Species Diversity 25: 183–188 Published online 8 August 2020 DOI: 10.12782/specdiv.25.183

A New Species of Bathyal Nemertean, Proamphiporus kaimeiae sp. nov., off Tohoku, Japan, and Molecular Systematics of the Genus (Nemertea: Monostilifera)

Natsumi Hookabe1,2,5, Shinji Tsuchida3, Yoshihiro Fujiwara3, and Hiroshi Kajihara4 1 Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan E-mail: [email protected] 2 Present address: Misaki Marine Biological Station, School of Science, The University of Tokyo, Miura, Kanagawa 238-0225, Japan E-mail: [email protected] 3 Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa 237-0061, Japan 4 Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan 5 Corresponding author (Received 21 October 2019; Accepted 15 April 2020)

http://zoobank.org/71D0D145-2CE7-4592-BD82-C67EAA0A41E5

The monostiliferous hoplonemertean Proamphiporus kaimeiae sp. nov. is described based on a single specimen collect- ed from the bottom of the Northwest Pacific, 262 m deep, off Tohoku in Japan, by use of a remotely operated vehicle during a cruise organized by Tohoku Ecosystem-Associated Marine Sciences (TEAMS) research project in 2019. The position of the cerebral organs in the new species, being posterior to the proboscis insertion, is unusual for Eumonostilifera, which is one of the diagnostic traits of the so-far monospecific Proamphiporus Chernyshev and Polyakova, 2019, and Amphiporus rectangulus Strand, Herrera-Bachiller, Nygren, and Kånneby, 2014. The latter is herein transferred to Proamphiporus to yield a new combination, Proamphiporus rectangulus comb. nov., based on the reported internal morphology. Molecular phylo- genetic analyses based on 16S rRNA, cytochrome c oxidase subunit I, 18S rRNA, 28S rRNA, and histone H3 genes placed P. kaimeiae in a clade comprised of Proamphiporus crandalli Chernyshev and Polyakova, 2019 and Proamphiporus rectangulus. Key Words: Amphiporidae, COI, deep sea, Eumonostilifera, Ofunato, ROV, taxonomy.

cytochrome c oxidase subunit I, 18S rRNA, 28S rRNA, and Introduction histone H3 genes.

The eumonostiliferous amphiporid genusProamphipo - rus Chernyshev and Polyakova, 2019 is currently mono- Materials and Methods specific. It was established for Proamphiporus crandalli Chernyshev and Polyakova, 2019, which was found from A single specimen of nemertean was collected from soft the abyssal zone of the Kuril-Kamchatka Trench at a depth sediment at a depth of 262 m, off Ofunato (39°06.70′N, of 5495 m (Chernyshev and Polyakova 2019). The internal 142°06.44′E), Iwate Prefecture, Tohoku, on 19 July 2019 by morphology of P. crandalli is generally similar to that of use of a remotely operated vehicle (ROV) Crambon dive Amphiporus Ehrenberg, 1831 (Chernyshev and Polyakova #35 during KM19-05C cruise of R/V Kaimei organized by 2019), although most of the characters in Amphiporus are Tohoku Ecosystem-Associated Marine Sciences (TEAMS) not genus-specific (Strand et al. 2014). Several phylogenetic project. The living specimen was anaesthetized with a studies have shown polyphyly of Amphiporus (e.g., Andrade MgCl2 solution isotonic to seawater and photographed et al. 2012; Kvist et al. 2014). One of the main features that with a digital still camera OM-D E-M1II (Olympus, Tokyo, characterize Proamphiporus among other Amphiporidae is Japan). The posterior tip was preserved in 99% ethanol the position of the cerebral organs, being entirely situated for molecular work, while the rest of the body for internal alongside the brain like in cratenemertids and reptantians morphological observation was pre-fixed in 10% formalin (Chernyshev and Polyakova 2019), instead of anteriorly to in seawater, post-fixed in Bouin’s fluid for 24 h, and pre- the brain in most members in the Eumonostilifera. served in 70% ethanol. Serial sections of 5-µm thickness In this paper, we provide a description of a new species of were stained with Mallory’s trichrome method. Holotype Proamphiporus and elucidate the phylogenetic relationship specimen has been deposited in the Invertebrate Collection within the genus based on genetic distances and phyloge- of the Hokkaido University Museum (ICHUM), Sapporo, netic trees reconstructed by partial sequences of 16S rRNA, Japan.

© 2020 The Japanese Society of Systematic Zoology 184 Natsumi Hookabe et al.

Total DNA was extracted from a piece of the ethanol- Nygren, and Kånneby, 2014, Nemertovema hadalis Cherny- fixed posterior end of the body, using a DNeasy Tissue Kit shev and Polyakova, 2018, N. norenburgi Chernyshev and (Qiagen Japan, Tokyo, Japan). PCR amplification was per- Polyakova, 2019, and Proamphiporus crandalli (Table 1). formed with primer pairs LCO1490/HCO2198 (Folmer et Alignment of the 16S, 18S, and 28S rRNA gene sequences al. 1994) for partial sequence of cytochrome c oxidase sub- was conducted by use of MAFFT ver. 7 (Katoh and Standley unit I gene (COI), 16Sar-L/16br-H (Palumbi et al. 1991) 2013); alignments of COI and H3 sequences were straight- for the 16S rRNA gene (16S), 1F/9R (Giribet et al. 1996) forward and lacked indels. Ambiguous sites were removed for the 18S rRNA gene (18S), LSU5/rd5b (Littlewood 1994; using Gblocks ver. 0.91b (Castresana 2000), which resulted Schwendinger and Giribet 2005) for the 28S rRNA gene in a final dataset comprised of 16S (454 bp), COI (657 bp), (28S), and H3aF/H3aR (Colgan et al. 1998) for the histone 18S (1773 bp), 28S (1077 bp), and H3 (326 bp). To infer the H3 gene (H3), with an Applied Systems 2720 thermal cy- phylogeny, maximum-likelihood (ML) and Bayesian infer- cler. The PCR protocol was as follows: preheating at 94°C ence (BI) analyses were performed. The best-fit partition for 2 min; 35 cycles of 94°C for 40 s, 52°C for 75 s, and 72°C scheme for ML was GTR+G+I model for concatenated se- for 60 s; then a final extension at 72°C for 7 min. Nucleotide quences according to PartitionFinder ver. 2.1.1 (Lanfear et sequencing was performed using internal primers in addi- al. 2017) employing the greedy algorithm. The ML analysis tion to the same primer pairs with an ABI BigDye Termi- was performed with RAxML ver. 8.0.0 (Stamatakis 2014). nator ver. 3.1 Cycle Sequencing Kit and an ABI 3100 Avant Nodal values were derived from 1000 bootstrap pseudo- Genetic Analyzer (Applied Biosystems Japan Ltd., Tokyo, replicates. The BI was performed using MrBayes ver. 3.2.3 Japan). Internal primers used in this study were as follows: (Ronquist et al. 2012) launching two independent Metropo- 3F/5R (Giribet et al. 1996) and 18Sbi/S2.0 (Whiting et al. lis-coupled analyses with four Markov chains for 107 genera- 1997) for 18S, and LSU3/D2F (Littlewood 1994), 28Z (Hillis tions, sampling every 100 generations from the chain, based and Dixon 1991), Sa (Whiting et al. 1997) for 28S. Sequenc- on GTR+G model selected by PartitionFinder ver. 2.1.1. es newly obtained in this study have been deposited in the Run convergence was assessed by Tracer ver. 1.7 (Rambaut DNA Data Bank of Japan (DDBJ) under accession number et al. 2018); for all parameters, effective sample sizes were listed in Table 1. above 200. Along with the newly determined sequences, a concat- Uncorrected pairwise genetic distances and Kimura two- enated dataset was created from GenBank entries for eight parameter (K2P) genetic distances were calculated based on species in Amphiporidae: Amphiporus formidabilis Grif- 657 bp of COI by MEGA ver. 7 (Kimura 1980; Kumar et al. fin, 1898;A. imparispinosus Griffin, 1898,A. lactifloreus 2016). (Johnston, 1828), A. rectangulus Strand, Herrera-Bachiller,

Table 1. List of taxa included in the phylogenetic analyses with GenBank accession numbers for 16S rRNA, COI, 18S rRNA, 28S rRNA, and histone H3 genes.

Species 16S rRNA COI 18S rRNA 28S rRNA Histone H3 Source Amphiporus formidabilis Griffin, 1898 KF935498 KF935547 KF935331 KF935387 KF935443 Kvist et al. (2014) Amphiporus imparispinosus Griffin, 1898 JF277618 HQ848612 JF293029 HQ856878 JF277696 Andrade et al. (2012) Amphiporus lactifloreus (Johnston, 1828) JF277617 HQ848611 JF293018 HQ856876 — Andrade et al. (2012) Nemertovema hadalis Chernyshev KY296891 KY296912 KY296895 KY296903 KY296921 Chernyshev and and Polyakova, 2018 Polyakova (2018) Nemertovema norenburgi Chernyshev MN211507 MN205522 MN211411 MN211461 MN205483 Chernyshev and and Polyakova, 2019 Polyakova (2019) Nemertovema norenburgi Chernyshev MN211506 MN205523 MN211412 MN211462 MN205484 Chernyshev and and Polyakova, 2019 Polyakova (2019) Proamphiporus crandalli Chernyshev MN211505 MN205524 MN211413 MN211463 MN205485 Chernyshev and and Polyakova, 2019 Polyakova (2019) Proamphiporus kaimeiae sp. nov. LC505452 LC505451 LC520132 LC505453 LC520133 present study Proamphiporus rectangulus — KC812587 — — — Strand et al. (2014) (Strand et al., 2014) comb. nov. Proamphiporus rectangulus — KC812588 — — — Strand et al. (2014) (Strand et al., 2014) comb. nov. Proamphiporus rectangulus — KC812589 — — — Strand et al. (2014) (Strand et al., 2014) comb. nov. Proamphiporus rectangulus — KC812590 — — — Strand et al. (2014) (Strand et al., 2014) comb. nov. Proamphiporus rectangulus — KC812603 — — — Strand et al. (2014) (Strand et al., 2014) comb. nov. Proamphiporus rectangulus — KC812604 — — — Strand et al. (2014) (Strand et al., 2014) comb. nov. Proamphiporus rectangulus — KC812605 — — — Strand et al. (2014) (Strand et al., 2014) comb. nov. Bathyal nemertean Proamphiporus kaimeiae sp. nov. 185

Description. External features (based on photographs Systematics of living specimen). Body 3.6 mm in length and 0.7 mm in maximum width; background body color generally whit- Genus Proamphiporus Chernyshev and Polyakova, 2019 ish (Fig. 2), epidermis semitransparent ventrally; internal [New Japanese name: Fukamizo-himomushi-zoku] organs (intestine, rhynchocoel, proboscis) visible through Proamphiporus rectangulus (Strand, Herrera-Bachiller, body wall (Fig. 2B). Dorsal surface with brownish pigments Nygren, and Kånneby, 2014) comb. nov. randomly distributed and aggregated to form single mid- [New Japanese name: Fukamizo-himomushi] dorsal longitudinal stripe of about 5/6 to 2/3 of body width (Fig. 2A); ventral surface without pigments (Fig. 2B). Ante- Amphiporus rectangulus Strand et al., 2014: 2–6, figs 2–5. rior tip of head devoid of pigment. Head slightly narrower than middle part of body. Single pair of cephalic furrows ex- Remarks. In Amphiporus rectangulus, the cerebral or- tending posteriorly on dorsal side, meeting at mid-line and gans are situated alongside the brain (Strand et al. 2014: forming steeply angled V-shape, ventrally forming trans- fig. 5F) just as in Proamphiporus crandalli (type species of Proamphiporus) (Chernyshev and Polyakova 2019), instead of being anterior to the brain as in most of the Eumonostil- ifera, including Amphiporus lactifloreus (type species of Am- phiporus) (Berg 1972). The angle of the V-shape formed by the cephalic furrows on the mid-dorsal body surface in A. rectangulus is as acute as that in P. crandalli, and much more acute than that in A. lactifloreus (Gibson 1994; Strand et al. 2014; Chernyshev and Polyakova 2019). Based on these unique similarities, we consider it reasonable to transfer A. rectangulus from Amphiporus to Proamphiporus.

Proamphiporus kaimeiae sp. nov. [New Japanese name: Kaimei-fukamizo-himomushi] (Figs 2, 3)

Material examined. Holotype: ICHUM 6058, male, transverse sections of anterior body fragment, 6 slides, 19 Fig. 1. Map showing the collection site (solid circle) of the speci- July 2019, collected at a depth of 262 m, off Ofunato, Japan. men examined in the present study.

Fig. 2. Proamphiporus kaimeiae sp. nov., holotype, ICHUM 6058, photographs taken in living state. A, Whole body, dorsal view; B, ventral view. Abbreviations: cf, cephalic furrow; co, cerebral organ. Scale bar: 500 µm. 186 Natsumi Hookabe et al.

Fig. 3. Proamphiporus kaimeiae sp. nov., holotype, ICHUM 6058, photomicrographs of transverse sections. A, Body wall in brain region; B, anterior part of cerebral organs; arrowheads pointing to the radial canal; C, posterior part of cerebral organs; arrowhead pointing to the lateral canal; D, brain; arrowhead pointing to vascular plug; E, proboscis; F, testis; G, excretory collecting tubule (arrowhead). Abbreviations: ag, acidophilic glands; br, brain; de, dermis; dg, dorsal ganglion; ep, epidermis; ln, lateral nerve cord; mlm, middle longitudinal muscle layer of body wall; ocm, outer circular muscle layer of body wall; pb, proboscis; pe, proboscis epithelium; pic, proboscis inner circular muscle layer; poc, proboscis outer circular muscle layer; rh, rhynchocoel; st, stomach; te, testis; vg, ventral ganglion. Scale bars: A = 20 µm, B = 75 µm, C, G = 50 µm, D = 100 µm, E, F = 40 µm.

Fig. 4. Phylogenetic trees of the genus Proamphiporus based on concatenated 16S rRNA (454 bp), COI (657 bp), 18S rRNA (1773 bp), 28S rRNA (1077 bp), and histone H3 genes (326 bp). A, Phylogenetic tree inferred by maximum likelihood (ML) analysis, showing support values generated by a separate partitioned ML bootstrap analysis with 1000 replicates; B, phylogenetic tree inferred from Bayesian analysis, showing posterior probability of a separate partitioned Bayesian analysis. verse line in front of brain (Fig. 2B). Paired cerebral ganglia longitudinal muscle layer of body wall 50 µm in maximum visible through body wall as orange spots (Fig. 2B). No eyes thickness. Esophagus with ciliated epithelium. Stomach epi- evident. thelium with glands and cilia. Intestinal diverticula deeply Internal morphology. Epidermis 20–40 µm in thick- branched (Fig. 2B). Mid-dorsal vessel penetrating rhyn- ness, containing numerous red and yellow staining ciliated chocoel wall behind ventral cerebral commissure, forming cells (Fig. 3A). Dermis up to 10–15 µm thick. Outer circu- single vascular plug (Fig. 3D). lar muscle layer of body wall 10–20 µm in thickness. Inner Proboscis pore terminal. Rhynchocoel long, more than Bathyal nemertean Proamphiporus kaimeiae sp. nov. 187

Table 2. Range of genetic distance within the genus Proamphipo- rus (a single specimen for P. kaimeiae and P. crandalli; seven speci- mens for P. rectangulus). Values below diagonal indicate K2P, while Acknowledgments values above diagonal indicate uncorrected p-distance. We thank Professor Alexei V. Chernyshev (A.V. Zhirmun- P. kaimeiae P. crandalli P. rectangulus sky National Scientific Center of Marine Biology, Russia) for P. kaimeiae — 0.141 0.133–0.135 providing us valuable suggestions on the present study. NH P. crandalli 0.158 — 0.104–0.107 also thanks Dr. Kensuke Yanagi (Natural History Museum P. rectangulus 0.149 0.112–0.116 0.000–0.005 and Institute, Chiba, Japan), Dr. Naoto Jimi (National Insti- tute of Polar Research, Japan), and all the other participants in the TEAMS research project for generous help in collect- two-thirds of body length, with thin wall comprised of few ing the sample. Furthermore, we are grateful to the captain muscle layers (Fig. 3B, D). Proboscis with outer circular, and the crew of R/V Kaimei. This work was supported by middle longitudinal, and inner circular muscle layers (Fig. the research project Tohoku Ecosystem-Associated Marine 3E); proboscis epithelium with developed papillae contain- Sciences from the Ministry of Education, Culture, Sports, ing basophilic glandular cells (Fig. 3B, D, E); ten probos- Science, and Technology. cis nerves (Fig. 3E). Lateral nerves with neither accessory nerves nor neurocords (Fig. 3G). Cerebral organs entirely situated alongside brain region, with forked chamber; References chamber bifurcating into an anterior radial and a posterior lateral canal, both ciliated (Fig. 3B, C); acidophilic glands Andrade, S. C. S., Strand, M, Schwartz, M., Chen, H., Kajihara, H., von developed in posterior region of cerebral organs (Fig. 3C). Döhren, J., Sun, S., Junoy, J., Thiel, M., Norenburg, J. L., Turbeville, Eyes absent. Testes between intestinal diverticula (Fig. 3F). J. 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