Hidden burrow associates: macrosymbiotic assemblages of subtidal deep-burrowing invertebrates in the northern part of the Sea of Japan

The activity of deep-burrowing macrofauna strongly influences all biogeochemical processes in sublittoral soft sediments. Despite this key role, these organisms are difficult to sample and, thus, often remain ignored in environmental studies. This study is the first in comprehensively exploring the diversity of the macrosymbiotic communities associated with the dominant subtidal deep-burrowing invertebrates from the southern part of the Russian coast of the Sea of Japan, represented by the species of the genera Upogebia Leach, 1814 (Arthropoda: Crustacea: Decapoda) and Urechis Seitz, 1907 (Annelida: Polychaeta: Echiura). The associated symbiotic communities mostly consist of obligate, host-specific species, while those species found in burrows of both hosts are probably using them just as refuges. Most symbionts occurred solitary or in heterosexual pairs, likely due to aggressive and strictly territorial behavior. This is certainly a hidden biodiversity, as more than half of the species reported here were not previously known from these “relatively simple and well-studied” boreal marine ecosystems. Our findings also allowed us to describe a new species belonging to the symbiotic genus Hesperonoe Chamberlin, 1919 (Annelida: Polychaeta: Polynoidae), based on morphological and molecular evidences, the latter being here presented for this genus for the first time.


Introduction
Soft sediment communities are very diverse and play an important ecological role in local biogeochemical cycles (e.g., Rhoads 1974;Meysman et al. 2006). The composition and structure of these communities are greatly influenced by the activity of burrowing macrofauna, which modulates biogeochemical processes such as organic matter processing oxygenation or microbial activity stimulation (Levinton 1995;Aller and Aller 1998;Glud 2008) and are considered as ecosystem engineers in sublittoral sandy or muddy marine bottoms (e.g., Tamaki and Ueno 1998;Dworschak 2000;Sandnes et al. 2000;Felder 2001;Webb and Eyre 2004;Pillay and Branch 2011).
Numerous symbiotic species use burrows of larger marine invertebrates as protected habitats to escape predators, access food, and/or support less environmental stress (Itani 2002;Atkinson and Taylor 2005;Anker et al. 2005Anker et al. , 2015Goto and Kato 2012;Seike et al. 2012;Itoh and Nishida 2013;Henmi and Itani 2014a, b;Marin 2014;Lavesque et al. 2016;Henmi et al. 2017;Moyo et al. 2017). For example, more than 100 symbiotic species, from protozoans to fishes, are known to inhabit the burrows of large crustaceans (i.e., Gebiidea and Axiidea) along the US Pacific coast (e.g., Campos et al. 2009), while the fauna associated with echiuroids (spoon worms) include more than 50 specific symbiotic species, such as bivalves, polychaetes, brachyuran crabs, alpheid shrimps, copepods, and fishes (Anker et al. 2005). In Russia, the study of the diversity of burrowing crustaceans and their associated fauna has just begun, and numerous symbionts including crustaceans (Marin 2010(Marin , 2013(Marin , 2015Marin et al. 2011Marin et al. , 2013Marin and Turbanov 2016) and a new phoronid species (Temereva and Chichvarkhin 2017) have recently been reported. The parasitic fauna of Upogebia major in Vostok Bay, namely the bopyrid isopods Gyge ovalis (Shiino, 1939) and Progebiophilus sp. (Crustacea: Isopoda: Bopyridae) and the rhizocephalan Sacculina upogebiae Shiino, 1943 (Crustacea: Rhizocephala: Sacculinidae), also represent new records for the Russian coast of the Sea of Japan. However, a large number of the small inhabitants of these communities still remain unknown likely because they are simply not caught by standard sampling gears. The importance of the associations between the burrowing macrofauna and their symbionts in a given ecosystem is difficult to estimate so that having new data on their diversity and biology will help to take these animals into account in future ecological studies.
Deep-burrowing crustaceans and spoon worms are two of the main groups of soft-bottom engineer species in Peter the Great and Posjeta Bays of the Sea of Japan, which are known to occur in large concentrations, for example, in Vostok Bay (Selin 2013(Selin , 2014(Selin , 2015(Selin , 2017. The shrimp Nihonotrypaea japonica (Ortmann, 1891) (Decapoda: Callianassidae) may reach up to 200 inds/m 2 , which represent about one-third of the total local macrozoobenthic biomass (Selin 2015). Virtually all bottoms from 0.2-to 3-m depth in the area are excavated by the several meters long burrows of the ghost shrimp Upogebia major (De Haan, 1841) (Decapoda: Gebiidea: Upogebiidae) (Nickell and Atkinson 1995;Kinoshita 2002), which may reach up to 117 mm in body length (Selin 2017), as well as the spoon worm Urechis unicinctus (Drasche, 1880) (Polychaeta: Echiura: Urechidae), reaching about 300 mm in body length (pers. observ.). Nevertheless, the symbiotic assemblages inhabiting these burrows in the region were out of scientific interest.
This article is a part of the project attempting to evaluate the biodiversity of shallow-water infaunal organisms, which revealed the highly diverse symbiotic communities mostly composed of undescribed symbiotic species. Moreover, a careful morphological observation and molecular analysis revealed a new species of the rare symbiotic genus Hesperonoe Chamberlin, 1919 (Polychaeta: Polynoidae), known exclusively from the Northern Pacific (Skogsberg 1928;Hartman 1968;Averincev 1990;Buzhinskaja 2013;Hong et al. 2017;Uschakov and Wu 1965).

Sample collection and treatment
Samples were mainly collected in the estuary of the Volchanka River in the Vostok Bay, near the scientific station "Vostok" (42°51′ 14.48″ N, 132°46′ 47.24″ E), and in the Troitza Bay (42°38′ 60.0″ N, 131°07′ 27.8″ E) (see Fig. 1), where the burrowing infauna was dominated by the crustaceans Nihonotrypaea japonica and Upogebia major (Marin and Kornienko 2014;Selin 2015Selin , 2017Selin , 2019, at least in summer from 2009 to date, as well as in the Astafieva Bay (42°36′ 52.2″ N, 131°12′ 01.1″ E), where a large population of Urechis unicinctus was studied from 2009 to 2012 (Fig. 1). Hosts and the associated symbiotic community were collected subtidally by scuba diving using a bait suction pump (yabbypump) (Eleftheriou and McIntyre 2005), which did not allow us to measure the length and volume of the burrows, neither to obtain quantitative estimates of the number of symbionts. To obtain reliable data on a qualitative analysis of the symbiotic communities, at least 50 burrows of each of the host species were examined.
Once preserved, the specimens were photographed under a Leica M165C stereomicroscope linked to a Leica IC80HD digital camera. Scanning electron microscope (SEM, Tescan Vega TS5130MM) micrographs were made after critical point drying and coating with 300 Ǻ of gold, at the Laboratory of Electronic Microscopy of the A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences. All symbionts were measured to the nearest 0.1 mm using a calibrated ocular micrometer under a Leica M165C stereomicroscope.
The similarity of the communities was measured with the Sørensen Index (SI) (Sørensen 1948) as SI = 2С/(S1 + S2), where C is the number of species the two communities have in common, S1 is the total number of species found in community 1, and S2 is the total number of species found in community 2; Jaccard Index (JI) (Jaccard 1908(Jaccard , 1912 was calculated as JI = sc/(sa + sb + sc), where sa and sb are the numbers of species unique to samples a and b, respectively, and sc is the number of species common to the two samples; Sørensen-Dice Index was calculated as SD = 2a/(a + b) + (a + c) (Dice Lee 1945;Sørensen 1948).

Molecular analysis
Total genomic DNA was extracted from muscle tissue using the innuPREP DNA Micro Kit (Analytik Jena, Germany) following the manufacturer's protocol. The gene marker of mitochondrial cytochrome c oxidase subunit I (COI mtDNA) was amplified with the help of primers «m13polylco» (TGTAAAAC GACGGCCAGTGAYTATWTTCAACAAATCATA AAGATATTGG) and «m13polyhco» (CAGGAAAC AGCTATGACTAMACTTCWGGGTGACCAAARAATCA) (Carr et al. 2011), mitochondrial 16S small subunit rRNA (16S rRNA) with the help of +16SA (′CGCCTGTTTATCAA AAACAT′) and -16SH (′CCGGTCTGAACTCAGATCACG′), and nuclear 28S large subunit rRNA (28S rRNA) with the help of +C1 (′ACCCGCTGAATTTAAGCAT′) and -D2 (′TCCGTGTTTCAAGACGG′). All obtained sequences are deposited in GenBank (NCBI) database (https://www. ncbi.nlm.nih.gov/genbank/). Consensus of complementary sequences was obtained with MEGA 7.0. The best evolutionary substitution model was determined using MEGA 7.0 and jModeltest2.1.141 via the CIPRES Science Gateway V. 3.3 (http://www.phylo.org/). Kimura's two-parameter (K2P) (Kimura 1980) substitution model was calculated using MEGA 7.0 for pairwise comparisons of sequence divergence between species based on the number of nucleotide substitutions. Phylogenetic analysis was performed for COI using RAxML v.8.0.0 with GTR+I+G evolutionary model for maximum likelihood (ML) analysis. Additional dataset of COI mtDNA sequences of the representatives of the family Polynoidae and related taxa was taken from GenBank (NCBI) database. Unfortunately, no sequences of species of Hesperonoe were presented in any of genetic databases to date.
The type material and vouchers are deposited in the collection of Zoological Museum of Moscow State University, Moscow (ZMMU), and the Laboratory of Ecology and Evolution of Marine Invertebrates (LEMMI) of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Moscow, Russia.

Ecological results
Eleven symbiotic species were found in the burrows of Upogebia major, Upogebia issaeffi (Balss, 1913), and Urechis unicinctus (Table 1; Figs. 1 and 2). The respective symbiotic communities did not differ and, thus, will be considered together in further analyses. All symbiotic species, indicated for each host, belong to approximately the same size class and were found together in the burrows. The large individuals of Hesperonoe urechis sp. nov. were found inhabiting the burrows alone with the host spoon worm.
Five over the eleven symbiotic species were found solitary or in heterosexual pairs. Pinnixa rathbuni was found in groups of up to 30 mature and young individuals inside the burrow of spoon worm, while 1, 2, or (rarely) 3 turbellarian specimens were found inhabiting the same burrow of U. unicinctus and Upogebia.

Hesperonoe japonensis
Diagnosis (based on Hong et al. 2017 and present study). Color red in vivo. Bilobed prostomium with sharply tapering cephalic peaks. Median and lateral antennae and all cirri with scattered papillae. A row of conical macrotubercles along posterior edge of elytra in adults (≥ 1.9 mm in body width without parapodia). Microtubercles and papillae over most of elytral surface. Marginal fringe of filiform papillae on posterior and outer-lateral elytral edges. Notochaetae at basal lobes of tentacular cirri. Both thick and thin notochaetae strongly serrated.
Taxonomic remarks. The specimens from Peter the Great Bay matched very well the original description based on the type specimens from Japan and that of "Hesperonoe hwanghaiensis" described by Sato et al. (2001Sato et al. ( , 2016 and Hong et al. (2017). The only intraspecific differences in our specimens are palps varying in length (Figs. 6a, c and 7a). Hesperonoe hwanghaiensis differs from H. japonensis in having a row of conical macrotubercles in elytra of adults, papillae on the surface of the median and lateral antennae, the thick and thin notochaetae being markedly serrated and notochaetae at cirrophores of tentacular cirri (Hong et al. 2017). GenBank accession numbers. COI mtDNA -MT237710, MT237711; 16S rRNA -MT241162; 28S rRNA -MT241160.
Geographic distribution. Type locality: Akkeshi Bay, Hokkaido Island, Japan. Present in the western part of the Northern Pacific, from Peter the Great Bay of the Sea of Japan (East Sea) to the southern island of Japan (Kyushu). Diagnosis. Prostomium bilobed, with prostomial peaks reduced to small processes. All antennae and cirri without papillae. Tentaculophores without chaetae. Elytra smooth, without or with median microtubercles, without marginal papillae. Distinct dorsal tubercles present. Upper thick notochaetae with minute serration. Yellow-brown in vivo, with a metallic sheen.
Description. Holotype fragmented in two parts, anterior with 28 segments and posterior with 8 segments; 71 mm in total length and 7 mm wide without parapodia, 15 mm wide with setae. Body dorsoventrally flattened. Dorsum yellow brown in vivo, with a metallic sheen, orange when preserved (Fig. 8).
Prostomium bilobed, slightly wider than longer, with cephalic peaks reduced to small processes on frontal prostomial margin, with two palps and three antennae (Figs. 8b and 9a). Two pairs of dorsal black eyes, ovate in shape, equal in size, anterior pair near middle of widest prostomial region, posterior pair on rear prostomial margin. Style of median antenna slender, 1.5 times longer than lateral ones, same length as prostomium, inserted anteriorly in median notch, with distinct ceratophore (Figs. 8b and 9a). Lateral antennae inserted ventrally, with short tapered styles, shorter than median antennae and prostomium length, with distinct ceratophores (Figs. 8b and 9a). All antennae not papillated, without subdistal inflation. Palps stout, 2.5 times longer than prostomium length, not papillated (Fig. 9a).
Taxonomic remarks. Hesperonoe urechis sp. nov. can be distinguished from all other species of the genus by its relatively long body (up to 7 cm in mature specimens). It resembles H. adventor (Skogsberg in Fisher and MacGinitie 1928), but differs in: (1) the absence of papillae on all antennae, palps and cirri (heavily ciliated in H. adventor); (2) microtubercles, if present, located in the median area of elytra (on the edge of ones as in H. adventor); (3) thick upper notochaetae with faint serrations (distinct pectination in H. adventor); and (4) rounded prostomium with reduced cephalic peaks (tapering lateral peaks in H. adventor, according to Hartman (1968)). The original description indicated the presence of two mammilliform anterior processes, which may almost be absent (Skogsberg 1928).
The new species is also resembling Hesperonoe andriashevi Averincev, 1990, but differs in having (1) small processes on prostomium (lacking in H. andriashevi); (2) up to 45 notochaetae and 70 neurochaetae (5-7 and~15 in H. andriashevi); (3) two types of neurochaetae (only one in H. andriashevi); and (4) median elytral microtubercles (absent in H. andriashevi). The elytra of H. andriashevi were reported as non-completely covering dorsum. However, its description was based on one elytron (Averincev 1990). Hesperonoe andriashevi differs from all other species of Hesperonoe in having 13 pairs of elytra and neurochaetae that cannot be clearly subdivided into two types so that its position within Hesperonoe was cast in doubt by Hong et al. (2017), but not by Averincev (1990). Upper and lower neurochaetae of H. urechis sp. nov. are different, but there are some transitional chaetae between them. A similar distribution of neurochaetae occurs in other species of Hesperonoe, such as Hesperonoe coreensis Hong, Lee & Sato, 2017. The fact is that it may be difficult to clearly subdivide neurochaetae into two types, thus supporting the necessity to modify the generic diagnosis of the genus as suggested by Averincev (1990).
Habitat and ecology. The specimens of the new species were collected from intertidal muddy sand flats. They are commensals of the shallow-water spoon worm Urechis unicinctus, which lives in large U-shaped burrows in muddy and sandy sediments in inter-and subtidal zones in the Sea of Japan and the Yellow Sea (Abe et al. 2014;personal observation). The symbiotic fauna of U. unicinctus in Peter the Great Bay includes turbellarian Stylochus/Paraplanocera sp., the pinnotherid crab Pinnixa rathbuni, an amphipod Liljeborgia sp., the copepod Goidelia cf. japonica Embleton, 1901, and gobiid fish Gymnogobius heptacanthus (Hilgendorf, 1879) (Marin 2016; Table 1; Fig. 1).
Etymology. The species is named after the generic name of its host -Urechis unicinctus.
Geographic distribution. Hesperonoe urechis sp. nov. were found in the southern part of Peter the Great and Posjeta Bays of the Sea of Japan (Russian Federation) (Fig. 11). Although the distribution of the new species is probably related with that of Urechis unicinctus (see Abe et al. 2014), it has never been collected from burrows in the northern part of Peter the Great Bay (Vostok Bay and adjacent areas), which have been intensive sampled with the same method. Similarly, no records are known from the southern region of the distribution.
Burrows of large invertebrates are very attractive for symbionts as they are stable and long-lasting habitats, which are long-time maintained in good conditions (MacGinitie and MacGinitie 1968). Most specialized symbionts use these burrows as a shelter from predators, but are trophically unrelated with their hosts, except those species that steal food from the spoon worm filtering net (Itoh and Nishida 2013;Henmi and Itani 2014b;Henmi et al. 2017;Burukovsky and Marin 2018).
The symbiotic communities associated with deepburrowing invertebrates in the Russian waters of the Sea of Japan are also very rich and diverse and still include many undescribed species, as very probably the turbellarian Stylochus/Paraplanocera sp. and the amphipod Liljeborgia sp. (Marin, 2020), while our findings of representatives of Hesperonoe are firstly recorded for the area and even for the Sea of Japan. At the same time, the brachyuran Sestrostoma balssi is firstly recorded from the Russian coasts of the Sea of Japan as a symbiont of Urechis unicinctus, as it was previously recorded from only burrows of Upogebia spp. only (Marin et al. 2011). However, the species was previously recorded from burrows of both hosts in Japan (Itani 2002(Itani , 2004Itani et al. 2002aItani et al. , 2005. Other symbiotic crustaceans were previously recorded from the area (see Marin 2010Marin , 2016Marin et al. 2011;Marin and Kornienko 2014;Marin and Sinelnikov 2016). The horseshoe worm Phoronis embryolabi was described as living commensally inside burrows of the callianassid shrimp Nihonotrypaea japonica (see Temereva and Chichvarkhin 2017), but it is also very abundant in our samples in the sediments around the burrows of Upogebia spp., so we may suggest a symbiotic association with this host too. Moreover, the relative Phoronis species are known as symbionts of burrowing shrimps of the genus Upogebia (Thompson 1972;Santagata 2004).
The species of Hesperonoe are among the most noticeable symbionts of various burrowing invertebrates. Despite the current lack of knowledge on their diversity and ecology, it is possible to conclude that relatively large-sized species, such as H. adventor and H. urechis sp. nov., tend to be associated with spoon worms, while the relatively small-sized species, namely H. complanata, H. coreensis, and H. japonica, tend to live associated with different ghost shrimps (Thalassinidea) (MacGinitie and MacGinitie 1968; Morris et al. 1980;Ricketts et al. 1985;Ruff 1995a;Britayev 1998, 2018;Sato et al. 2001Sato et al. , 2016Hong et al. 2017) (see Fig. 11). Usually one single specimen of H. urechis sp. nov. occurs in each burrow, from where other conspecific individuals are actively expelled, likely due to food rather than to space limitations. The scale worm feeds mainly on detritus trapped in the host mucous net (Fisher and MacGinitie 1928;Ricketts et al. 1985), and its particular feeding mode is now being studied using stable isotopes (Marin, unpublished data). This agile worm is rarely, if ever, found outside the innkeepers' burrows, and often dwells in the immediate proximity of the host. The juveniles associated with burrowing shrimps are commonly attached to the ventral or lateral surface of the thorax or abdomen of the host. As soon as they become adults, they detach themselves from the host carapace to move freely on the inner surface of the host burrow (MacGinitie 1935;MacGinitie and MacGinitie 1968;Sato et al. 2001). Almost all species of Hesperonoe, especially the small-sized H. japonica, H. coreensis, and H. complanata, have a uniform bright red coloring (Sato et al. 2001), probably as a result of being rich in blood pigments like other symbionts living in relatively oxygen-poor conditions Britayev 1998, 2018).
As part of this investigation, we were able to obtain molecular genetic data of the species of the polynoid polychaete genus Hesperonoe Chamberlin, 1919 (Polychaeta: Polynoidae) for the first time. Several gene markers were amplified, sequenced, and compared. The obtained molecular genetic data (Fig. 12) confirms the relationships between two described species, although they greatly differ in their appearance and size. The divergence for more than 15% by COI mtDNA (e.g., Carr et al. 2011) clearly supports the interspecific differences between the species within the genus Hesperonoe. The genus Hesperonoe is well isolated on the general phylogenetic reconstruction (tree) of the Polynoidae family (Fig. 12) and is associated with a clade including such genera as Harmothoe Kinberg, 1856;Enipo Malmgren, 1865;Gattyana McIntosh, 1897;Eunoe Malmgren, 1865;and Grubeopolynoe Pettibone, 1969. However, the taxonomy of these genera is rather complicated, and many of them could be polyphyletic. Nevertheless, the phylogenetic relationships of the genus Hesperonoe lie beyond the aims of our research. The autapomorphy of the clade Hesperonoe-Grubeopolynoe is the presence of the setae (notosetae) of two types, short blunt and slender tapering.
Finally, we conclude that, in the relatively simple boreal ecosystem, the existence of hidden biodiversity is confirmed by the finding of at least three undescribed species, which correspond to a 30% of the total observed symbiotic species. If we take into account the recent records and redescription of Fig. 12 Molecular phylogenetic (COI mtDNA) reconstruction (tree) of the genus Hesperonoe and all representatives of the family Polynoidae from NCBI (GenBank). Nodes indicated support based on ML algorithm two species of symbiotic decapods (Marin 2010;Marin et al. 2011) and the recently described phoronid species (Temereva and Chichvarkhin 2017), the number of new species discovered in these communities will increase up to 60%. The symbiotic assemblages associated with burrowing shrimps (Gebiidea) and spoon worms (Echiura) are quite different at the species level, but coincident at higher taxa level, which may support a common origin for these associations. There are also clear differences in species composition of the symbiotic assemblages of boreal and tropical ecosystems. Related host species of Upogebia are inhabited by the same species of symbionts with low host specificity in boreal ecosystems (Table 1), whereas the tubes of related host species of Chaetopterus spp. are inhabited by different, although some closely related, associates, showing higher specificity of symbionts in relation to the host in tropical ecosystems (Britayev et al. 2017).
The presence of such diverse symbiotic communities seems not only to be associated with the ability to escape in burrows from predators but also to receive/steal food from their host without leaving holes. Accordingly, we suggest that the symbiotic assemblage may be more abundant and diverse when associated to Urechis unicinctus, as the host feeding can also be a food resource for the symbionts (Fisher and MacGinitie 1928;Anker et al. 2005;Burukovsky and Marin 2018). In turn, the species of Upogebia only pump water through their burrow to filter food particles with their appendages (Dworschak 1981(Dworschak , 1983(Dworschak , 1987Kinoshita 2002). In fact, the only possibility to obtain food that the symbionts of Upogebia has is either to creep over the host's body (crabs and polychaetes) or attach on its carapace (the bivalves Neaeromya rugifera (Carpenter, 1864) or the species of the genus Peregrinamor Shôji, 1938 (Mollusca: Bivalvia: Lasaeidae) (Kato and Itani 2000)) to be able to steal the food that gets stuck in the bristles of appendages and body of Upogebia (Fig. 5d, e).
It is also known that symbiotic assemblage is influenced by physical factors and habitat requirements. Species richness and community composition associated with the spoon worm Ochetostoma erythrogrammon Leuckart & Rüppell, 1828 (Annelida: Echiura: Thalassematidae) in the Ryukyu Archipelago, Japan, are greatly influenced by the granulometric characteristics of the sediment, separating them for shrimp-(Alpheus barbatus Coutière, 1897) and scale worm-dominant (Lepidonotus sp.) sites (Goto and Kato 2012).