Natsumi HABANABE (Doctoral Student, Department of Biological Sciences)

Hiroshi Kaihara (Professor, Hokkaido University)

Naoto Jimi (Assistant Professor, Nagoya University)

Alexei V. Chernyshev (Professor, Zhirmunsky National Scientific Centre of Marine Biology)

Tsutomu Ueshima (Associate Professor, Department of Biological Sciences)

Key points of the presentation

• Discovery of 10 new species of damselfly in the genus Nipponemertes (Chordata: Monopoda) from the intertidal to deep-sea region of the northwestern Pacific coast (Honshu, Japan - Vladivostok, Russia).
• Molecular phylogenetic analysis based on multiple gene regions reveals three distinct lineages characterized by head morphology.
• It was suggested that the smaller body size in the genus Nipponemertes may be an adaptation to the deep-sea environment.

Summary of presentation

An international research team led by Natsumi Habuabe, a graduate student (3rd year, Ph.D., JSPS Research Fellow, Department of Biological Sciences, Graduate School of Science, The University of Tokyo), and their colleagues, through comprehensive collection surveys using scuba diving, ROV ^{(Note 1) and} dredge ^{(Note 2)} from the intertidal to deep sea areas along the northwest Pacific coast Ten new species of the genus Nipponemertes were discovered and described.

Molecular phylogenetic analysis based on multiple genetic regions using the collected specimens revealed that they are divided into three lineages characterized by head morphology. The clade ^{(Note 3)}, consisting of a deep-sea species and some shallow-sea species newly found in this study, was also found to differ in other ways besides head morphology, such as mature individuals being significantly smaller. It is suggested that the smaller body size in this group may be an adaptation to the deep-sea environment where food resources are limited for carnivorous cordiform animals.

Contents of Presentation

Research Background
The phylum Cordata (cordata) ^{(Note 4)}, of which approximately 1,300 species are known from around the world to date, is a group of coralline animals ^{(Note 5)} that inhabit marine to freshwater areas as well as terrestrial habitats. As the name suggests, many species have soft, smooth, string-like bodies and are carnivorous or carrion-eating. Although about 130 species of cyprinids have been reported from Japan, most of them are found in the intertidal zone, and their diversity has not yet been studied in deep to deep water areas where scuba diving is required. Only four known species of the genus Nipponemertes, all of which are shallow-water species, were known from the study area along the northwestern Pacific coast of Japan.

Research Details
From 2012 to 2021, a research group led by Natsumi Hazakubu, a graduate student in the Department of Biological Sciences, Graduate School of Science, the University of Tokyo, conducted collection surveys in the intertidal to deep-sea areas along the Northwest Pacific coast with researchers from the University of Tokyo, Hokkaido University, Nagoya University, Hiroshima Shudo University, National Museum of Nature and Science, JAMSTEC, and other institutions (Figure 1). (Figure 1). Specimens were collected comprehensively using several methods including intertidal collection, scuba diving, dredging, and ROV.

Figure 1: Collection surveys were conducted along the coasts of Honshu and Vladivostok (A-D). In addition to intertidal collections, SCUBA dives were used to collect under boulders (E), and ROV surveys were used to collect from the deep sea floor (F, G).

The external morphology of the collected Nipponemertes specimens was recorded during life, and the internal morphology was observed by preparation of serial tissue sections, and molecular phylogenetic analysis based on the maximum likelihood and Bayesian method ^{(Note 6)} was conducted by sequencing multiple gene regions of mitochondrial and nuclear origin. As a result, 10 new species were newly discovered and described from the Northwest Pacific coast (Figure 2).

The molecular phylogenetic analysis revealed that there are three lineages in this genus characterized by their head morphology (Figure 2). The clade containing only the species newly discovered in this study (Figure 2; Clade C), which includes deep-sea and some shallow-sea species, had significantly smaller body sizes of mature individuals than the species in the other clades. Shallow-sea species in this clade have secondarily expanded from deep-sea areas into shallow water (Figure 2), suggesting that the smaller body size in the genus Nipponemertes may be the result of adaptation to deep-sea environments with limited food resources in carnivorous cordiform animals.

Figure 2: Phylogenetic analysis of the collected specimens revealed the existence of three lineages in the genus Nipponemertes. The morphology of the head (acf, anterior cephalic furrow; cp, cephalic patch; cr, mid-dorsal cephalic ridge; oc, ocelli; pcf, posterior cephalic furrow; scg, secondary The species in Clade C (blue) are all smaller in body size than the other clades.

Significance and Prospects
In previous studies, 18 species of the genus Nipponemertes have been described from around the world, but the high species diversity revealed in this study suggests that there may be many species yet to be discovered. The diversity and taxonomic studies of the nipponemerteans have not progressed, especially in deep waters, due to the small population of researchers and the need for proper specimen handling ^{(Note 7)}. Many ecological and evolutionary aspects remain unresolved. It is hoped that more species will be discovered in the future, and that the ecology and evolution of caterpillars will be unraveled as the understanding of their diversity progresses.

Members of this research team:

Natsumi Habanobe	D. Student, Department of Biological Sciences, Graduate School of Science, The University of Tokyo
Hiroshi Kaihara	Professor, Department of Biological Sciences, Graduate School of Science, Hokkaido University
Alexei V. Chernyshev	Professor, Zhirmunsky National Scientific Centre of Marine Biology
Naoto Jimi	Assistant Professor, Sugashima Misaki Marine Biological Station, Graduate School of Science, Nagoya University
Naohiro Hasegawa	D. Department of Natural History, Graduate School of Science, Hokkaido University
Hisanori Kouzuka	Technical Specialist, Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo
Masanori Okanishi	Assistant Professor, Faculty of Human Environment, Hiroshima Shudo University
Kenichiro Tani	Chief Researcher, Department of Earth Science, National Museum of Nature and Science
Yoshihiro Fujiwara	Senior Researcher, Marine Biological Environment Impact Research Center, Global Environment Division, JAMSTEC
Shinni Tsuchida	Associate Senior Researcher, Marine Biological Environment Effects Research Center, Global Environment Division, JAMSTEC
Tsutomu Ueshima	Associate Professor, Department of Biological Sciences, Graduate School of Science, The University of Tokyo

Journal

Journal name	Frontiers in Marine Science
Title of paper	Molecular phylogeny of the Genus Nipponnemertes (Nemertea: Monostilifera: Cratenemertidae) and description of 10 new species, with notes on small body size in a newly discovered clade. body size in a newly discovered clade.
Authors.	Natsumi Hookabe*, Hiroshi Kajihara, Alexei V. Chernyshev, Naoto Jimi, Naohiro Hasegawa, Hisanori Kohtsuka, Masanori Okanishi, Kenichiro Tani, Yoshihiro Fujiwara, Shinji Tsuchida, Rei Ueshima
DOI Number	10.3389/fmars.2022.906383

Terminology

1 ROV

Remotely operated vehicle. In this study, manupilators and slurp guns were used to collect cyprinids from deep waters. ↑up

Note 2 Dredge (biological dredge)

A bottom trawl-like device to collect benthic organisms living on the seafloor by lowering a metal cage and weight to the seafloor with a wire and towing the cage by boat. It has long been widely used in Research Students for deep-sea Benthic Animals, including flatworms. ↑up

Note 3 Clade

A group that includes one common ancestor and all taxa derived from the common ancestor. ↑up

Note 4 Cordata

A group of cord worms. They have a smooth worm-like body without segmental structures or appendages. They have a rostral cavity separate from the digestive tract, which contains a proboscis used for feeding and migration. ↑up

Note 5: Cordate animals

A group of invertebrates that includes the phylum Cordata, the phylum Platyhelminthes (planaria), the phylum Annelida (gobies, leeches, worms), the phylum Mollusca (shellfish, squids, octopi), and the tentacled coralline animals (chamissonids, borers, and borers). ↑up

Note 6: Maximum Likelihood Method and Bayesian Method

The maximum likelihood method (Felsenstein 1981) and Bayesian method (Yang and Rannala 1997) are known as analysis methods widely used for constructing molecular phylogenetic trees. ↑up

Note 7: Appropriate specimen processing

For accurate species identification of cyprinids, it is necessary to record body color and pattern, head and rostral stinger apparatus morphology when alive, anesthetize the specimens with magnesium chloride solution or other solution, and prepare ethanol specimens for DNA extraction and Buan's solution or formalin specimens for tissue observation. ↑up