A new species of the genus Liljeborgia Spence Bate, 1862 (Crustacea: Amphipoda: Liljeborgiidae) associated with the burrows of the spoon worm Urechis unicinctus in the Sea of Japan

A new symbiotic species of liljeborgiid amphipods, Liljeborgia associata sp. nov., is described from the burrows of the spoon worm Urechis unicinctus (Drasche, 1880) (Annelida: Polychaeta: Echiura: Urechidae) in the southern part of Peter the Great Bay and Posjeta Bay in the Sea of Japan. The new species is mostly similar and probably related to Liljeborgia geminata Barnard, 1969, known from the Californian coasts of the USA, and Liljeborgia serratoides Tzvetkova, 1967, described from Posjeta Bay in the Sea of Japan, but can be clearly distinguished from all congeners by morphological features of mouthparts, appendages and telson. The new species is only the fourth in the family Liljeborgiidae to be described from the Russian coast of the northwestern Pacifi c and the fi rst in association with spoon worms (Echiura).


Introduction
A variety of symbiotic communities associated with soft-bottom deep-burrowing invertebrates were investigated in the Peter the Great Bay and Posjeta Bay in the Sea of Japan. The sampling of infaunal animals and symbiotic assemblage was carried out using a bait suction pump (yabby pump), which allowed us to study the diversity of burrowing crustaceans and their associates in Russian waters (e.g., Marin 2010Marin , 2015Marin , 2016Marin , 2018aMarin , 2018bMarin et al. 2011Marin et al. , 2013Marin & Kornienko 2014) with the description of several new crustacean (Marin 2013(Marin , 2017 and even a new phoronid species (Temereva & Chichvarkhin 2017). Numerous specimens of Liljeborgia sp. were also collected from the burrows of the spoon worm Urechis unicinctus (Drasche, 1880) (Annelida: Polychaeta: Echiura: Urechidae). Previously, this species was identified as L. serratoides/geminata in accordance with its similar morphology and distinctive coloration, however, the presence of several specific morphological features has allowed its separation into a new species described herewithin.

Specimen sampling and imaging
Sampling was carried out in the estuary of Volchanka River in Vostok Bay near the scientific station "Vostok" (42°51′14.48″ N, 132°46′47.24″ E) and in Astafieva Bay (42°36′45.6″ N, 131°12′24.8″ E), both within Peter the Great Bay, as well as Troitza Bay (42°38′60.0″ N, 131°07′27.8″ E) located within Posjeta Bay in Sea of Japan (see Fig. 1). These bays are well known by large populations of the common spoon worm U. unicinctus and other burrowing animals. The hosts and symbiotic community were collected using a bait suction pump (yabby pump), which is actively used in the sampling of deep burrowing animals (e.g., Eleftheriou & McIntyre 2005). Unfortunately, in our study it was not possible to measure the length or volume of the host's burrows. The collected specimens were photographed alive in situ using a Canon G16 digital camera and then fixed with 90% ethanol solution. The drawings were made using camera a lucida attached to an Olympus SZX10 stereo microscope.

Molecular study
To study the molecular genetic diversity, a fragment of the mitochondrial gene coding for cytochrome c oxidase subunit I (COI mtDNA) gene marker was amplified, sequenced and compared. Two female specimens (LEMMI) from Vostok Bay were used for the moleculargenetic examination. Total genomic DNA was extracted from abdominal and pereiopod muscle tissue using the innuPREP DNA Micro Kit (AnalitikJena, Germany) following the manufacturer's protocol. The COI gene marker was amplified with the help of primers «m13polylco» (TGTAAAACGACGGCCAGTGAYTATWTTCAACAAATCATAAAGATATTGG) and «m13polyhco» (CAGGAAACAGCTATGACTAMACTTCWGGGTGACCAAARAATCA) (Carr et al. 2011); 16S with the help of +16SA ('CGCCTGTTTATCAAAAACAT') and -16SH ('CCGGTCTGAACTCAGATCACG'); 28S with the help of +C1 ('ACCCGCTGAATTTAAGCAT') and -D2 ('TCCGTGTTTCAAGACGG'). PCR products were performed on amplificator T100 (Bio-Rad, USA) under the following conditions: initial denaturation at 96°C for 1.5 min followed by 42 cycles of 95°C for 2 min, 49°C for 35 seconds, and 72°C for 1.5 min, followed by chain extension at 72°C for 7 min. The volume of 10 µL of reaction mixture contained 1 µL of total DNA, 2 µL of 5 × PCR mix (Dialat, Russia) and 1 µL of each primer. The amplification products were separated by using gel electrophoresis of nucleic acids on a 1.5% agarose gel in 1 × TBE, and then stained and visualized with 0.003% EtBr using imaging UV software. DNA nucleotide sequences were determined using Genetic Analyzer ABI 3500 (Applied Biosystems, USA) and BigDye 3.1 (Applied Biosystems, USA) with direct and reverse primers. Unfortunately, the number of sequences of the representatives of the family Liljeborgiidae deposited in genetic databases is still very small. As the phylogenetic relations of studied species are out of our interest, the obtained DNA (COI mtDNA) data are presented in the paper without any analysis.

Type material and morphological study
The type material is deposited in the collection of Zoological Museum of Moscow State University, Moscow, Russia (ZMMU) and the Laboratory of Ecology and Evolution of Marine Invertebrates (LEMMI), A.N. Severtzov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia.
The body length (bl, mm), defined as the dorsal length from distal margin of head to the posterior margin of telson without the length of uropod III and antennae, is used as a standard measurement.
The terminology and the general model of the description are used in the study after d'Udekem d 'Acoz & Vader (2009) and d'Udekem d' Acoz (2010).

A1
= antenna 1 A2 = antenna 2 bl = body length Ep1-3 = epimeral plates 1-3 Diagnosis A1 with stout articles 2 (about as long as wide) and 3 (about 1.5 times as long as wide), article 2 with dorsal projection produced into a rounded lobe; palp of Md with long and slender articles, article 3 almost equal to articles 1 and 2, about 5 times as long as wide; palp of Mx1 with broad shovel-shaped article 1; stout articles 3 and 2 of A2, article 3 about as long as wide and article 2 about 3 times as long as wide; P1 and P2 with short and wide merus, about 3 times as long as wide; PP5-7 with slender propodal segments, about 6, 7 and 10 times as long as wide, respectively; posterodorsal area of Pleonites 1 and 2 produced into 3 small teeth of which the median one is the longest; Urosomites 1 and 2 with wellmarked dorsal crest; Telson with long distal teeth, accompanied by 2 interdental long and slender spines.

Etymology
The species is named after its symbiotic lifestyle.
antenna 2 (Fig. 2e). Article 3 about as long as wide, unarmed; article 2 about 3 times as long as wide, with 4 small dorsal spines, with 1 long distodorsal and 2 long simple distoventral spines, with simple setae ventrally; article 1 about 4-4.5 times as long as wide, with small dorsal spines, with 1 distoventral long spine, unarmed ventrally; flagellum with 13-15 articles. Labrum (Fig. 2a). Upper lip with labrum broader than long and smaller than epistome, apical margin sinuous. epistome (Fig. 2b). With rounded lobes, protruding in lateral view, armed with small setae dorsally. mandibLe (Fig. 2f). Lacinia mobilis large, anterior margin armed with 5 small strong teeth; palp consists of 3 long slender articles, similar in size, covered with long simple setae, article 1 almost equal to article 2, about 5-6 times as long as wide; article 2 about 5-6 times as long as wide; article 3 almost equal to articles 1 and 2, about 5 times as long as wide.   (Fig. 2g). Outer plate with 8 large slender spines, ventrally denticulated; inner plate with a single long plumose seta; palp consists of 4 articles, article 1 shovel-shaped, with broad median part, about 1.5-2 times as long as wide, with 8-9 sharp robust spines along anterior margin, and simple small setae along dorsal margin. maxiLLa 2 (Fig. 2h). Inner and outer plates distally rounded, robust, covered with numerous long simple setae along anterior and lateral margins. maxiLLiped (Fig. 2i). Palp consists of 4 articles: article 1 of palp unarmed, article 2 with a cluster of long setae distodorsally, small robust setae distally and long simple setae along the inner margin, outer margin unarmed; article 3 with a cluster of long simple setae along anterior border and distal part of inner margin, outer margin unarmed, article 4 (dactylus) slender, curved, about 0.8 times as long as article 3, unarmed; outer plate with 13-15 robust spines along medial border (distal spines are narrow and rather long); inner plate covered with small setae along anterior margin.
GnatHopod 1 (Fig. 3a). Coxa trapezoidal, with anterior medial setae only, posterior border weakly concave; basis slender, about 6-7 times as long as wide, with ventral projection in proximal part, with long simple setae; ischium about as long as wide, with long simple setae along distoventral border; merus about 1.5 times as long as wide, sharping distoventrally, with several groups of setae along ventral margin; carpus slender, with blunt distoventral process, reaching ⅓ of palm length, not reaching propodal group of strong spines, armed with several groups of setae; propodus about twice as long as wide both in male and females (Fig. 3b), with convex ventral margin, armed with small simple setae along almost all its length, with a depression and 3 long simple setae in proximal part level with end of dactylus, dorsal margin straight and unarmed; dactylus with 5-7 triangular teeth.
GnatHopod 2 (Fig. 3c). Very similar to Gn1 in shape and slightly larger in size; coxa triangulo-elliptic; basis slender, about 6 times as long as wide, with long simple setae; ischium about as long as wide, with long simple setae along distoventral border; merus about 1.5 times as long as wide, sharpening distoventrally, with several groups of setae along ventral margin; carpus slender, with blunt distoventral process, reaching ¼ of palm length, not quite reaching propodal group of strong spines, armed with several groups of setae; propodus about 2 times as long as wide in both male (Fig. 5a) and females (Fig.  3d), dorsal margin straight and unarmed, with convex ventral margin, armed with small simple setae along almost entire length, with a depression and 3 long simple setae in proximal part level with end of dactylus; dactylus with 10-11 triangular teeth. pereiopod 3 (Fig. 4a). Coxa elliptic and narrow; basis slender, about 8-9 times as long as wide, with straight margins, covered with long simple setae; ischium about as long as wide, with long simple setae along distoventral border; merus broad, about 3 times as long as wide, slightly longer than carpus and equal to propodus, dorsal margin produced forward, with long simple setae along dorsal and ventral margins; carpus about 3 times as long as wide, with straight margins, unarmed dorsally and with simple long setae along ventral margin; propodus about 4-4.5 times as long as wide, slightly curved, unarmed dorsally and ventrally, with several long simple setae at distodorsal angle; dactylus of normal length, slender, weakly curved, slightly shorter than propodus and equal to carpus in length. pereiopod 4 (Fig. 4b). Coxa wide, with parallel anterior and posterior border, ventral border with 3 wellmarked serrations; basis slender, about 7-7.5 times as long as wide, with straight margins, covered with long simple setae; ischium about as long as wide, with long simple setae along distoventral border; merus broad, about 3 times as long as wide, longer than carpus and slightly shorter than propodus, dorsal margin produced forward, with long simple setae along dorsal and ventral margins; carpus about 2.5-3 times as long as wide, with straight margins, unarmed dorsally and with long simple setae along ventral margin; propodus about 5-5.5 times as long as wide, slightly curved, unarmed dorsally, with several long simple setae at distodorsal angle, with 5 tiny spines along ventral margin; dactylus of normal length, slender, weakly curved, slightly shorter than propodus and slightly longer than carpus.
pereiopod 5 (Fig. 4c). Coxa subquadrate, with medially concave ventral margin, unarmed; basis broad, with anterior and posterior border convex; anterior border with 12-13 small spines accompanied by small setae, posterior border with well-marked serration; ischium about as long as wide, with large simple spine on anterodistal corner; merus about 4 times as long as wide, with 4 short anterior spines, 1 large distodorsal spine paired with a spinule, and 1 simple distoventral spine; carpus about 4 times as long as wide, slightly shorter than merus, unarmed posteriorly, with 3 small anterior spines, and with a distal pair of spines anteriorly and posteriorly; propodus (Fig. 4d) about 6 times as long as proximal width, slightly tapering distally, with 10 anterior spines and series of long medial setae; dactylus distinctly curved and of normal stoutness, with tip entire, about ½ of the length of propodus. pereiopod 6 (Fig. 4e). Coxa subquadrate, with medially concave ventral margin, unarmed; basis broad, with anterior and posterior border convex; anterior border with 10 small spines accompanied with small setae, posterior border with well-marked serration; ischium about as long as wide, with large simple spine on anterodistal corner; merus about 4 times as long as wide, with 5 short anterior spines, a pair of large distodorsal spines and 1 simple distoventral spine paired with a spinule; carpus about 4.5-5 times as long as wide, slightly shorter than merus, unarmed posteriorly, with 4 small anterior spines, and a distal pair of spines anteriorly and posteriorly; propodus (Fig. 4f) about 7 times as long as proximal width, tapering distally, with 4 long anterior spines and series of long medial setae; dactylus distinctly curved, sharp, about ⅓ of the length of propodus. pereiopod 7 (Fig. 4g). Coxa almost rectangular; basis greatly broad, almost round, with convex anterior border and strongly convex posterior border; anterior border with 12 small conical spines, posterior border serrated, distal border with strong long spine; ischium about as long as wide, unarmed; merus broad, about 2.5-3 times as long as wide, with 4 anterior small spines and pair of anterodistal simple spines, 3 posterior spines and a pair of long posterodistal long spines; carpus about 5.5 times as long as wide, slightly longer than merus, with single or paired anterior spines and long simple setae posteriorly; propodus about 10 times as long as wide, tapering distally, with 4 small anterior spines and long setae posteriorly; dactylus straight, very long and slender, entire, about ⅓ of the length of propodus.
CoLoration. General coloration of body and all appendages translucent white with large light and dark brown spots; some segments of appendages dark red or with dark red spots; eyes brightly white (Fig. 1). body size. Largest collected female = bl 6.5 mm; largest collected male mostly similar in size to largest female, with bl 6.5 mm.

Taxonomic remarks
The new species is distinctly morphologically similar to L. geminata and L. serratoides. At the same time, the new species can be clearly distinguished by several morphological features not known from the latter species.
From L. geminata (see description, presented by Barnard (1969)), the new species can be distinguished by the following characters: smaller dorsal crest on urosomites 1 and 2 (Fig. 5d); Mx1 with wide, shovelshaped article 3 of palp (Fig. 2h); shorter and wider merus of P1 and P2 (see Fig. 4a-b); slender propodal segments of PP5-7 (see Fig. 4c-g); absence of long proximal seta of peduncle of U1 (see Fig. 5f); shape of medial teeth of telson (Fig. 2k), which are significantly longer and accompanied by 2 long interdental spines. At the same time, L. geminata seems to be more closely related to the new species by its very similar body coloration, the presence of a small dorsal crest on urosomites 1 and 2, short articles of peduncle of A1, relatively stout distal segments of PP3-4 (especially merus), and other morphological features that clearly separate both species from their relative L. serratoides.
From L. serratiodes (after Tzvetkova 1967), the new species is easily separated by the following characters: presence of a dorsal crest on urosomites 1 and 2; stout articles of peduncle of A1 and A2 (Fig.  2d-e); wide distal article of palp of Mx1; stout and wider distal segments, especially merus, of PP1-2; slender propodal segment of PP5-7; length and shape of medial teeth of telson, which are significantly longer and accompanied by 2 long interdental spines. In addition, relatively stout and wide segments of PP3-4 are distinguishing features that separate the aforementioned species from tropical relatives, such as L. japonica and L. serrata (e.g., Nagata 1965; Azman & Othman 2013).
From L. japonica and L. serrata, known from the Seto Inland Sea in southern Japan (see Nagata 1965), the new species can be distinguished by longer rostrum, mandibular palp with longer and slender articles, especially distal one; shorter and stouter articles of peduncle A1 and A2; stouter distal segments of PP3-4, especially stout and wide merus of P3; significantly shorted dactyli of PP5-7; slender rami of U3 and longer median teeth of telson.
From Liljeborgia hwanghaensis Kim & Kim, 1990, known from the Korean coasts of the Yellow Sea (37°23′ N, 126°35′ E) (see Kim & Kim 1990), the new species can be clearly separated by the posterodorsal armature of pleonites 1 and 2 (3 teeth in the new species vs 5 teeth in L. hwanghaensis) and shorter median teeth of telson.
-Urosomites 1-2 without dorsal crest, peduncle of A1 with slender articles (article 3 about 2-2. 5 times as long as wide; article 2 about 1.5 times as long as wide). NW Pacific: Posjeta Bay in the Sea of Japan, intertidal ............................................................. Liljeborgia serratoides Tzvetkova, 1967 8 Some other species of the genus Liljeborgia are known from the region and the Sea of Japan (e.g., Ishimaru 1994;Ren 2007). For example, Ishimaru (1994) notes Liljeborgia aequabilis Stebbing, 1888 from the tropical part of the Seto Inland Sea influenced by the Kuroshio Current. This species is actually recognized in Australia, New Zealand and the Sulu Sea as a common associate of hermit crabs (Stebbing 1888;after Hurley 1954;see also Vader 1995) and by most morphological features, especially long and slender segments of PP3-4 and the form of telson (see above), belong to the tropical representatives of the genus, such as L. japonica and L. serrata, or possibly represent an undescribed species. These species are known from warm (tropical) waters and probably are not present in the boreal zone of the northern Pacific Ocean.

Discussion
The symbiotic communities associated with deep-burrowing invertebrates are very diverse and still include many undescribed species. Burrows of spoon worms of the genus Urechis as well as almost all large echiuroid worms are inhabited by various symbiotic animals, such as shrimps, crabs, bivalves and even fishes (e.g., Fisher & MacGinitie 1928;Anker et al. 2005Anker et al. , 2015Itani et al. 2005;Goto & Kato 2012;Marin 2014;Goto et al. 2017 Liljeborgia associata sp. nov. is probably only associated with the burrows of the spoon worms, since no free-living specimens of the genus Liljeborgia or any other liljeborgiid amphipods were sampled on the surface of muddy or sand-bottom substrates in the Peter the Great Bay and Posjeta Bay in the Sea of Japan during the present or previous studies (e.g., Gurjanova 1951;Tzvetkova 1967). Moreover, Tzvetkova (1967) described a quite common L. serratoides from mussel communities, overgrowing rocks and hard substrates in Posjeta Bay of the Sea of Japan, while Liljeborgia species were not recorded on muddy and soft-bottom substrates that dominate the bay. This fact indirectly proves that Liljeborgia associata sp. nov. rarely emerges from the host's burrows and does not inhabit the surrounding softbottom substrates as a free-living species.
At the same time, representatives of the family Liljeborgiidae have been observed in association with other marine invertebrates. Often Liljeborgia spp. are observed is association with hermit crabs (Taylor 1979;Vader 1995 fig. 1), similar to other symbiotic species of the family (Barnard 1959;Mills 1962: fig. 2 (Mills, 1962) was found and described inside the tube arrays of the polychaete worm Clymenella torquata (Leidy, 1855) (Polychaeta: Maldanidae) in the intertidal flats of Barnstable Harbor, Massachusetts (Mills 1962;Sanders et al. 1962). These amphipods inhabit about 20-25% of tubes with live polychaetes (Mills 1962) and show a positive response to polychaetes mucus (Batcheler & Mills 1965). Also, Idunella albina (J.L. Barnard, 1959) was commonly collected from dense beds of echiuroid worms off Californian coasts (Barnard 1959), however, the direct relationship of this species with spoon worms is not shown. An undescribed species of the genus Idunella is reported from the burrows of the mud shrimp Upogebia affinis (Say, 1818) (Crustacea: Decapoda: Upogebiidae) (Fox & Bynum 1975), while individuals of the genus Idunella (= Listriella) are common inhabitants of maldanid and terebellid polychaete tubes (Barnard 1969;Bousfield 1973) and are reported with holothurians (Fox & Bynum 1975;Vader 1978). It is probable that associations of these animals with marine invertebrates are much more common than is currently known.
As mentioned above, according to morphological data, the new species is closely related to L. geminata. Unfortunately, minimal genetic data on the family Liljeborgiidae are presented in GenBank (NCBI) database, and genetic data are not available for the relative congeners of the described species. At the same time, it can be assumed that Liljeborgia associata sp. nov. and L. geminata represent a pair of closely related vicariant taxa known as boreal amphi-Pacific species. Such species had a wide distribution area in the northern part of the Pacific Ocean during the Pliocene-Middle Pleistocene period, characterized by a higher water temperature and sea level, which allowed the boreal fauna to move from east to west and even through the Arctic basin [as Great Trans-Arctic Biotic Interchange (GTAI)] (Einarsson et al. 1967;Durham & MacNeil 1967;Briggs 2003). During the temperature decreasing in the Sea of Okhotsk and the Bering Sea in the Late Pleistocene (about 2 Ma ago), the ranges were subsequently divided into the western and the eastern distribution ranges (Zenkevitch 1963;Briggs 2003;Maggs et al. 2008;Marin 2018a). Further genetic data will allow a more detailed description of the phylogenetic relationships within the representatives of the family from the northern part of the Pacific Ocean.