Diversity of Pacific Agathotanais (Peracarida: Tanaidacea)

Agathotanais is one of the seven genera classified into the family Agathotanaidae. So far, 12 species have been described for the genus, seven of which are known from the Pacific. However, considering the present poor state of knowledge on deep-sea environments, a much higher number of Agathotanais species than currently known can be suspected. Among the studied material, collected from below 1,000 m during five deep-sea expeditions in different parts of the Pacific Ocean, we identified eight species: two of them were already known to the science and five species were identified as new to knowledge and their formal description is presented in the paper: two from the North West Pacific (the Sea of Okhotsk and Kuril-Kamchatka Trench), two from the Central Pacific (Clarion-Clipperton Fracture Zone), and one from the Australian slope. The eighth Agathotanais species in our material was determined using a molecular approach, but it was represented by only one partially destroyed individual and could therefore not be formally described. The proportion of Agathotanais collected at the Sea of Okhotsk was the highest (22%), whereas the numbers were substantially lower for the Kuril-Kamchatka Trench, and the Central and the Southern Pacific. Molecular analyses confirmed the monophyly of Agathotanais and Paragathotanais and a close relationship between both genera. Moreover, a close relationship between the two Australian species was revealed. As a result of our findings, the number of species known from the Pacific increased from 5 to 11, with the total number of species in this genus increasing from 12 to 17. An updated identification key for Agathotanais species is given.


INTRODUCTION
Genus Agathotanais was established to allocate Agathotanais ingolfi Hansen, 1913, discovered off Iceland during the Danish Ingolf Expedition (Hansen, 1913). It was described and marked as the most aberrant among all tanaids due to its rudimentary antenna, cheliped attached directly to the cephalothorax, the appearance of the pleopods in juvenile males (Hansen, 1913), and the setulose surface of its body (Larsen, 2005;Jóźwiak and Jakiel, 2012;Kakui and Kohtsuka, 2015). Hansen (1913) placed Agathotanais within the family Tanaidae Dana (1849), the only existing tanaidacean family at that time, but few decades later the Agathotanais, together with Paragathotanais, was transferred to the newly erected family Agathotanaidae Lang, 1971. The family was successively supplemented by newly erected genera and new species 1. A total of 634 specimens were collected in the Sea of Okhotsk during the German-Russian expedition SokhoBio (Sea of Okhotsk Biodiversity Studies) aboard the RV Academic M.A. Lavrentyev between July and August of 2015 (Malyutina et al., 2015). Specimens of Agathotanais were found in twelve epibenthic sledge (EBS) samples. 2. A total of 70 specimens were collected in the KKT and the adjacent abyssal zone during two expeditions: KuramBio (Kuril-Kamchatka Biodiversity Study, NW Pacific abyssal, and western KKT slope) and KuramBio II (KKT slope) in 2012 and 2016, respectively aboard the RV Sonne. Agathotanais was present in 12 EBS samples taken during KuramBio and KuramBio II. 3. A total of 21 specimens were collected in 2015 in the Central Pacific (Clarion-Clipperton Fracture Zone, CCZ) during the EcoResponse (SO-239) cruise, one of the Joint Project Initiative Oceans (JPIO) expeditions. From a total of eleven EBS deployments, Agathotanais was found in the following License Areas: Bundesanstalt fur Geowissenschalfen und Rofstoffe (BGR, Germany); Interoceanometal Joint Organisation (IOM); Global Sea Mineral Resources NV, Belgium (GSR); Areas of Particular Environmental Interest 3 (APEI3). 4. Five specimens were collected off the SE Australian coast during the ABYSS (Sampling the Abyss) expedition in 2017 from aboard the RV Investigator. Agathotanais were found in three EBS samples. 5. Two specimens collected in the continental slope off SE Australia, during the SLOPE campaign in 1988 and 1994. Agathotanais was found in two samples collected by dredging.
Except for the SLOPE collection that was preserved in formalin, all specimens were fixed in 96% ethanol. Specimens from the Sea of Okhotsk, the KKT, and Central Pacific have been loaned from the Zoological Museum Hamburg (ZMH), Natural History Museum in Frankfurt (NHM) and A.V. Zhirmunsky National Scientific Center of Marine Biology in Vladivostok (MIMB). Material from the Australian and Tasmanian coast has been loaned from the Museum Victoria in Melbourne (catalog numbers start with J and NMV).

Phylogenetic and Genetic Distance Analyses
The analyses included 16 specimens from the Sea of Okhotsk (SokhoBio collection), 16 from the KKT (KuramBio collection), 21 from the Central Pacific (JPIO collection), and two specimens from Australia (ABYSS collection). For the DNA extraction, the whole specimen was taken as starting material using sterile needles and following the Chelex (InstaGene Matrix, Bio-Rad) method as in Palero et al. (2010). The ribosomal RNA18S and the histone H3 genes were amplified using a 25 µl-volume reaction containing 13 µl AccuStart II GelTrack PCR SuperMix, 10 µl H 2 O, 0.5 µl of each primer (10 pmol/µl) and 2 µL DNA template. The 18S fragments were amplified using the universal primers SSU_F04 and SSU_R22 (Blaxter et al., 1998) following the protocol: 95 • C for 2 min, 95 • C for 1 min, 57 • C for 45 s, 72 • C for 3 min, for 35 cycles, and a final elongation of 10 min at 72 • C. The H3AF and H3AR fragments (Colgan et al., 1998) were obtained according to the protocol: 95 • C for 3 min, 95 • C for 30 s, 50 • C for 30 s, 72 • C for 1 min, for 35 cycles, and a final elongation of 15 min at 72 • C. A 2 µl-aliquot of the PCR product was visualized in a Midori Green-stained (Nippon Genetics) 1.5% agarose gel to verify its quality and length. PCR purification and sequencing using forward and reverse primers were carried out by MACROGEN (Amsterdam, Netherlands). Consensus sequences were built using Geneious version 9.1.3 1 and compared with the GenBank database with BLAST (basic local alignment search tool, NCBI) (Altschul et al., 1990) to discard contamination from non-arthropod sources. Sequences were aligned using the option L-INSi of MAFFT (multiple alignment using fast Fourier transform) (Katoh and Standley, 2013), as implemented in Geneious. Genetic distances were calculated using pairwise distances and runon p-distance model using MEGA 7 (Molecular Evolutionary Genetics Analysis, Pennsylvania State University) (Kumar et al., 2018). Before running molecular phylogenetic analyses, the most suitable nucleotide substitution model was selected according to the AICc (The Akaike information criterion) and BIC (the Bayesian information criterion) criteria as implemented in MEGA 7 (Kumar et al., 2018). The aligned sequences and selected evolutionary model were then used to obtain a Maximum Likelihood phylogenetic tree in BEAST (BEAST Developers) (Drummond et al., 2012). Node support was evaluated with, bootstrap replicates. MCMC (Markov chain Monte Carlo) analyses were set for 10 million generations whereas all Effective Sample Size (ESS) values were calculated with Trace Analysis Tool (Tracer v1.5.0) software (Rambaut et al., 2018). To analyze the MCMC outputs Tree Annotator v1.7.5 (Drummond and Rambaut, 2007) was used with the default parameters.

Species Description
Chemically sharpened tungsten needles were used for the dissection of the individuals selected for detailed morphological analyses. The dissected appendages were placed on a microscope slide on a drop of glycerine, protected with a cover glass, and sealed with a ring of melted paraffin . Drawings were prepared using a light microscope (Nikon Eclipse 50i, Japan) equipped with a camera lucida. Publication-quality illustrations were prepared using a digital tablet and Adobe Illustrator (Adobe inc.) (Coleman, 2003). Total body length (BL) was measured along the main axis of symmetry from the frontal margin to the end of the telson. Body width (BW) was measured at the widest point along the main axis of symmetry. The measurements of specimens were made with the help of a camera connected to the microscope (Nikon Eclipse Ci-L) using 1 www.geneious.com NIS-Elements View software 2 . The morphological description follows Jakiel et al. (2019), where the expression 'Nx' replaces 'N times as long as' and 'N L:W' replaces 'N times as long as wide. ' For appendage ornamentation, we have followed the classification according to Garm and Watling (2013). Following types were used: (1) simple setae; (2) plumose setae; (3) serrated setae; (4) setules; (5) spines. For more specific ornamentation we used the following definition: (6) penicillate seta -with a tuft of setules located distally and with a small knob on which a seta is fixed to the tegument; and (7) spinule -short, tiny spine.
The stages recognized among the studied individuals were: manca (II and III), neuter, and a juvenile male. Specifically, the term 'manca' describes juveniles with (manca III) or without (manca II) buds on pereopod-6. 'Neuter' is retained for the development stage after manca that cannot be classified as either female or juvenile male. Juvenile male refers to individuals with initially developed pleopods.

Confocal Laser Scanning Microscopy
Two individuals from the SokhoBio collection (neuter: ZMH K-61187, juvenile male: ZMH K-61184) and two individuals from the KuramBio collection (neuter: ZMH K-61178) were used for imaging. Pictures were obtained with a confocal laser scanning microscope CLSM 780 (Zeiss) equipped with a Plan-Apochromat 10x/0.47 M27 objective and the InTune tunable excitation laser system (set to excitation at wavelength 555 nm).
Ethanol-preserved specimens were stained for 48 h with a mixture of equal volumes of saturated aqueous solutions of Congo red and acid fuchsin. Animals were sequentially washed in 80% glycerol and 100% glycerol and kept in 100% glycerol. Fluorescence was registered in the emission range 560-696 nm. Scan images were collected for further editing. Images were pseudo-colored in gold and reconstructed into a 2D stack image with maximum intensity projection using ZEN 2012 (Zeiss).

Scanning Electron Microscopy
One individual from the SokhoBio material (neuter: ZMH K-61187) and two individuals from the Clarion-Clipperton Fracture Zone material (neuter: ZMH-K-61146, neuter: ZMH-K-61156) were used for imaging with a Phenom ProX microscope. Specimens for the scanning electron microscopy analysis were initially rinsed with distilled water to remove the ethanol from their surface and tissues and then transferred to the SEM stub mounted in a temperature-controlled sample holder and frozen at -10 • C.

Diversity, Abundance, and Distribution
A total of 736 specimens of Agathotanais were classified into eight species, five of which were new to science: A. beatae n. sp., A. frutosae n. sp. A. jani n. sp., A. oharai n. sp., A. paleroi n. sp. One of the species was represented by only one individual as was FIGURE 1 | Evolutionary relationships of Agathotanaidae species inferred by using the Histone H3 (A) and 18S (B) sequences and the Maximum Likelihood method. The percentage of trees in which the associated taxa clustered together (bootstrap support) is shown next to the branches. Only values above 0.6 are shown.
not formally described (Agathotanais sp. abyss-1). Only two of the sampled species: A. hadalis and A. spinipoda, from the Japan Trench (NW Pacific) and the SE Australian Slope, respectively, were already known .
Most of the species studied here were represented by several specimens. However, A. frutosae was extremely abundant in the Sea of Okhotsk, represented by 634 specimens. The majority of them were found in the Kuril Basin, the deepest part of the Sea, Frontiers in Marine Science | www.frontiersin.org and only ten specimens were collected on the outer slope of Kuril Island (st. 9-7). In the KKT and the adjacent abyssal, we have recorded two species. One of them, A. hadalis, was identified on the basis of only three specimens, but A. paleroi, was relatively abundant and represented by 67 specimens that were all collected from the western side of the KKT, except for one specimen, found on the eastern side (st. 3-9). In the Central Pacific (CCZ), Agathotanais was represented by 21 specimens: four of them were identified as A. beatae and 17 as A. jani. Finally, six individuals were found off the Australian coast: two specimens were assigned to the species A. spinipoda, three to A. oharai, and one was classified as Agathotanais sp. abyss-1.

Phylogenetic and Genetic Distance Analysis
A total of four H3 and three 18S different haplotypes were obtained (Figure 1), representing Agathotanais species. The sequence alignments spanned 298 bp for H3 and 550 bp for 18S. For H3 the Kimura 2-parameter (K2+G+I) model showed the lowest AICc (AICc = 3614.52) and BIC (BIC = 3837.83) scores. The non-uniformity of evolutionary rates among sites was modeled using a Gamma distribution (+G = 1.39). The rate variation model revealed that some positions were evolutionarily invariable (+I = 44% sites). The Maximum Likelihood tree with the highest log-likelihood value (lnL = -1,770.90) was obtained.
For 18S the Kimura 2-parameter (K2+G) model showed the lowest AICc (AICc = 1313.07) and BIC (BIC = 1475.82) scores, which is considered to describe the best substitution pattern. Non-uniformity of evolutionary rates among sites was modeled using a Gamma distribution (+G = 0.32). The Maximum Likelihood tree with the highest log-likelihood value (lnL = -624.84) is shown in Figure 1.
All Agathotanais species included in the analyses were grouped into a well-supported clade. Additionally, the genetic clustering in the ML trees of the obtained haplotypes agrees with the morphological identification of taxa (see below).
The pairwise genetic p-distances between all the agathotanaid specimens ranged between 5.4 and 17.4% for H3, while for 18S sequences they ranged between 1 and 4% (Supplementary Table 2). The intraspecific genetic variation was very low, as expected given the limited sample size per species, represented in all cases by one haplotype for both markers. The evolutionary divergences for sequence pairs for H3 were largest between A. frutosae and both Australian species (A. frutosae -Agathotanais sp. abyss-1 1.174 ± 0.023 and A. frutosae -A. oharai 0.161 ± 0.022), while the lowest divergences were observed between Agathotanais sp. abyss and A. oharai (0.054 ± 0.012). Divergences between 18S sequences are shown in Supplementary Table 2.

Taxonomic Description
Genus: Agathotanais Lang, 1971 Diagnosis (amended after Larsen, 2005). Body strongly calcified, with a pitted surface. Pleon narrower or similar in width to pereon or pleotelson. Antennule with three articles. The antenna is usually one-articled (except A. manganicus antenna two-articled). The mandible molar process conical, left mandible lacinia mobilis is absent or reduced. Labium with the spiniform distal process and lateral process. Maxilliped bases partially fused. Cheliped slender, attached ventrally to cephalotorax, carpal sclerite absent (basal lobe truncated). Marsupium (where known) with four pairs of oostegistes. Pereopod coxa present. Uropods short, exopod reduced and fused with the basal article, endopod one-or two-articled. All appendages are covered with dense setules apart from the distal part of cheliped and pereopod dactyli.
Etymology: Species is named after Mrs. Beata Pełczyńska, mother of AP.
Cheliped ( Figure 4A) basis 1.5 L:W, naked; merus with two midventral setae; carpus 2.6 L:W, slightly shorter than propodus and fixed finger combined, with one midventral and one dorsodistal setae; palm 1.3 L:W, with two short spines near dactylus insertion, fixed finger subequal palm, with ventral seta and numerous long setules, cutting margin irregular, with one seta visible, terminal spine large and sharp; dactylus as long as fixed finger, with short dorsoproximal seta, cutting margin with two teeth; unguis robust and sharp.
Pereopod-5 was damaged in the dissected specimen, but in other individuals like pereopod-4.
Uropod ( Remarks. One-articled antenna distinguishes A. beatae n. sp. from A. ahyongi and A. manganicus, which have a fully reduced or two-articled antenna, respectively . Pereonites 4-5 longer than wide allow to separate A. beatae from A. brevis having those pereonites wider than long , and A. ingolfi that has these pereonites subequal in length (Hansen, 1913). A longer than wide pereonite-6 differentiates A. beatae from A. hadalis, A. frutosae, A. paleroi, and A. spinipoda, which have the pereonite-6 as long as wide (A. frutosae, A. paleroi, and A. spinipoda) or clearly wider than long (A. hadalis).
A serrated ventral margin of the unguis in pereopods 4-6 separates A. beatae from A. ghilarovi, A. hanseni, A. oharai, and Agathotanais sp. abyss-1, for which the unguis is unarmed (Lang, 1971;, and from A. jani which has two or three distinct teeth on the unguis. Furthermore, the new species differs from A. misakiensis by having a carapace shorter than the combined length of the two succeeding pereonites (for A. misakensis the carapace is subequal to pereonites 1-2). A two-articled uropodal endopod observed in A. cilacapicus separates this species from A. beatae that has a one-articled uropod ( Table 1).
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Etymology: Species is dedicated to Dr. Inmaculada Frutos (University of Łódź), our colleague and peracarid specialist, who collected tanaids during the SokhoBio expedition to the Sea of Okhotsk.
Left mandible (Figure 6D) incisor with a blunt tooth, lacinia mobilis short and rounded.
Right mandible ( Figure 6E) incisor with rounded tooth. Labium ( Figure 6F) with the spiniform distal process and lateral process, covered by numerous setae.
From the Agathotanais with elongated palm, A. frutosae can be distinguished by unarmed dactylus and unguis of pereopods 4-6. Unguis is serrated in A. hadalis, A. misakiensis, and A. toyoshioae; A. cilacapicus has a setulose unguis; A. jani has distinct teeth on unguis, and A. ingolfi has a small accessory spine on its dactylus, near the unguis. An unarmed dactylus and unguis are also present in A. hanseni, A. ahyongi and A. brevis. However, A. frutosae differs from A. hanseni and A. ahyongi in the length of the dorsodistal carpal seta of pereopods 2 and 3. It is 0.6 in relation to propodus in A. frutosae, and 0.8 and 1.0 in A. hanseni; A. ahyongi has only two short carpal setae in pereopods 2 and 3 (about 0.1 of propodus length) ( Table 1).
Left mandible ( Figure 9C) incisor with three small rounded teeth; lacinia mobilis rounded and short.
Right mandible ( Figure 9D) incisor with three rounded teeth. Maxillule endite (Figures 9E,E') with eleven robust distal spines of various lengths and numerous setules along outer and inner margin; palp not observed.
Maxilla ( Figure 9F) elongated and simple.  Labium ( Figure 9G) with the spiniform distal process and long lateral process, covered by numerous setae.
Maxilliped ( Figure 9H) endite with gustatory cups seta and numerous fine setae on distal margin; palp article-1 1.3 L:W, naked; article-2 1.4 L:W, with inner seta and numerous fine setae of various lengths on inner and outer margins; article-3 2.4 L:W, with two inner subdistal plumose setae, margins with numerous fine setae; article-4 1.7 L:W, with one subdistal and five serrated distal setae. Endites short with one midlength seta and numerous fine setae distally.
Cheliped ( Figure 10A) basis 0.9 L:W, naked; merus with midventral seta; carpus 3.0 L:W, marginally shorter than propodal palm and fixed finger combined, with two midventral setae and two dorsal (one proximal and one subdistal) setae; palm 2.5 L:W, with robust seta at dactylus insertion; fixed finger subequal to palm, with long ventral seta, cutting edge gently undulated with three setae, distal spine robust; dactylus as long as a fixed finger, with dorsoproximal short seta, cutting edge with proximal seta; unguis robust.
Intraspecific variation: Manca III: length 1.3-1.8 mm. Remarks. The presence of distinct pointed ventral teeth on the unguis in pereopods 4-6 is a unique character that allows distinguishing A. jani from congeners. In A. hadalis, A. misakiensis, A. toyoshioae, A. paleroi, and A. beatae only a weak serration is present Kakui and Kohtsuka, 2015), while in other Agathotanais species the unguis in those pereopods is unarmed (Table 1).
Etymology: The species is named in honor of Tim O'Hara, senior curator of the Marine Invertebrates Section in Museums Victoria (Melbourne) and specialist in biogeography.
Right mandible (Figure 12D) incisor with a broad triangular tooth.
Cheliped ( Figure 12G) basis 0.8 L:W, with small subventral seta; merus with midventral seta; carpus 2.5 L:W, marginally shorter than propodus and fixed finger combined, with one middorsal and one midventral setae; palm 1.2 L:W, with seta near dactylus insertion and a row of ventral setae; fixed finger 0.8× palm, with one ventral seta, cutting edge with three tubercles and three setae, terminal spine small; dactylus as long as a fixed finger, curved, naked; unguis moderate size.
Cheliped ( Figure 13I) basis 1.2 L:W, naked; merus with midventral seta; carpus 2.8 L:W, marginally shorter than propodus and fixed finger combined, with two midventral setae; palm 1.2 L:W, with seta near dactylus insertion and a row of ventral setae; fixed finger as long as palm, with one ventral seta, cutting edge with two tubercles and three setae; dactylus as long as a fixed finger, curved, naked; unguis moderate size.
Intraspecific variation: Juvenile male: maxilliped palp article-4 with four-five distal setae on the left and right palp, respectively.
Remarks. Agathotanais oharai belongs to the group of Agathotanais with a short cheliped palm (L:W 1.0-1.2), together with A. ghilarovi, A. splendidus, and A. paleroi (Table 1). It differs from A. paleroi in the unguis of pereopod-6, which is unarmed in A. oharai and serrated in A. paleroi. Additionally A. oharai has a pleon narrower than pereonite-6, what distinguishes it from A. splendidus and A. ghilarhovi, which have the pleon as wide as peronite-6 ( Table 1).
Etymology: Species is dedicated to Dr. Ferran Palero (University of Valencia), a great colleague, fellow, and peracarid specialist.
Left mandible ( Figure 15D) incisor with two blunt teeth, lacinia mobilis rounded and short-fused with incisor.
Right mandible ( Figure 15E) incisor with blunt tooth distally. Labium ( Figure 15F) with a spiniform distal process (broken, not pictured) and lateral process, covered by numerous setae.
Maxillule endite (Figure 15G) with ten distal spines and distal fine setae of different lengths.
Epignath (Figure 15I) elongated, tipped with plumose setae. Cheliped ( Figure 15J) basis 1.0 L:W, naked; merus with midventral seta; carpus 2.6 L:W, marginally shorter than propodus and fixed finger combined, with one middorsal and one midventral setae; palm 1.0 L:W, with seta near dactylus insertion and with a row of setae ventrally; fixed finger similar in length to palm, with midventral seta, cutting edge with tubercles and three setae, terminal spine sharp; dactylus as long as a fixed finger, cutting edge with two spines; unguis slender.
Antenna ( Figure 16I) one-articled, 3.4 L:W, tipped with a seta. Cheliped ( Figure 16J) basis 1.0 L:W, with the dorsodistal seta; merus with midventral seta; carpus 2.5 L:W, 1.6× palm, with midventral seta; chela palm 1.5 L:W, with seta near dactylus insertion and with row of ventral setae; fixed finger with ventral seta, cutting edge with two inner setae, and with three tubercles distally, distal spine sharp; dactylus as long as a fixed finger; unguis slender.
Juvenile male: length 2.4-2.9 mm; cheliped fixed finger cutting edge with one/three inner setae.
Remarks. Agathotanais paleroi n. sp., collected from the KKT, belongs to the species of Agathotanais with a short cheliped palm (L:W 1.0-1.2). It differs from other short-palm species by the appearance of the unguis of pereopod-6: serrated in A. paleroi but unarmed in A. ghilarovi, A. splendidus, and A. oharai. Furthermore, A. paleroi has a pleon narrower than pereonite-6, distinguishing it from A. splendidus and A. ghilarhovi with a pereonite-6 that is as wide as pleon (Table 1).
Left mandible (Supplementary Figure 1F) incisor with two blunt teeth, lacinia mobilis small and fused with incisor.
Right mandible (Supplementary Figure 1G) incisor with a blunt tooth.
Labium (Supplementary Figure 1H) with a spiniform distal process (broken, not pictured) and lateral process, covered by numerous setae.
Cheliped (Supplementary Figure 2A) basis 1.0 L:W, naked, rectangular; merus with midventral seta; carpus 2.2 L:W, marginally shorter than propodus and fixed finger combined, with two midventral, one dorsoproximal, and one dorsosubdistal setae; chela palm 1.4 L:W, with seta near dactylus insertion and with row of ventral setae; fixed finger 0.8× palm, cutting edge with three setae; dactylus as long as a fixed finger, cutting edge with short proximal seta.
Remarks: Agathotanais sp. abbys-1 belongs to the species of Agathotanais with a moderate elongated cheliped palm (L:W 1.3-1.4) ( Table 1). It differs from A. beatae by an unarmed pereopod-4 unguis (serrated in A. beatae). Moreover, in Agathotanais sp. abbys-1 the pleonites are narrower than pereonite-6, whereas in A. beatae they are similar in width. On the other hand, it can be distinguished from A. manganicus by a one-articled antenna (two-articled in A. manganicus). Finally, Agathotanais sp. abbys-1 differs from A. spinipoda by the propodus of pereopod-6. It is smooth in Agathotanais sp. abyss-1 but has a row of spines in A. spinipoda (Table 1).

DISCUSSION
The Pacific is a vast ocean with a high variety of benthic environments and ecosystems (Gage and Tyler, 1991). Many of its regions have never been explored, and its fauna and diversity remain simply unknown. This deficient biological recognition is particularly evident when it comes to the identification of a group of organisms that is particularly poorly understood (Błażewicz-Paszkowycz and Bamber, 2012) and it is seen in the proportion of the new taxa discovered (e.g., Larsen and Shimomura, 2007;Błażewicz-Paszkowycz et al., 2013;Bird, 2015;Jakiel et al., 2019Jakiel et al., , 2020. This work presented findings on just one genus of small macrobenthic peracarids, which was collected from several locations of the North Pacific. Only two of eight identified species were previously described, regardless of the fact that some areas had been previously investigated, e.g., the KKT Larsen and Shimomura, 2007) or the Clarion Clipperton Fracture Zone . As a result of our study, the number of species known from the Pacific has raised from five to ten, and the total number of species classified into this genus increases from 12 to 17.

Phylogenetic and Genetic Analyses
The current research presents the first results from studies on the extensive collections of Agathotanais performed in the frame of an integrative taxonomy approach, combining molecular and morphological techniques. So far, only five agathotanaid sequences were deposited at GenBank and only two of them were identified down to species level -A. ingolfi . In the present study, we upgrade the number of sequences to 12, adding three fragments of 18S from species: A. frutosae, A. jani, and A. paleroi, and four H3 sequences from A. frutosae, A. oharai, A. paleroi, and Agathotanais sp. abyss-1. These results, based on two markers, should be considered merely as the first step into more complex phylogenetic studies in the future. Nevertheless, the results allow us to confirm the monophyletic character of Agathotanais, as well as of another agathotanaid genus -Paragathotanais. Moreover, both genera grouped within the same clade in both obtained phylogenetic trees, confirming their close relationship, although for full phylogenetic resolution and testing of the monophyletic character of Agathotanaidae more genetic data including also other genera are needed.
Within the Agathotanais, A. oharai from the Australian slope and Agathotanais sp. abbys-1 from the Tasmanian slope showed a close relationship (Figure 1). Both species present similar body appearance, with similar sizes, a gently rounded lateral margin of the carapace, pereonites rectangular in dorsal view. They both reveal similar proportions of the antennule articles and ratios of length to width in antenna and uropod. The next similarities are the appearance of the pereopods, with elongated carpus and propodus in pereopods 1-3 and unarmed unguis in pereopods 4-6. The molecular and morphological similarities of both species are supported by a relatively small geographic distance between their known distributions.
Agathotanais oharai and Agathotanais sp. abbys-1 were located on the tree close to A. paleroi. Agathotanais paleroi is separated by several thousands of kilometers from the two species A. oharai and Agathotanais sp. abbys-1. The taxonomical characters that support genetic similarities are the appearance of the cheliped and the length of the distroventral setae on pereopods 2-3. All three species are characterized by a short or moderately short cheliped palm (L:W less than 1.4) and long dorsodistal setae on the carpus of pereopods 2 and 3 (0.7-0.8× propodus). These features distinguish these species from A. frutosae, with a long cheliped palm (1.6 L:W) and short setae on the carpus (0.4-0.6× propodus). Moreover, A. frutosae bears a pair of setae on the dorsal surface of the first pereonite, a character that is unique among Agathotanais species. The pereopods of A. frutosae are thicker and armed with strong setae (e.g., carpus and propodus of pereopods 1-3). The place of occurrence of A. frutosaea semi-enclosed sea -may influence the isolation of the species and the evolution of characters different from other Agathotanais species.

Distribution
The genus Agathotanais is a cosmopolitan taxon. It was recorded in temperate and tropical zones of the Atlantic, Indian, and Pacific Oceans (Kakui and Kohtsuka, 2015;Chim and Tong, 2021). So far, the genus is absent only from the south of the Antarctic Polar Front (Błażewicz-Paszkowycz and Siciński, 2014;Pabis et al., 2014), although one undescribed species of Agathotanais was registered for the slope of the Scotia Sea (Pabis et al., 2015). Since the slope and the abyssal zone of the Southern Ocean are vast and still unexplored areas (Brandt et al., 2007) it can be assumed that the distribution of Agathotanais in the Antarctic may be wider, although yet to be discovered.
Regardless of the wide zoogeographical distribution of the genus, each species of Agathotanais usually has a narrow zoogeographical range (with the exception of A. hanseni and A. ingolfi; see Kakui and Kohtsuka, 2015;Chim and Tong, 2021). They are often limited to a defined basin (e.g., sea or trench), although in the case of A. jani the boundary is not physically obvious. That species was, however, present only at the closest stations of the Central Pacific (CCZ), separated by a maximal distance of less than 1,000 km (Figure 2). The mechanisms which support the connectivity in deep-sea populations are not fully understood yet. The data on population genetics combined with biophysical transport models and trace-element signatures that scrutinized the dispersal potential of deep-sea fauna is just one order of magnitude larger than for shallow water fauna (Baco et al., 2016). Those findings question the paradigm of unlimited distribution of deep-sea species. It is not clear how tubebuilding small tanaids can sustain genetic connectivity for their low numbers and sparsely distributed populations. The presence in the deep-sea population of mobile males "swimming"  along with favorable hydrological regimes and near-bottom currents adds to the rationale of this phenomenon, although it does not explore the problem. Moreover, neither physical nor hydrological connections warrant an unlimited distribution. A. frutosae is known from the Sea of Okhotsk, isolated from the open Pacific by the Kuril Islands. Although hydrological contact between the sea and the Pacific is sustained by numerous straits (Bussol Strait is 2,300 m deep), they do not perform as a zoogeographical passage that would allow the species to disperse. trench, similar to other tanaids and isopods (Lörz et al., 2018;Bober et al., 2019;Jakiel et al., 2019).