Investigation of the Amathillopsidae (Amphipoda, Crustacea), including the description of a new species, reveals a clinging lifestyle in the deep sea worldwide

Abstract Amathillopsidae is a widely distributed, but rarely sampled family of deep-sea amphipods. During a recent expedition to the North Atlantic, specimens were filmed clinging to a polychaete tube in situ at abyssal depths by a Remote Operated Vehicle and then sampled for further study. The species was new to science and is described in detail herein. A barcode sequence is provided. Further investigations of photographic and video records revealed the genus Amathillopsis to be more widely distributed, both geographically and bathymetrically, than indicated by current literature records, and that these species occur at abyssal depths in all oceans. Specimens of Amathillopsis are reported clinging to a variety of different organisms whose erect structures provide the means to raise these charismatic deep-sea predators above the seafloor facilitating feeding opportunities.


Introduction
During the third expedition of the Icelandic Genetics & Evolution (IceAGE) project on the RV 'Sonne' (Cruise SO267) to the North Atlantic from June to July 2020, large numbers of amphipod crustaceans were collected using a variety of methods. Using the Remotely Operated Vehicle (ROV) KIEL 6000, an interesting amathillopsid amphipod was observed sitting as a pair clinging to an onuphid worm tube at 4600 m depth at the Porcupine Abyssal Plain. The amathillopsid species was successfully sampled and proved to be new to science. A single specimen of the new species, collected at the Porcupine Abyssal Plain Sustained Observatory site at 4844 m, was also found within the Discovery Collections at the National Oceanography Centre, Southampton, UK. This new species, amongst the deepest confirmed record of the genus, is described herein. Lowry (2006) included the parepimeriids and the genus Cleonardopsis K.H. Barnard, 1916 in the family Amathillopsidae and created three subfamilies: Amathillopsinae, Parepimeriinae and Cleonardopsinae. The new species of Amathillopsis described herein is classified within the Amathillopsinae.
Three species of Amathillopsis and one species of Cleonardopsis have been reported from the North Atlantic to date: Amathillopsis affinis Miers, 1881; Amathillopsis spinigera Heller, 1875; Amathillopsis atlantica Chevreux, 1908; and a probable new species of Cleonardopsis which was first reported from off the coast of eastern Greenland by Stephensen (1944) as Cleonardopsis carinata K.H. Barnard,1916, and may be the same species as the Cleonardopsis sp. sequenced by Jażdżewska et al. (2018).
The Amathillopsidae are rarely collected, and very little is known of their biology and ecology. Most studies of the family relate to the description of new species based on material from a single or very few specimens, from a single locality.
In recent years, the increased use of ROVs to capture high-resolution footage of deep-sea ecosystems has provided an opportunity for the study of poorly known and rarely captured organisms (Macreadie et al. 2018). This is certainly the case with the Amathillopsidae. We provide collated records of ROV footage of a number of as yet unidentified species of Amathillopsis, from the deep sea, worldwide; thus confirming that despite the paucity of records and specimens, the genus is cosmopolitan and relatively common.

Collection methods and locations
During the IceAGE 3 expedition on the RV 'Sonne', the ROV KIEL 6000 sampled the Porcupine Abyssal Plain at station 133-4 (49°47.969'N, 015°12.975'E, 4622 m, 20 July 2020), via photo and video transects, as well as physical sampling. The specimens of the new species of Amathillopsis were initially photographed and filmed in situ, after which attempts were made to use the ROV suction to collect them. When this failed the specimens were scooped up by the ROV operator arm using a net and then placed into a sampling box.
Once on board, the single specimen collected was immediately photographed and then placed in RNAlater. The left first pleopod was then dissected and placed in a separate tube to be used for DNA extraction. The whole specimen and the dissected pleopod sample were then both transferred to the -20 °C freezer for later study.
The type localities and holotype materials of all known species of Amathillopsis were collated to aid in future studies of the genus (Table 1). Additional photographic records of other Amathillopsis specimens were collated from a variety of sources (Table 3), along with associated metadata, allowing the mapping of specimen localities.

Taxonomic methods
The adult male holotype specimen (ZMH K 60236) was photographed in situ by the ROV KIEL 6000, photographed on board by a Nikon D5 camera with an objective Nikon AF-S Mikro-Nikkor 105 mm 1:2.8, and dissected appendages were photographed using a Keyence 7000 microscope. A video of the Amathillopsis in situ can be found in the Suppl. material 1.
Initial observations and photographs were made on board of the RV 'Sonne'. The pencil drawings were conducted using a LeicaM125 and an Olympus BX53. Pencil drawings were scanned and inked digitally using Adobe Illustrator and a WA-COM digitiser tablet (Coleman 2003(Coleman , 2009. Some setae are omitted from the illustrations for clarity. Type material is deposited in the Zoological Museum Hamburg (ZMH) and the Natural History Museum, London (NHMUK).

Genetic methods
Isolation of DNA was performed on board using the NucleoSpin tissue extraction kit from MACHEREY-NAGEL GmbH & Co. KG according to the manufacturer's protocol. A fragment of the COI gene (ca. 670 bp fragment) was amplified using primers LCO1490-JJ CHACWAAYCATAAAGATATYGG Forward (Astrin and Stüben 2008) and HCO2198-JJ AWACTTCVGGRTGVCCAAARAATCA Reverse (Astrin and Stüben 2008).
The PCR reaction mixes were prepared to a final volume of 25 µl containing 12.5 µl AccuStart II PCR ToughMix (Quanta Bio), 0.5 µl of each primer (10 pmol/µl), 9.5 µl dH2O and 2 µl template DNA. PCR settings for amplifying CO1 sequences consisted of initial denaturing of 4 min at 95 °C, 5 cycles of 45 s at 95 °C, 90 s at 45 °C, 60 s at 72 °C, following 35 cycles of 45 s at 95 °C, 60 s at 51 °C, 60 s at 72 °C, and final extension 3 min at 72 °C. PCR products were purified using the Exonuclease-I/Shrimp Alkaline Phosphatase (Thermo Fisher) method and were sequenced at Macrogen Inc. Europe. Sequences were edited using Geneious 9.1.8 resulting in a sequence of length of 626 bp excluding primers. Relevant voucher information, taxonomic classifications and sequences are deposited in BOLD.
Remarks. Amathillopsis is the type genus of the family Amathillopsidae and the genus has a cosmopolitan distribution (Wakabara and Serejo 1999). It currently contains 12 species, five of which are known from the Pacific, A. annectens (Holmes, 1908), A. australis Stebbing, 1883, A. grevei J.L. Barnard, 1961, A. takahashiae Tomikawa &Mawatari, 2006 andA. pacifica Gurjanova, 1955 (and the sub species A. pacifica margo J.L. Barnard, 1967),; two from the Antarctic, A. roroi Coleman, 2008 andA. charlottae Coleman, 1998; two from the Indian Ocean, A. septemdentata Ledoyer, 1978 andA. comorensis Ledoyer, 1986; and three from the North Atlantic and Arctic, A. affinis Miers, 1881, A. spinigera Heller, 1875and A. atlantica Chevreux, 1908. Amathillopsis is a deep-sea specialist, with the shallowest records of the large species, A. spinigera, coming from colder Arctic waters at 248 m. All other species are found at depths of 500 to 3580 m. The new species, A. inkenae, described here, provides the deepest confirmed records of an Amathillopsis to date, at 4622 m and 4844 m. Table 1 summarises the known species in the genus along with the type locality and depth.
Mouthparts. Upper lip with weakly convex apical margin, bearing two groups of setae. Lower lip with outer lobes broad, setulose; inner lobes indistinct, fused. Mandibles with left incisors bearing eight teeth, left lacinia mobilis with four teeth; accessory setal row with nine setae, some bearing a row of minute protuberances. Molar developed, triturative. Palp articles 1, 2, and 3 in length ratio of 1.0: 5.0: 7.1, article 1 lacking setae, article 2 with marginal and submarginal setae, and article 3 with six marginal and three terminal setae. Maxilla 1 with inner plate ovate and bearing four plumose setae; outer plate rectangular, with 11 serrate, robust setae; palp two-articulate, longer than outer plate, terminally with seven long robust setae. Maxilla 2 inner plate slightly broader than outer plate, bearing row of long plumose setae. Maxilliped, inner plate reaching base of palp, with three robust nodular setae on the distomedial margin, distolateral margin with apical robust setae; outer plate exceeding distal margin of palp article 1. Maxillipedal palp long, raptorial, four-articulate; article 2 and 3 heavily setose and widened medially; dactylus as long as article 3. subchelate, basis posterior margin with row of robust setae, posterodistal lobe absent; ischium and merus short; carpus 0.68 × as long as propodus, ventral lobe broad, concave, allowing propodus to retract; propodus stout, tapering distally, with four groups of robust setae, palmar margin with long and short robust setae; dactylus as long as palmar margin, sickle-like. Gnathopod 2 subchelate, basis with posterodistal lobe present, posterior margin with row of robust setae; carpus 0.67 × as long as propodus, ventral lobe broad, concave, allowing propodus to retract; propodus stout, tapering distally, with four groups of robust setae, palmar margin with long and short robust setae; dactylus as long as palmar margin, sickle-like. Pereopod 3 basis with row of robust setae along weakly convex posterior margin, ischium short, as long as wide; merus margins subparallel with slight anterior curvature. Pereopod 4 similar to pereopod 3. Pereopods 5-7 anterior and posterior margins of basis sub-parallel, linear, posterior lobe lacking; ischium short, as long as wide; merus margins subparallel with slight anterior curvature. Carpus, propodus and dactylus missing from pereopods 3-7. Uropods. Uropod 1 long, peduncle length 0.88 × inner ramus; medial margin of peduncle with robust setae, inner ramus, lateral and medial margins with robust setae, outer ramus 0.88 × as long as inner, lateral and medial margins with robust setae. Uropod 2 with peduncle length 0.57 × inner ramus, lateral margin with robust setae, dorsomedial margin with one robust seta distally; inner ramus, lateral and medial margins with robust setae; outer ramus 0.64 × inner, lateral and medial margins with robust setae. Uropod 3 peduncle length 0.74 × inner ramus; dorsomedial margin of peduncle with three robust setae distally; inner ramus with lateral and medial margins bearing robust setae, outer ramus 0.64 × as long as inner, lateral, and medial margins with robust setae. Telson length 1.44 × width, cleft 22%. Each lobe bearing terminal setae.
Paratype male, 14 mm: As for holotype except the dorsal processes are more pronounced and acute on pereonites 5-7 and pleonites 1 and 2 (Fig. 5c).
Etymology. The name is dedicated to Dr. Inken Suck, the pilot who flew the ROV and sampled the specimen, to honour her dedication to deep-sea biology.
Remarks. In live condition, Amathillopsis inkenae sp. nov. has a white coloured body and antennae, the last three segments of both gnathopods as well as the mouthparts are red. Eyes are clearly visible, solid white, in live and fresh condition, but fade after a few days of fixation. Care should be taken in use of the relative sizes of the dorsal processes as these are likely to vary ontogenetically, as for the two specimens available here, where the larger male paratype has more pronounced, acute processes than the smaller male holotype. This is also likely to occur in other species in the genus. The specimens reported by Wakabara and Serejo (1999) as A. atlantica are likely to belong to a new species, and the authors point out that the mid-dorsal processes and the telson show some variation. Other characters, such as the reduced lobes on the basis of the gnathopods, also indicate that this is probably a new taxon.
Amathillopsis inkenae sp. nov. differs from known species of Amathillopsis by the characters listed in Table 2. Amathillopsis inkenae sp. nov. is most similar to Amathillopsis comorensis Ledoyer, 1986, which was collected in the Indian Ocean near the Comoros Islands at 2500 m. The new species has a similarly cleft telson, and a similar development of the dorsal processes and of the lobe on the basis of gnathopod 2 only. Amathillopsis inkenae differs from A. comorensis in having carination on urosomites 2 and 3, with posteriorly directed dorsal processes (lacking in A. comorensis), the stronger acute tooth on epimeron 3 posterodistal corner (smaller on A. comorensis), the curved article 2 of the mandible palp (straight in A. comorensis), and the medially widened articles 2 and 3 of the maxilliped palp (subparallel sided in A. comorensis). The barcode of Amathillopsis inkenae sp. nov. is deposited in BOLD:AEF9286 and GenBank MW726208.
Depth range. 4622-4844 m. Distribution. Only known from the North East Atlantic Ocean, Porcupine Abyssal Plain, between 4622-4844 m.

Discussion
We have described a new species of Amathillopsis collected from abyssal depths and differentiated this new species from the known species found globally. Only A. grevei, A. roroi, and A. pacifica have been collected at abyssal depths; all other Amathillopsis species were collected shallower than 2000 m. However, photographs and video captured by ROVs are now able to show that the genus is relatively common at bathyal and abyssal depths. Amathillopsis species have now been observed by ROVs and other camera systems on a number of occasions, clinging in pairs (and occasionally in larger numbers), to a tubular or stalk-like structure erected from soft substrate, and also on corals attached to hard substrates. We have collated these records and present them alongside the type localities of known Amathillopsis species (Table 3, Fig. 7).
All photographic records of Amathillopsis collated here are from the Pacific (Fig. 7, purple diamonds), while Amathillopsis species are distributed globally (Fig. 7, orange stars). The NOAA Office of Ocean Exploration and Research Benthic Deepwater Animal Identification Guide includes a number of images of amphipods of the genus Amathillopsis from the central Pacific at the Northern Mariana Islands (Fig. 6c) and the Pacific Remote Islands Marine National Monument, northeast of Kingman Reef (Fig. 6f ). There a number of images of pairs of Amathillopsis from abyssal depths in the Eastern Clarion Clipperton Zone (Fig. 6a) from Kiribati waters, and from bathyal depths of the northwest Pacific at the Emperor Seamounts (Fig. 6b) which were collected during AUV and ROV surveys of these areas.
The ROV KIEL 6000 captured images of pairs of Amathillopsis clinging to sponges below 4000 m during the DISCOL expedition on RV 'Sonne' to the southeast Pacific in 2015 (Fig. 6d) and to the abyssal plains North of New Zealand (southwest Pacific) in 2017 (Fig. 6e). We have also included in Table 3 and Fig. 7 a recently published record of a specimen of Amathillopsis. The specimen shown in Brandt et al. (2018: fig.  6a) is probably a specimen of A. pacifica, since it conforms to the illustrations of that species by Gurjanova (1955), and it was collected from a similar geographic locality and depth. We have not made any further attempts to identify the specimens in these images to species, since it is likely that there are new species involved and therefore specimens will be needed for more detailed analyses. It is remarkable that the type localities and the photo localities (see Fig. 7) are so different. While dragged (towed) gear has coincidently sampled occasional specimens of Amathillopsis, the advent of technology has resulted in numerous additional records of these animals in situ, work which has been largely focussed in the Pacific Ocean. Amathillopsis inkenae sp. nov.
was observed with two individuals clinging to a polychaete worm tube (video, Suppl. material 1) at 4622 m depth on the Porcupine Abyssal Plain. During the observation time of 20 minutes, the amphipods did not alter their position on the tube. The  Table 3 for detailed information. tube belongs to a polychaete worm of the family Onuphidae (Alexandra Kerbl, pers. comm., August 2020). Unfortunately, we only managed to retrieve one of the two specimens. Nevertheless, this is the first confirmed record of an Amathillopsis species observed in situ which has been subsequently collected and deposited in a natural history collection. Stills camera and video technology have developed in recent years to a level that enables high resolution images at bathyal and abyssal depths. Until now, the habits of species of Amathillopsis could only be postulated, on the basis of their possession of posterior pereopods adapted to a clinging lifestyle. The functional morphology enabling the gripping of thin structures is expressed by the strongly curved pereopodal dactyli and the pereopods being flexed backwards. These adaptations were described by McCloske (1970) as well-adapted for grasping, in reference to Dulichia clinging onto sea urchin spines. While clinging behaviour is known from a number of amphipod families in shallow waters, such as Caprellidae, Podoceridae, and Dulichiidae (e.g., Takeuchi and Hirano 1995;Guerra-García et al. 2002), it is only now that we are able to report that amathillopsids express this behaviour at bathyal and abyssal depths of all oceans. We assume the stem they cling to is simply a means to expose them higher in the water column for feeding. Amathillopsis inkenae was found on a worm tube, whereas other Amathillopsis species have been photographed clinging to a variety of other organism structures, including sponges and corals. Based on the raptorial structure of the mouthparts and gnathopods we assume Amathillopsis to be predators (or micropredators), capturing their prey, such as zooplankton or small suprabenthic crustaceans from the water column. The red colour of gnathopods and mouthparts may result from the consumption of carotinoids from prey. Amathillopsids have never been caught in baited traps, and therefore we exclude the possibility of them being scavengers. Also remarkable are the well-developed eyes of Amathillopsis specimens living below 3000 m. It is probable that they rely on bioluminescence as communication, either for catching prey, avoiding predators or finding mating partners.  Table 3 for detailed locality information.