First records of Hubrechtella ijimai (Nemertea, Hubrechtiiformes) from Korea and China

Hubrechtella ijimai is reported for the first time from South Korea (East China Sea) and China (Yellow Sea), about 260 and 930 km from the nearest locality in Japan. Additional morphological data, confocal laser scanning microphotographs, and DNA data (COI sequences) are provided. This species possesses high intraspecific genetic COI p-distances for nemerteans (1.6–6.3%).


Introduction
The nemertean order Hubrechtiiformes with 2 families, Hubrechtiidae (sometimes incorrectly referred to as Hibrechtidae) and Hubrechtellidae, occurs mainly on a mud or sand substrate at shallow depths of boreal, tropical, and subtropical waters.The family Hubrechtiidae is monotypic and includes the single species, Hubrechtia desiderata (Kennel, 1891), inhabiting the Mediterranean Sea.The family Hubrechtellidae consists of 3 genera: Hubrechtella Bergendal, 1902 (14 or 15 species from the Pacific, Atlantic, and Indian Oceans), Sundbergia Gibson, 2002 (1 species from New Zealand), and Tetramys Iwata, 1957 (1 species from Japan).According to Kajihara (2017), the genus Tetramys Iwata, 1957 is almost certainly a junior synonym of Hubrechtella.
The species was originally described from the Pacific coast of Honshu, Japan as Coeia ijimai Takakura, 1922 and was subsequently placed into the genus Hubrechtella (Kajihara 2006).
The present paper reports the first find of H. ijimai from Korea and China, identified by molecular and morphological data.

Methods
Samples were collected from 2 sites (see below) (Fig. 1).One specimen from China was fixed for morphological study in Bouin's fluid, dehydrated in 100% EtOH, cleared in xylene, and embedded in paraffin wax (m.p. 56-57 °C).Serial sections of 6-8 µm thickness were stained with Mallory's trichrome method (with the addition of chromotrope 2R).For confocal laser scanning microscopy, pieces of the body and the proboscis of the specimen from Korea were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) and stained for 10 h at room temperature with phalloidin-Alexa Fluor 633 (Invitrogen, USA) diluted (1:200) in 1% triton X-100 (Serva, German) in PBS.Pieces of the proboscis and body were immersed in Mowiol 4-88 (Sigma, USA) and mounted on glass slides.The specimens were examined under light Imager.Z2 and LSM-780 confocal microscopes (Carl Zeiss, Germany).The studied specimens are deposited in the Museum of the National Scientific Center of Marine Biology (MSCMB), Vladivostok, Russia.
Total genomic DNA was extracted from the ethanol-preserved tissues (the posterior part of the Korea specimen; the second specimen from China) using a DNA-sorb-B-100 Blood and Tissue Kit (CMD, Moscow) according to the manufacturer's protocol.The mitochondrial cytochrome c oxidase subunit I (COI) gene fragment was amplified using the primer pair LCO1490 ⁄HCO2198 (Folmer et al. 1994).Thermal cycling was initiated by denaturation at 94 °C for 2 min followed by 35 cycles at 94 °C for 40 s, annealing at 50 °C for 40 s, and extension at 72 °C for 1 min.After cycling, the reaction was completed with an extension phase at 72 °C for 7 min, and the reaction products were visualized in a 1% agarose gel and purified through enzymatic reaction with Exo-SAP-IT (Fermentas, Lithuania).The purified PCR products were sequenced in forward and reverse directions under conditions recommended by the manufacturer, using a BigDye Terminator ver.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the same primers as for PCR.DNA sequences were edited and overlapping fragments assembled into consensus sequences using ChromasPro ver.1.34.To align the sequences, MEGA ver.6.0 (Tamura et al. 2013) was used.The sequences were submitted to GenBank with the accession numbers # KY986686 and # KY986685.The uncorrected pairwise distances (p) between the COI sequences within the species were calculated in MEGA ver.6.0.We reconstructed the haplotype networks based on the COI gene sequences using SplitsTree 4 version 4.12.3 (Huson and Bryant 2006).
Korea and # KY986685 for the specimen from China).
Diagnosis.This species can be distinguished from the other species of the genus Hubrechtella by the following combination of characters: body 3-20 cm long, translucent, whitish, spirally coiling when contracted, with caudal cirrus.Head rounded and wider than neck foregut.Lacunar network present, U-shaped blood lacuna absent in foregut region, mid-dorsal blood vessel penetrates rhynchocoel wall.Longitudinal body wall musculature lacks zigzag muscles.Proboscis with muscle cross, proboscis epithelium with "spherical bodies".
Comments.The so-called "spherical bodies" in the proboscis epithelium of H. ijimai are spherical pseudocnidae 10-13 μm in diameter and with central filament ( Figs 2E,  G).The same pseudocnidae were described in H. atypica Senz, 1992, H. globocystica Senz, 1993, and H. kimuraorum Kajihara, 2006 (see Kajihara 2006).Confocal laser scanning microscopy revealed several new characters for H. ijimai: crisscrossed diagonal muscles are present between the outer circular and the inner longitudinal body musculature in the anterior part of the body (Fig. 3B); the muscle "cross" is present between the rhynchocoel musculature and the circular body wall muscle layer (Figs 3E); the foregut musculature consists of inner circular and interwoven outer longitudinal muscles (Fig. 3F); isolated bundles of circular muscles have been found outside the brain (Fig. 3C); the proboscis musculature consists of 4 layers, as described for Hubrechtella juliae Chernyshev, 2003(Chernyshev et al. 2013), with one irregular muscle cross, but the weakly developed second muscle cross is also sometime visible (Fig. 3D).

Discussion
According to our morphological observations, the present specimens are identified as H. ijimai.Like most Hubrechtella species, H. kimuraorum Kajihara, 2006 can be distinguished from H. ijimai by the lack of the muscle cross in the proboscis, by the spacious U-shaped blood lacunar connection below the foregut, and the shape of the head, which is narrower than the trunk in H. kimuraorum (Kajihara 2006).Unfortunately, only 18S DNA sequence is available for H. kimuraorum, and this sequence is identical to that of H. ijimai.The presence of the muscle cross in the proboscis of H. ijimai is a unique feature among hubrechtiid nemerteans.A confocal laser scanning microscopy observation showed that the proboscis in H. juliae possesses "internal muscle crosses" (the crisscrossed zone in the outer diagonal musculature), which are not visible in histological sections (Chernyshev el al. 2013).The muscle cross in H. ijima is similar to that in most heteronemerteans, but it has an irregular structure and consists of a few irregular diagonal muscle strands.We detected not only a dorsal but also a low observable ventral cross.
The uncorrected pairwise distances between the mitochondrial COI sequences of the specimens from Korea and China and that of 2 specimens of H. ijimai (Kvist et al. 2014, Sundberg et al. 2016) obtained from Japan (Gen-Bank Accession # KF935520 and KU840155) were from 1.6 to 5.6% (Table 1).The largest p-distance (6.3%) was observed between the samples from Korea and China.It should be noted that the genus Hubrechtella has 2 sibling species, H. dubia and H. juliae, with COI p-distances of 6.0-7.5% (mean: 6.6%), but the intraspecific p-distances among 26 samples of H. dubia are only 0.0-1.2%(mean: 0.4%).We calculated these p-distance values using Gen-Bank COI sequences.At the same time, the p-distances among three specimens of H. ijimai (from Japan and Korea) are 1.6 and 2.2%, that is, the p-distances among sequences of H. ijimai do not show any clues of potential gaps.The Korean and Chinese samples have a higher distance but both are included in one haplotype network (Fig. 4) and have a very similar internal morphology.The specimen from China is distinguished by the short caudal cirrus, which is long in H. ijimai (Kajihara 2006), but the caudal cirrus could be damaged.Therefore we are confident that the specimen from China can be referred to as

H. ijimai
Based on the literature data, the geographical distribution of H. ijimai seems to be limited to the coasts of Japan (from Fukue to Asamushi), Korea, and China.This species has not been found in Peter the Great Bay (the best studied bay in the Sea of Japan), where the other species, H. juliae, is very common (Chernyshev 2014).

Figure 4 .
Figure 4.The haplotype network based on COI for 4 Hubrechtella ijimai specimens.Hah, hypothetical ancestral haplotype; the number of blank circles corresponds to the number of nucleotide substitutions between the haplotypes.
Sequences.Two COI sequences were deposited in Gen-Bank (accession # KY986686 for the specimen from