Integrative systematics and ecology of a new deep-sea family of tanaidacean crustaceans

A new family of paratanaoidean Tanaidacea – Paranarthrurellidae fam. nov. – is erected to accommodate two genera without family classification (Paratanaoidea incertae sedis), namely Armatognathia Kudinova-Pasternak, 1987 and Paranarthrurella Lang, 1971. Seven new species of Paranarthrurella and two of Armatognathia are described from material taken in different deep-sea areas of the Atlantic and Pacific oceans. The type species of Paranarthrurella — P. caudata (Kudinova-Pasternak, 1965) — is redescribed based on the paratype. The genus Cheliasetosatanais Larsen and Araújo-Silva, 2014 originally classified within Colletteidae is synonymised with Paranarthrurella, and Arthrura shiinoi Kudinova-Pasternak, 1973 is transferred to Armatognathia. Amended diagnoses of Armatognathia and Paranarthrurella genera are given. Choosing characters for distinguishing and defining both genera was supported by Principal Component Analysis. Designation of the new family is supported by molecular phylogenetic analysis of COI and 18S datasets. The distribution of all species currently included in the new family was visualised and their bathymetric distribution analysed.


Methods taxonomic analyses.
A total of 87 individuals of nine new species was examined morphologically with a Leica M125 stereomicroscope. For all specimens, the body length (BL) was measured from the tip of the rostrum to the distal edge of the pleotelson.
Seven life stages are recognized for the studied individuals: two stages of manca, one neuter, two stages of females, and two stages of males. The terms "manca-2" and "manca-3" refers to specimens without or with buds of pereopod-6, respectively 26 ; 'preparatory female' and 'ovigerous female' are bearing oostegites buds either fully developed oostegites, respectively; 'juvenile male' and 'mature male' (swimming) 27 show incompletely or completely developed sexual dimorphic characters, respectively. Finally, the term 'neuter' is retained for the stage developed from manca-3 that cannot be classified as precopulatory female with oostegites buds (including non-ovigerous females), or juvenile male. Appendages from the chosen specimens were dissected in a glycerine solution using chemically-sharpened tungsten needles, mounted in glycerine on slides, and sealed with paraffin wax. For staining, methylene blue or chlorazol black were used. Initial drawings were made using a Nikon Eclipse 50i microscope combined with a camera lucida; they were then digitally inked as proposed by Coleman 28 .
The general morphological terminology follows that proposed by Jóźwiak et al. 16 . The body length-to-width ratio was assessed dividing a measurement of total body length (see above) and by a measurement the widest part of cephalothorax. Length of the articles and segments was measured along the central axis, whereas width was assessed at the mid-length of the article. As proposed by Bird and Bamber 29 , the 'spines' are called the articulated and unflexible cuticular structures while 'setae' are flexible and bristle or hair-like articulated structures; the apophyse (or teeth) are reserved for the nonarticulated cuticular outgrowth. The short, weakly calcified, round tip setae in mandibule molar of Paranarthrurella are called finger-shape setae.
All of the measurements were performed with Leica M205C and LAS V4.5 software. To simplify species descriptions, the expression 'Nx' replaces 'N times as long as' and 'N L:W' replaces 'N times longer than wide' .
Photographs were made using the focus stacking method on a Leica M205C stereo-microscope combined with a DFC295 camera and LAS V4.5 software. For the redescription of Paranarthrurella arctophylax, confocal microscopy imaging was used. Images were registered with the confocal laser scanning microscope LSM 780 (Zeiss) equipped with EC Plan-Neofluar 10x/0.30 M27 objective, 405 nm laser diode and and InTune tunable excitation laser system (set to excitation wavelength 595 nm). Natural autofluorescence of the specimen was enhanced by chemical crosslinking by transient incubation in formalin. Otherwise unstained, ethanol-fixed animal was imaged in 100% glycerol. Autofluorescence was registered sequentially in two emission channels: 410-580 nm (405 nm excitation) and 600-735 nm (595 nm excitation). In 3 × 3 tile scan area (3188.22 µm × 3188.22 µm), images were collected for stitching along with optimal number of Z-frames, each in 2048 × 2048 pixels format with 3.15 µs pixel dwell and 2 × line averaging. Images in both channels were then combined, pseudo-colored in gold and reconstructed into 2D image stack by maximum intensity projection using ZEN 2012 software (Zeiss).
The type material is deposited in the Museum of Comparative Zoology (MCZ, United States), Zoological Museum of Hamburg (ZMH, Germany) and Melbourne Museum (NMH, Australia). The material for the redescription of Paranarthrurella arctophylax is deposited in the Icelandic Institut of Natural History (NI, Iceland). DNA extraction, alignment, and phylogenetic analysis. Single appendages (chela or pereopod-1) were taken from each specimen for DNA extraction using sterile needles, and transferred to buffer solution. Extraction of DNA was performed at the Smithsonian Laboratories for Analytical Biology 25 . Two markers were sequenced: the nuclear ribosomal small subunit (18S), and the mitochondrial cytochrome c oxidase subunit I (COI). Polymerase Chain Reactions (PCR) protocols, primer sequences, and sequencing protocols were according to Riehl et al. 25 .
Sequencing reads were assembled in Geneious v. 11.1.4 and checked by hand to resolve ambiguities and remove primer sequences. To place sequences of Paranarthrurella in the proper phylogenetic context, nine sequences of 18S and 14 of COI from other genera within superfamily Paratanaoidea were obtained from GenBank (Agathotanaidae, Akanthophoreidae, Cryptocopidae, Paratanaidae, Leptocheliidae, Nototanaidae, Typhlotanaidae; Table 2 27,[30][31][32][33][34][35]. Finally, a gammarid species for which both COI and 18S sequences were accessible in GenBank. Alignment of 18S was performed with the online MAFFT server v7 36 , followed by deletion of poorly-aligned regions using the online Gblocks server 37 , employing all three options for less-stringent selection. Alignment of COI was performed on DNA codons using the ClustalX algorithm 38 in BioEdit (written by Tom Hall, Ibis Theraputics). Both alignments were trimmed to remove large front-and back-end blocks of gaps. Twenty-nine  Table 2. Details on the tanaidacean taxa used for genetic analysis applying COI and 18S markers.
principal component analysis. PCA divides the taxa onto three groups (Fig. 3). The first, second and third axes explain 52.7%, 15.3% and 8.4% of total variation, respectively. Axes 4 and 5 (not illustrated on Fig. 3) explain together only 11.4% of a variation. At first glance, two groups of features clearly differentiate the two genera, Armatognathia and Paranarthrurella, along axis 1. The genus Armatognathia is characterised by: presence of spine on antennular article-2, presence of spines on the mandible molar, rounded gustatory cusps (tubercles) on the maxilliped endites, robust chela, cheliped distal part long, presence of pleopods in females, and article-1 of the uropod endopod longer than article-2. The genus Paranarthrurella is characterised by: presence of a seta on antennulal article-2, finger-shape setae on the mandible molar, slender gustatory cusps (spine-like) on the maxilliped endites, slender chela, cheliped distal part short, absence of pleopods in females, and subequal articles 1 and 2 of the uropod endopod. Axes 2 and 3 cluster Paranarthrurella into two species groups. The first group, which includes P. spinimaxillipeda, P. samba sp. nov., P. polonez sp. nov., P. tango sp. nov., P. kizomba sp. nov., P. caudata and P. dissimilis, has a longer body (except P. tango sp. nov.), uropod endopod/exopod length ratio lower and absence of hyposphaenium on pleonites 2 and 3. Additionally, P. polonez sp. nov. and P. samba sp. nov. have an elongated cheliped carpus. In the second group, P. rocknroll sp. nov., P. arctophylax, P. corroboree sp. nov. and P. moonwalk sp. nov. have short bodies, presence of hyposphaenium on pleonites 2 and 3 (only in P. rocknroll the hyposphaenium on pleonite 2 is absent), uropod endopod/exopod length ratio higher and the cheliped carpus L/W ratio is smaller. Paranarthrurella voeringi is an outlier in the distinct presence of small hyposphaenium in pleonite 4 and large hyposphaenium on 3. Additionally, features localised on pereopods as follows: pereopod 4-6 propodus spine, pereopod-1 merus/carpus spines were correlated with PC Axes 4 and 5, are not illustrated on Fig. 3. They do not play a significant role in morphological differentiation of the two genera, but are important for discrimination of particular species.
Male unknown.
Distribution. The species is known only from the type locality -Bougainville Trench from 7974-8006 m depth.
Remarks. Paranarthrurella caudata was described by Kudinova-Pasternak based on five specimens of length from 3.2 mm to 4 mm. From this collection, only the holotype (marked by Kudinova-Pasternak on the original label) exists and was available for our studies. For this reason, we took the advantage and partly dissected the specimen to redescribe this inadequately-described species. P. caudata is the only member of the genus with a relatively slender, elongated and almost rectangular pleotelson and with three spines on carpus of the pereopods 4-6. Those two characters combined allow P. caudata to be distinguished from the other members of the genus, which have a rounded pleotelson ("onion-shape" in the description of Kudinova Pasternak 2 ) and four spines on carpus of the pereopods 4-6. Moreover, the apex of the pleotelson in P. caudata is robust and directed backward, but small and directed downward in all other Paranarthrurella species.
Six species: P. caudata, P. dissimilis, P. kizomba sp. nov. P. spinimaxillipeda, P. polonez sp. nov. and P. samba sp. nov. (see below) are the only known species of the genus with elongated body habitus whose body is eight or more times longer than wide. Yet, none of them has a pleonite hyposphaenium (see remarks on page 6). Paranarthrurella caudata has strongly serrated dorsodistal seta on the propodus of pereopods 4-6. This character is present in a few members of the genus (described below), as well as in P. dissimilis, although the latter has four spines on carpus of pereopod 4-6, while P. caudata has only three.  Distribution. The species was described for the NE Atlantic based on one specimen dredged during the Porcupine expedition at 2524 m depth between Ireland and Rockall. Later on, during the Ingolf expedition, new records in the David Strait and subsequently during BIOGAS expeditions in the northern Bay of Biscay, extended its depth range between 1970 and 4720 m ( Table 5, Fig. 39). The species has been also collected in the Iceland Basin during BIOICE cruises, and it has been also recorded in the Ibero-Moroccan Gulf (Bird, pers. comm.). www.nature.com/scientificreports www.nature.com/scientificreports/ Remarks. Paranarthrurella arctophylax is the only Paranarthrurella species in the North Atlantic that has well developed hyposphaenium only on pleonite-3 80 . Another species that also occurs at higher latitude in the North Atlantic is P. voeringi, whose hyposphenia is present also in pleonite 2 and 4 as well. Those two taxa have distinct temperature preferences conditioning their geographical distribution (see Table 5; also remarks under Paranarthrurella sp.2). The individual collected during Chain Cr. 106, is a fully mature male, so certain identification is not possible. However, because this individual was collected in the distributional area where P. arctophylax occurs 81 , we anticipate it probably represents this species, until abiotic data could corroborate it.  etymology. In Aboriginal language 'corroboree' is an event during which the particular interaction with the Dreamtime is reached through dance and music. Noun in apposition.
Distribution. Paranarthrurella corroboree sp. nov. is known from SE Australia (Bass Strait slope) from depths of 1450-1975 m.

Remarks.
Paranarthrurella corroboree, is one of four species (see below) with a short body habitus and well developed hyposphaenium on pleonite-3 and small hyposphaenium on pleonite-2. Two other species with large hyposphaenium on pleonite-3 are: P. voeringi that has also small on pleonite-4, and P. moonwalk n. sp (see below) whose hyposphaenium on pleonite-3 is directed backward. Additionally, P. corroboree lacks a projection on the inner margin of the cheliped dactylus that is well developed in P. voeringi 16 and has a slender endopod in uropod that is about four times as long as wide (endopod uropod is only three times as long as wide in P. moonwalk). Another species with a hyposphaenium on pleonite-3 is P. arctophylax, but it lacks hyposphaenia on pleonites 2 and 4.

Paranarthrurella dissimilis (Lang, 1972)
For synonyms see Jóźwiak et al. 16 . Diagnosis (amended) 16  etymology. The species name is given for a dance with African origin in Angola. Noun in apposition.
Antenna (Fig. 14A) 0.7 times length of antennule; article-1 fused with body; article-2 1.8 L:W, 1.4x article-3, with short distodorsal seta, situated on apophysis in right angle to axis of the article; article-3 1.7 L:W, 1.3x article-4, with short distodorsal seta situated in right angle to axis of the article; article-4 5.5 L:W, 2.0x article-5, with microtrichia on dorsal margin, and at least seven short distal and subdistal setae; article-5 4.3 L:W, with distal seta; article-6 as long as wide, with four long and one short distal setae.

etymology. The species name is given for a dance of Polish origin. Noun in apposition.
Description of neuter, length 4.3 mm. Body (Fig. 21A,B)  www.nature.com/scientificreports www.nature.com/scientificreports/ Antenna (Fig. 22B) 0.7 times length of antennule; article-1 fused with body; article-2 2.5 L:W, 1.5x article-3, with distodorsal seta, set at right angle to axis of the article; article-3 1.25 L:W, 0.3x article-4, with distodorsal seta, situated in right angle to axis of the article; article-4 5.8 L:W, 1.5x article-5, with three simple and three penicillate distal setae; article-5 4.1 L:W, with distal seta; article-6 as long as wide, with four long and two short distal setae.
Cheliped (Fig. 26A) sclerite large triangular; basis 1.6 L:W; merus ventral margin longer than that of carpus, ventral seta not seen; carpus wider medially, 1.4 L:W, with two ventral setae, dorsal setae not seen; chela 1.3x carpus, propodus (palm) with one longer and four short setae on inner side and seta near dactylus insertion; fixed finger with two simple setae ventrally and three setae on cutting edge, incisive margin well calcified, with uneven blunt teeth; fixed finger and dactylus unifacial; dactylus weakly bent downward, with weak spines on cutting margin.
Uropod  www.nature.com/scientificreports www.nature.com/scientificreports/ etymology. The species that was found off Brazilian coast is called after a samba -Brazilian musical genre and dance style. Noun in apposition.
Distribution. Paranarthrurella samba sp. nov. is known so far only from the type locality off Brazilian coast, at depth of 3459 m.

Remarks.
Paranarthrurella samba is one of five Paranarthrurella species with an elongate body habitus and swollen pleotelson with its apex directed downward. In contrast to P. polonez has smooth lateral margins on the pleonites. The species can be distinguished from P. spinimaxillipeda by the length of pereonite-1 that is 1.7 times as long as wide (clearly shorter in P. spinimaxillipeda). Finally, the new species is distinguished from P. dissimilis by lack of strongly serrate dorsodistal spine in pereopods 4-6 propodus (present in P. dissimilis, see Lang 1972, p. 234).
Distribution. The species is known only from the type locality located off the Argentinian coast at the depth 2440-2707 m.
Remarks. Paranarthrurella tango sp. nov. is the third species with short body and hyposphaenium only on pleonite-3; this hyposphaenium is small, distinguishing it from P. arctophylax (see remarks page 15). Another species with a small hyposphaenium on pleonite-3 is P. rocknroll. Those two species can be distinguished by character of the distal seta on the propodus of pereopods 4-6 that is strongly serrate in P. rocknroll and serrated in P. tango.

Paranarthrurella voeringi (G.O. Sars, 1877)
Tanais  Remarks. P voeringi is the only species that has hyposphaenia on pleonites 2 to 4. Remarks. Paranarthrurella sp.1 and Paranarthrurella sp.2 are genetically discriminated species, but both are morphologically identical with P. voeringi. The latter species was considered cold-water taxon 8 recorded in relatively shallower waters (<2000 m) than the congener -P. arctophylax, which although known from the depth (>2000 m), was considered a "warm" water species (Table 5, Fig. 39). In the material studied by us, all of the individuals had distinctive hyposphaenia on pereonites from 2-4, thus morphologically are similar to P. voeringi  Table 5) and current study. (2019) 9:18720 | https://doi.org/10.1038/s41598-019-53446-1 www.nature.com/scientificreports www.nature.com/scientificreports/ and both occur at the depths typical for P. voeringi (St 1155 and 1191, depth 1577-1578 and 2177-2174 m, respectively; Table 5). Generally, we might assume that one of our two genetically distinguished species is P. voeringi. However, the decision which of them would truly represent the latter requires further molecular investigations which will include individuals of P. voeringi from a type locality.

Discussion
Diversity. The new family Paranarthrurellidae is so far represented by 16 nominal species classified to two genera. It is a rather less diverse family that holds 14 th place on the list of 21 tanaidomorph families gathering 1.8% of all tanaidomorph species ( Table 6). The two paranarthrurellid genera, Paranarthrurella and Armatognathia, which were removed from Paratanaoidea incertae sedis group, reduced the number of the taxa with uncertain family classification by seven species (<1%). Nevertheless, the group Paratanaoidea incertae sedis still contains 52 species and 29 genera (5.8% and 15.3%, respectively).  www.nature.com/scientificreports www.nature.com/scientificreports/ In case of the species analyzed by us, small but noticeable morphological differences distinguishing each species can be pointed out. In our material, most of them belonging to historical collections 2,88 , we were able to discriminate all the species based only on morphology according to phenotypic condition for species delimitation 89 . In many cases, morphological study is insufficient for identification of various groups of Peracarida crustaceans [90][91][92][93] including tanaids 94 (Jakiel et al. unpublished data).
The finding of previously undescribed species, as well as considerable cryptic diversity (i.e. high genetic divergence associated with high morphological similarity) is quite common in the vast and under-explored deep-sea [95][96][97] . Deep-sea peracarids are highly diverse 98 , and, in particular they appear to exhibit high degrees of cryptic and genetic diversity 99 including amphipods 91 ; isopods 92,93 ; tanaidaceans 94,100 , Jakiel et al. (submitted). In the material examined herein, high levels of genetic diversity in 18S and COI similarly revealed cryptic species whose morphology is indeed different upon thorough re-examination (i.e. Paranarthrurella sp.1, Paranarthrurella sp.2). phylogeny. Obtaining reliable quality DNA sequences for the deep-sea small crustaceans is a big challenge.
The lack of a reasonable amount of genetic data is a main obstacle for comprehensive phylogenetic analysis that could confidently corroborate the relationship between higher tanaidacean taxa and shed light on origin and radiation place of deep-sea tanaidaceans. So far, the only phylogenetic approach implies, that colonising the deep sea by tanaids might have happened more than once in the Paratanaoidea 31 . Yet, the long branches suggest an ancient diversification of the two main clades: (1) Tanaidae + Neotanaidae and (2) Paratanaoidea with Leptocheliidae and Nototanaidae as ancestors. In our limited molecular approach, all taxa representing here the superfamily Paratanaoidea cluster together in a clade in consistence with the results of Kakui et al. 31 . The Paranarthrurellidae nest within the families showing the most distinctive apomorphic characters, rather than with the Leptocheliidae, a presumed plesiomorphic group.
Distribution and bathymetry. The distribution of all taxa included in the Paranarthrurellidae indicates clearly the cosmopolitan and deep-water nature of the family (Figs 49 and 50). Tanaidacea are an abundant and diverse component of the benthic assemblages in all depths [100][101][102] with 15 families of the superfamily Paratanoidea represented in the deep-sea: i.e. Agathotanaidae, Akanthophoreidae, Anarthruridae, Colletteidae, Cryptocopidae, Leptocheliidae, Leptognathiidae, Nototanaidae, Paratanidae, Pseudotanaidae, Pseudozeuxidae, Tanaellidae, Tanaopsidae, Tanaissuidae, and Typhlotanaidae. The Paranarthrurellidae, which is absent on the shelf, would be the sixteenth family within this group. Its occurrence in the deep-sea shows a distributional