New species of Trophoniella from Shimoda, Japan (Annelida, Flabelligeridae)

Abstract Trophoniella hephaistos sp. n. was collected from a tank irrigated with seawater pumped directly from Nabeta Bay, Japan. This species is discriminated from other Trophoniella by having dorsal tubercles, a tongue-shaped branchial plate, a tunic covered with large sediment grains dorsally and ventrally, having eyes, and anchylosed neurohooks starting from chaetigers 17–20. This is the first record of Trophoniella from Japanese waters. Identification keys to species of Trophoniella and four gene sequences (COI, 16S, 18S, 28S) of this species are provided. Phylogenetic analysis was conducted to clarify phylogenetic position of Trophoniella in Flabelligeridae using four genes.


Introduction
Trophoniella Hartman, 1959 belongs to the family Flabelligeridae and currently consists of 25 species and one undescribed species (Salazar-Vallejo 2012b). Trophoniella polychaetes live in sediments from shallow water to the deep sea in tropical or subtropical regions (Salazar-Vallejo 2012b). This genus is characterized by having anchylosed neurohooks in the median or posterior chaetigers, bidentate or bifid tips, a thick tunic, a tongue-shaped branchial lobe (except for Trophoniella enigmatica), and longitudinal rows of elongated single papillae along the body (Salazar-Vallejo 2012b). Trophoniella resembles Piromis and Pycnoderma in having a thick tunic, often with sediment grains, a tongue-shaped branchial lobe, and multiarticulated notochaetae. However, it is distinct from Piromis and Pycnoderma by having anchylosed neurohooks in the median or posterior chaetigers (Salazar-Vallejo 2011b).
Phylogenetic analyses of Flabelligeridae were conducted several times by using morphological and molecular data sets (Burnette et al. 2005;Osborn and Rouse 2008;Salazar-Vallejo et al. 2008). A morphological analysis suggested that Trophoniella was similar to Piromis. However, the molecular data was unable to robustly resolve the phylogenetic position of Trophoniella; this is likely an artefact of limited taxon sampling within the genus.
During benthos sampling in an aquarium in the Shimoda Marine Research Center (SMRC), University of Tsukuba, we collected undescribed species of Trophoniella. Here, we describe Trophoniella hephaistos sp. n. and cytochrome c oxidase subunit I (COXI), 16S ribosomal RNA (16S), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) gene sequences to contribute to the DNA barcoding of the Flabelligeridae. A phylogenetic analysis was conducted using four genes to clarify relationships of Trophoniella within the family Flabelligeridae. To the best of our knowledge, this is the first report of Trophoniella from Japanese waters.

Material and methods
Worms were collected by hand from a tank (MF-5000S, aquaculture system, Japan. 2.4 m in diameter and 1.1 m in depth) installed in the SMRC, University of Tsukuba, Shizuoka (34°40.045'N; 138°56.145'E) (Fig. 1). The tank contained sandy mud and sea water and the worms lived between 0 and 30 cm below the sediment surface. Seawater in the tank was drawn only from Nabeta Bay, directly in front of the SMRC, from a depth of 3 m (location of the head gate: 34°39.950'N; 138°56.283'E). Several samplings were conducted in Nabeta Bay and other surrounding sites at depths between 2 and 386 m by the first author and members of the SMRC but there was no individual of Trophoniella discovered except in the tank. All the specimens were first anesthetized with menthol and then fixed and preserved in 70% ethanol. The anesthesia duration differed among samples. Preserved specimens were observed under stereoscopic MZ 16F (Leica, Germany) and E600 (Nikon, Japan) microscopes. All specimens were deposited in the National Museum of Nature and Science, Tokyo (NSMT), Japan.
Etymology. The worm is coated with sediment particles, resembling armor. Hephaistos (Ἥφαιστος) was the name of the ancient Greek god of blacksmiths who forged the armor worn by Achilleus. Hephaistos is also spelled Hephaestus. The Japanese name is derived from the type locality (Shimoda), Japanese armor (Yoroi), and flabelligerids in Japanese (Habouki).
Distribution. This new species is currently only known from the tank of the type locality. The seawater in the tank was drawn only from Nabeta Bay from a depth of 3 m directly facing the SMRC. The natural habitat of this species remains unknown. Due to the location of the head gate, T. hephaistos could be a shallow-water species. However, several sublittoral (~50-60 m) invertebrates were collected from this tank (Dr. Hiroaki Nakano, pers. comm.). Additional sampling efforts in Nabeta Bay will clarify the natural habitat of this species.
Phylogenetic analysis. The final lengths of the aligned sequences were 669 bp (COXI), 485 bp (16S), 1893 bp (18S), and 910 bp (28S). The bootstrap value of 98% in ML analysis strongly supported the monophyly of Flabelligeridae, but internal relationships of Flabelligeridae were not resolved (Fig. 6). The sister group of Trophoniella was Piromis. The bootstrap value in ML analysis (100%) demonstrated the monophyly of this clade (Fig. 6).
Remarks. Trophoniella hephaistos sp. n. resembles T. enigmatica Salazar-Vallejo, 2012 and Trophoniella indica (Fauvel, 1928) in having dorsal tubercles at the anterior chaetigers, a tunic covered with large sediment grains dorsally and ventrally, and anchylosed neurohooks starting from chaetiger 14 or posterior. However, T. hephaistos is discriminated by the presence of anchylosed neurohooks starting from chaetigers 17-20, whereas those of T. enigmatica start from chaetiger 40, and of T. indica from chaetiger 14. Additionally, T. enigmatica does not have a tongue-shaped branchial plate and T. indica does not have eyes. Chaetiger number of T. hephaistos was more than twice as many as that of T. indica. Trophoniella hephaistos has dorsal body papillae in two longitudinal rows, whereas T. enigmatica in three and T. indica in five.
The phylogenetic analysis showed Trophoniella to be the closest relative of Piromis in Flabelligeridae supported by a high bootstrap value (See Fig. 6). Our findings are consistent with previous morphological studies that indicated a close relationship between Trophoniella and Piromis based on their shared characters such as tongue-shaped lobe, multiarticulated notochaeta, and thick tunic (Salazar-Vallejo 2011b; Salazar-Vallejo et al. 2008).

Key to species of the genus of Trophoniella
The key by Salazar-Vallejo (2012b) is amended with the addition of this new species at couplet 20.