of the type specimen (male) of Opisthoteuthis hardyi Villanueva et al. Reference Villanueva, Collins, Sanchez and Voss2002.
Molecular data
New nucleotide sequences generated in this study were deposited in GenBank under accession numbers FJ785403 and FJ785404. Indels (insertion/deletion events) were necessary to align 16S rDNA. The final alignment was 476 base pairs in length.
The phylogenetic tree resulting from ML and Bayesian analysis of 16S rDNA is rooted using Grimpoteuthis sp. (Figure 2) as a previous study using this gene has shown Grimpoteuthis to be a suitable outgroup to Opisthoteuthis (Piertney et al., Reference Piertney, Hudelot, Hochberg and Collins2003). The two Opisthoteuthis specimens from the Patagonian shelf grouped together with the holotype of Opisthoteuthis hardyi in a strongly supported monophyletic clade (bootstrap (BS) = 87, posterior probability (PP) = 88). The O. hardyi holotype sequence (AF487302) from Piertney et al. (Reference Piertney, Hudelot, Hochberg and Collins2003) contains eight base pairs which are unknown and are thus designated ‘N’. Apart from these unknown bases, one of the two newly sequenced specimens was identical to the holotype, while the other differed in a single base pair within a highly variable loop region of 16S rDNA.
Fig. 2. Maximum likelihood tree depicting the phylogenetic relationship of six species (nine individuals) of Cirrata. The analysis employed a portion of the mitochondrial gene 16S rDNA. Bayesian posterior probability support values are indicated above the nodes and maximum likelihood bootstrap values with 50% support or greater are indicated below the nodes.
Opisthoteuthis depressa and O. californiana are highly supported sister taxa (BS = 97, PP = 99). Opisthoteuthis massyae is the sister-taxon to O. calypso, although this relationship is only weakly supported (BS = 55, PP = 60). Higher level relationships between the O. hardyi clade, the O. californiana/O. depressa clade and the O. massyae/O. calypso clade were not supported.
MATERIAL EXAMINED
Holotype: mature ♂; 45 mm ML; FV ‘Argos Galicia’ Station SG97-25; 11 September 1997; 53.30°S 42.20°W; 800–1000 m; pelagic trawl; NMSZ1999158.088.
Other material: 22 ♂, 2 ♀; FV ‘Manuel Angel Nores’ Station 162; 21 November 2007; 53.40°S 59.42°W; 1391 m; bottom trawl; FIFD Collections. 1 ♀; ‘Manuel Angel Nores’ Station 127; 8 November 2007; 52.82°S 57.14°W, 997 m, bottom trawl; FIFD Collections. 5 ♂, 1 juvenile; FV ‘Jose Antonio Nores’ Station 128; 3 September 2006; 53.75°S 59.33°W; 1088 m; bottom trawl; BMNH 20080845 (2 ♂only). 1 ♂ FPRV ‘Dorada’ Station 2476; 16 July 2006; 53.34°S 59.54°W; pelagic trawl; 992 m; BMNH 20080843. 1 ♀; FPRV ‘Dorada’ Station 2491; 19 July 2006; 53.82°S 58.49°W; 630 m; pelagic trawl; BMNH 20080844. 1 ♂; FV ‘Heroya Primera’, 11 November 1994; 53.63°S 59.97°W; 1018 m; bottom trawl; SBMNH 143060.
DIAGNOSIS (AMENDED)
Medium sized species. First arms of mature males slightly thickened. Distal enlarged sucker field in males comprise 9–13 suckers beginning at about sucker 18, with suckers 22–24 usually largest. In mature males, the sucker enlargement in distal field is approximately equal on all arms. Maximum sucker diameter is slightly greater in the proximal than distal field. First cirrus usually occurs between suckers 3 and 4. Digestive gland entire. Basal portion of shell with highly concave outer surface, and convex inner surface. Single muscular nodules or multiple, trabecula-like muscular supports extending from ventral margins of arms absent. Arm sucker count in adults 54–67. Pigment-free spots not known to occur on skin.
DESCRIPTION
Modified from Villanueva et al. (Reference Villanueva, Collins, Sanchez and Voss2002) and based on specimens in Table 2. Medium-sized species, body semi-gelatinous, ovoid in form (Figure 3). Eyes and fins superior. Mantle moderately short, 18–27 (mean 21) % TL, and broadly rounded posteriorly. Head slightly wider than mantle, with no discernible constriction between head and mantle. Pallial aperture small, closely surrounding funnel. Funnel flaccid, moderately long, 63–92 (mean 75) % ML. Funnel organ not discernible. Olfactory organs rounded and prominent, located just within mantle aperture and to either side of funnel. Fins moderate in size, 66–110 (mean 93) % ML, depending on preservation state and positioned postero-laterally. Anterior and posterior margins of fins thin, slightly convex; without lobe near the anterior insertion; fin tip rounded. Each fin supported by an internal, flexible cartilage that extends from the shell sac to which it closely adheres. Fin cartilage thick basally, becoming progressively thinner and lanceolate as it extends out to fin. Eyes of moderate size, bulbous, 38–62 (mean 45) % ML, occupy entire sides of head. Optic lobes large, kidney-shaped, with 4 large bundles of optic nerves running to each eye. White body, closely associated with lobe, large, dark brown/purple. Optic lobes larger than semi-circular brain.
Fig. 3. Opisthoteuthis hardyi Villanueva et al. Reference Villanueva, Collins, Sanchez and Voss2002. Illustration of the whole animal (A), with the oral view of the arm of a female (B) and detail of the suckers and cirri (C). Modified from Villanueva et al. (Reference Villanueva, Collins, Sanchez and Voss2002). Scale bars: A = 50 mm; B = 40 mm; C = 10 mm.
Table 2. Opisthoteuthis hardyi. Details of the specimens on which the redescription is based.
NMSZ, National Museums of Scotland (Zoology); BMNH, British Museum (Natural History); FIFD, Falkland Islands Fisheries Department.
Arms long, 73–86% TL, and approximately equal in length. Arms moderately stout, with slight increased thickness in first (dorsal) arms of mature male. Single muscular nodules or multiple, trabecula-like muscular supports extending into web from ventral margins of arms absent. Arms enveloped in web, occupying approximately 2/3 of arm length. Web extends further on the dorsal arms, formula A = B > C = D > E. Single row of suckers deeply set in semi-gelatinous tissue of all arms. Sucker count 54–67. Mature male with two fields of markedly enlarged, bulbous suckers on all arms. Proximally, suckers 4–9 typically enlarged, with sucker 6 or 7 the largest. Sucker enlargement in proximal field approximately equal on all arms. Distal enlargement field comprises 9–13 suckers beginning at about sucker 18, with suckers 22–24 the largest. Sucker enlargement in distal field, approximately equal on all arms. Sucker diameter slightly larger in proximal field (mean 11% ML) than distal field (mean 10% ML). Cirri short to moderate, first appear between suckers 3 and 4 on most arms. Longest cirri on mid portion of arms, length 3.6–8.1 mm or 6.5–15.4% ML.
Gills, small, compact, spherical, with 7 lamellae. Median pallial adductor muscle thin and narrow. Branchial heart rounded, approximately half the size of the gill. Oesophagus and stomach deeply pigmented dark purple (Figure 4A). Two digestive ducts unite before emptying into caecum. Intestine wide and approximately same length as oesophagus. Anal flaps and ink sac absent. Digestive gland entire. Beak of typical Opisthoteuthis form (Figure 4B, C). Shell U-shaped, with short, flaring lateral wings (Figure 4D). Basal portion with edges highly inrolled, making outer surface highly concave; inner surface convex; cross-section U-shaped. Lateral wings with inrolled margins, tapering to acute points. However, the extreme inrolling of the shell may be a consequence of freezing. Skin surface smooth, orange-brown in fresh specimens, becoming reddish-brown with preservation. Oral surface of the web deeply pigmented centrally, contrasting sharply with the less pigmented arms. Pigment-free spots on skin absent.
Fig. 4. Opisthoteuthis hardyi Villanueva et al. Reference Villanueva, Collins, Sanchez and Voss2002. Illustration of internal anatomy. Digestive system (A), upper (B) and lower (C) beaks, lateral, dorsal and ventral views of the internal shell (D) and female reproductive system (E). Abbreviations: bcm, buccal mass; cae, caecum; dgg, digestive gland; dod, distal oviduct; oes, oesophagus; ovg, oviducal gland; ovy, ovary; pod, proximal oviduct. Scale bars: A, D, E = 30 mm; B, C = 10 mm.
Male genitalia composed of large, oval testis located in median portion of mantle cavity and dorsal in position; short vas deferens; large, convoluted seminal vesicle complex; three accessory glands, with gland 2 the largest; and with a short terminal organ (penis) projecting from accessory gland 3. Spermatophores disc-shaped (see Villanueva et al., Reference Villanueva, Collins, Sanchez and Voss2002) with length-range of 0.7–1.4 mm.
Female genitalia large (Figure 4E), occupying approximately 50% of visceral mass in mature specimens, composed of large ovary, single (left) proximal oviduct with thin walls that leads to large, two-chambered, acorn-shaped oviducal gland. Proximal chamber of gland striated, opaque in colour, slightly shorter than striated, dark-coloured distal chamber. Distal oviduct short, with muscular terminal section. The ovaries of mature females contained oocytes at different stages of development (see below).
Reproductive biology
The ovaries of three females (38, 45 and 45 mm ML) were examined in detail to estimate fecundity and oocyte/egg sizes (Figure 5). In all three females oocyte sizes ranged from approximately 0.3 mm–12 mm. The modal size of oocytes in two specimens was 0.5, but was larger in the third (1.0 mm). In the third specimen there was a single ripe egg (10.8 × 6.0 mm) in the oviduct. In one of the specimens (38 mm ML) 219 resorpting oocytes were identified (3.5–5 mm; Figure 5) which were slender and dark yellow in contrast to rounded whitish normal oocytes of the same size. No resorpting oocytes were found in the other females. The total number of oocytes ranged from 427 (45 mm ML) to 952 (38 mm ML). The largest ovarian oocytes had 21–23 follicular folds (counts 21, 23, 23, 23, 23 and 22), but they were not fully developed.
Fig. 5. Length–frequency of oocyte sizes from three mature or sub-mature female Opisthoteuthis hardyi. The upper panel shows a female in which some eggs were in the process or resorption.
DISCUSSION
The combination of morphology and molecular biology confirm that the Patagonian slope Opisthoteuthis specimens are conspecific with the single specimen from Shag Rocks that Villanueva et al. (Reference Villanueva, Collins, Sanchez and Voss2002) described as Opisthoteuthis hardyi. This, therefore, extends the known bathymetric and geographical distribution of O. hardyi and facilitated a more detailed description than was possible from the holotype alone, with the first description of the female of the species.
The 16S rDNA sequence of one of the newly sequenced Patagonian slope O. hardyi individuals (FJ785403) is identical to the sequence of the holotype of O. hardyi (AF487302) from Piertney et al. (Reference Piertney, Hudelot, Hochberg and Collins2003). Although the O. hardyi sequence (AF487302) does possess eight unknown bases each of these are in positions where the corresponding base does not differ across the nine individuals included in the study and, therefore, these bases are highly unlikely to be different within the O. hardyi holotype. The second newly sequenced O. hardyi individual (FJ785404) differs by only 1 base pair from the other two sequences. This sequence difference of a single base pair occurs in the highly variable loop region of the gene and is consistent with intraspecific differences seen in other cephalopod species (Strugnell et al., Reference Strugnell, Collins and Allcock2008a, Reference Strugnell, Rogers, Prodohl, Collins and Allcockb).
The gelatinous nature of the cirrates makes comparisons between species difficult and preservation can have a major effect on body form. The clearest morphological difference between O. hardyi and other Atlantic Opisthoteuthis species is in the location of the distal enlarged sucker fields in the males (see Villanueva et al., Reference Villanueva, Collins, Sanchez and Voss2002). The females are rather more difficult to distinguish from other Opisthoteuthis species.
Although Shag Rocks is 500 miles south-east of the Patagonian Shelf and south of the Antarctic Polar Front, there are a number of species that are found in both places. In fact the Shag Rocks shelf appears to be a transition zone between the distinctly Antarctic fauna at South Georgia and that of the Patagonian Shelf. For instance the small nototheniid Patagonotothen guntheri is abundant at Shag Rocks and on the Patagonian Shelf, but absent from South Georgia (Collins et al., Reference Collins, Shreeve, Fielding and Thurston2008), whilst other common South Georgia shelf species such as mackerel icefish (Main et al., Reference Main, Collins, Mitchell and Belchier2008) are also caught there. Patagonian toothfish are also found on the Patagonian Shelf and at Shag Rocks and South Georgia, although there is evidence that the populations are genetically distinct (Shaw et al., Reference Shaw, Arkhipkin and Al-Khairulla2004). Whilst the Polar Front is not likely to be a barrier to benthic or bentho-pelagic species, a deep trench does separate the Shag Rocks and Patagonian Shelves, but the North Scotia Rise may provide a stepping-stone between the two locations. The larvae of cirrates are direct developing and probably benthic, so there is little opportunity for planktonic dispersion (Collins & Villanueva, Reference Collins and Villanueva2006), so any movement between the two areas is more likely to be due to adult movement.
The twenty-four Opisthoteuthis hardyi caught in a single trawl are noteworthy and support the hypothesis that cirrates may have schooling behaviour (Vecchione et al., Reference Vecchione, Piatkowski and Allcock1998).
The reproductive biology of O. hardyi is similar to other cirrates, characterized by the presence of a range of sizes of oocytes, indicative of an extended spawning with eggs laid individually (Boyle & Daly, Reference Boyle and Daly2000; Daly et al., Reference Daly, Boyle and Collins1998; Villanueva, Reference Villanueva1992). No follicular sheaths, that indicate the release of eggs (Boyle & Daly, Reference Boyle and Daly2000), were identified in this study, which may indicate that the females had released few eggs. Potential fecundity estimates ranged from 427–952, which are slightly lower than O. massyae (Boyle & Daly, Reference Boyle and Daly2000), but similar to many other opisthoteuthids (see Collins & Villanueva, Reference Collins and Villanueva2006).
Whilst the extension of fishing into deeper waters represents a threat to deep-sea biodiversity it also represents an opportunity to obtain new material. It is important that the fishing industry and scientists work together to improve our knowledge of the deep-sea environment and understand the impacts of fishing activities.
ACKNOWLEDGEMENTS
Thanks to captain and crew of the FPV ‘Dorada’ and the fishing vessels ‘Manuel Angel Nores’ and ‘Jose Antionio Nores’. Thanks also to Elizabeth White for the illustrations.
