VENUS 76 (1–4): 19–28, 2018 DOI: http://doi.org/10.18941/venus.76.1-4_19First Record of lenkae in Japan ©The Malacological Society of Japan19

The First Record of Trinchesia lenkae Martynov, 2002 from Japan: Morphological and Molecular Comparison with the Material from the Type Locality in Russia (Nudibranchia: )

Tatiana Korshunova1,2, Toshihiko Fujita3 and Alexander Martynov2* 1Koltzov Institute of Developmental Biology RAS, 26 Vavilova Str., 119334 Moscow, Russia 2Zoological Museum, Moscow State University, Bolshaya Nikitskaya Str. 6, 125009 Moscow, Russia 3National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan

Abstract: The mollusc Trinchesia lenkae Martynov, 2002 is reported in the Japanese fauna for the first time. The newly collected Japanese specimens of T. lenkae are compared with those from the type locality of this species in Peter the Great Bay in the Sea of Japan, Russia, using external and internal morphological data and molecular analyses. The molecular study revealed high genetic homogeneity between Japanese and Russian populations of T. lenkae. The integrative morphological and molecular data thus clearly suggest that the same species occurs in Russia and Japan.

Keywords: Japanese fauna, molecular analysis, morphology, nudibranchs,

Introduction

The recently restored family Trinchesiidae Nordsieck, 1972 is a large and diverse group of predominantly small-sized aeolidacean nudibranchs (Korshunova et al., 2017a). Representatives of this family have been reported from Japan and included the genera Winckworth, 1941, “” Alder & Hancock, 1855 s.l., Bergh, 1874, Costa, 1866 s.str., and Trinchesia Ihering, 1879 (e.g., Baba, 1937, 1949, 1955, 1961, 1975, 1984; Hamatani, 1993; Hirano & Hirano, 1991; Nakano, 2004). During a research trip to Hokkaido in October of 2016, T. Korshunova and A. Martynov were able to collect nudibranch molluscs at Akkeshi Marine Station, Hokkaido University. Among the collected nudibranchs several a trinchesiid species previously unrecorded from Japan were found. After morphological and molecular analysis it was confirmed that it belongs to the species Trinchesia lenkae Martynov, 2002 that had been described from the Russian part of the Sea of Japan fifteen years previously (Martynov, 2002). Additionally, a single specimen of Trinchesia lenkae without any morphological information was recorded as “Tenellia” lenkae in Cella et al. (2016) also from the Russian side of the Sea of Japan. In this study the description of the Japanese specimens of T. lenkae and its comparison with specimens of T. lenkae from the type locality in Russia are presented.

* Corresponding author: [email protected] 20 T. Korshunova et al.

Materials and Methods

Collecting data and morphological methods Material for this study was obtained by T. Korshunova and A. Martynov during fieldwork at the Akkeshi Marine Station (Field Science Center for Northern Biosphere, Hokkaido University), Japan in 2016 and in the Far Eastern Marine Reserve in Peter the Great Bay, Russia in 2014. The morphology of the molluscs and their egg masses were studied under a stereomicroscope and using Nikon D-90 and D-810 digital cameras. For the description of internal features both preserved and fresh specimens were dissected under the stereomicroscope. The buccal mass of each specimen was extracted and processed in a weak solution of domestic bleach. The jaws were analysed under the stereomicroscope and scanning electron microscope. The gold and platinum sputter- coated radulae were examined using a scanning electron microscope (JSM 6380). The reproductive systems were also examined and drawn using the stereomicroscope.

Molecular methods Three specimens were successfully sequenced for the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S rRNA. Small pieces of foot tissue were used for DNA extraction with Diatom™ DNA Prep 100 kit by Isogene Lab, according to the manufacturer’s instructions. Extracted DNA was used as a template for the amplification of partial sequences of the COI and 16S. The primers that were used for amplification are LCO 1490 (GGTCAACAAATCATAAAGATATTGG (Folmer et al., 1994)); HCO 2198 (TAAACTTCAGGGTGACCAAAAAATCA (Folmer et al., 1994)); 16S arL (CGCCTGTTTAACAAAAACAT (Palumbi et al., 2002)); and 16S R (CCGRTYTGAACTCAGCTCACG (Puslednik & Serb, 2008). Polymerase chain reaction (PCR) amplifications were carried out in a 20-μL reaction volume, which included 4 μL of 5× Screen Mix (Eurogen Lab), 0.5 μL of each primer (10 μM stock), 1 μL of genomic DNA, and 14 μL of sterile water. The amplification of COI was performed with an initial denaturation for 1 min at 95°C, followed by 35 cycles of 15 s at 95°C (denaturation), 15 s at 45°C (annealing temperature), and 30 s at 72°C, with a final extension of 7 min at 72°C. The 16S amplification began with an initial denaturation for 1 min at 95°C, followed by 40 cycles of 15 s at 95°C (denaturation), 15 s at 52°C (annealing temperature) and 30 s at 72°C, with a final extension of 7 min at 72°C. DNA sequences of both strands were obtained using the ABI PRISM® BigDye™ Terminator v. 3.1. on an automated DNA sequencer (Applied Biosystems Prism 3700). COI sequences were translated into amino acids for confirmation of the alignment. All sequences were deposited in GenBank (Table 1, highlighted in bold). Original data and publicly available sequences were aligned with the MUSCLE algorithm (Edgar, 2004). Separate analyses were conducted for the following data sets: resulting alignments are 658 bp for COI, 442 bp for 16S, and 1,100bp for the concatenated dataset. Evolutionary models for each data set were selected using MrModelTest 2.3 (Nylander et al. 2004) under the Akaike information criterion (Akaike, 1974). The HKY+ I+ G model was chosen for COI, the GTR + I model was chosen for 16S, and the GTR + I + G model was chosen for the concatenated dataset. Two different phylogenetic methods, Bayesian Inference (BI) and Maximum Likelihood (ML) were used to infer evolutionary relationships. Bayesian estimation of posterior probability was performed in MrBayes 3.2. Markov chains were sampled at intervals of 1,000 generations. Analysis was started with random starting trees and 107generations. Maximum likelihood-based phylogeny inference was performed in GARLI 2.0 (Zwickl, 2006) with bootstrap in 1,000 replicates. The program Mega7 (Kumar et al., 2016) was used to calculate the uncorrected p-distances between all the sequences. Intra- and inter- group genetic distances were also examined. To evaluate the genetic distribution of the different haplotypes the haplotype network for the combined molecular data (COI + 16S) was reconstructed using the Population Analysis with Reticulate Trees (PopART, http://popart.otago.ac.nz) with the TCS network method. First Record of Trinchesia lenkae in Japan 21

Table 1. List of specimens used for molecular analyses.

GenBank accession nos. Species Voucher Locality COI 16S Diaphoreolis lagunae CAS179465a California KY128956 KY128749 (O’Donoghue, 1926) Diaphoreolis viridis ZMMU:Op-537 Russia, White Sea MG266028 MG266026 (Forbes, 1840) Trinchesia caerulea ZMMU:Op-622 Norway, Gulen MG266024 MG266022 (Montagu, 1804) Trinchesia lenkae Japan: Hokkaido (Pacific side), ZMMU:Op-619 MG242334 MG242333 Martynov, 2002 Akkeshi Bay Trinchesia lenkae Japan: Hokkaido (Pacific side), ZMMU:Op-620 MG242336 MG242335 Martynov, 2002 Akkeshi Bay Trinchesia lenkae Russia: Sea of Japan, Spaseniya ZMMU:Op-621 MG242338 MG242337 Martynov, 2002 Bay Trinchesia lenkae AC17-19* Sea of Japan, Sobol Bay KY129093 KY128884 Martynov, 2002 Trinchesia ornata CAS180344 Hawaii KY128967 KY128758 (Baba, 1937) Trinchesia sibogae CAS177489 Philippines KY128975 KY128767 (Bergh, 1905) Trinchesia speciosa CAS176913 South Africa KY128996 KY128788 (Macnae, 1954) Trinchesia speciosa CAS176954 South Africa KY128998 KY128790 (Macnae, 1954)

* The voucher number AC17-19 doesn’t belong to any of the museum collections mentioned in Cella et al. (2016).

Results

Taxonomy

Family Trinchesiidae Nordsieck, 1972 Trinchesia Ihering, 1879

Trinchesia lenkae Martynov, 2002

Trinchesia lenkae – Martynov, 2002: 48–50, fig. 1.

Material: Three specimens, ZMMU Op-619, Op-620 (Fig. 1), 5 October 2016, Japan, Hokkaido (Pacific side), Akkeshi Bay, 2–4 m depth, on brown algae Sargassum spp., leg. T. Korshunova and A. Martynov. Comparative material: Two specimens and egg mass, ZMMU Op-621 (Fig. 2), 10 September 2014, Russia, Spaseniya Bay, Peter the Great Bay, the Sea of Japan, intertidal, under stones, leg. T. Korshunova and A. Martynov. Description (based on specimens from Akkeshi Bay): External morphology. The maximum length of the examined specimens is 12 mm (Figs 1A–C). The body is moderately broad. The rhinophores are similar in size to the oral tentacles, and smooth. The cerata are relatively long, thin, and finger-shaped. Ceratal formula of the largest 12 mm specimen (ZMMU Op-619): right (2,3,4;5 22 T. Korshunova et al.

Fig. 1. Trinchesia lenkae. A, B, D–F. Specimen ZMMU Op-619 from Akkeshi Bay (Hokkaido, Japan), 12 mm length; (A) dorsal (B) ventral views of living specimen; (D) posterior part of the radula; (E) details of the radular teeth; (F) jaw; (G) denticles on the masticatory edge of the jaw (indicated by arrows); (H) penial stylet. C. Specimen ZMMU Op-620 from Akkeshi Bay (Hokkaido, Japan), 8 mm length. Scale bars: D, E, G, 10 μm; H, 20 μm; F, 100 μm. anus, 4,4,3,2,2,1) left (2,4,4;5 anus, 4,5,4,3,2,1). The foot is moderately broad, with well-defined long foot corners anteriorly (Fig. 1B). Colour. The ground colour is whitish. The digestive branches in the cerata are brownish (Figs 1A, B) to reddish (Fig. 1C). A few small opaque white spots are scattered on the ceratal tops. There First Record of Trinchesia lenkae in Japan 23

Fig. 2. Trinchesia lenkae. A, B, D–F. Specimen ZMMU Op-620 from Spaseniya Bay (Peter the Great Bay, the Sea of Japan, Russia), 12 mm length; (A) dorsal (B) ventral views of living specimen; (D) posterior part of the radula; (E) details of the radular teeth; (F) jaw; (G) denticles on the masticatory edge of the jaw (indicated by arrows); (H) penial stylet. C. Egg-mass from Spaseniya Bay (Peter the Great Bay, the Sea of Japan, Russia). Scale bars: D, E, G, 10 μm; H, 20 μm; F, 100 μm. are no white spots throughout most of the length of the cerata. The are bright white. The rhinophores are covered basally with orange pigment up to half or 2/3 of their lengths. Upper parts of the rhinophores and oral tentacles are covered with diffuse white pigment. Anatomy. Digestive system. The jaws are ovoid (Figs 1F, G). The masticatory processes of jaws bear a single row of very delicate denticles (Fig. 1G). The radular formula in the 12 mm 24 T. Korshunova et al.

Table 2. Uncorrected p-distances (%) ± Std. Err. amongst Trinchesia lenkae specimens for COI (highlighted in red) and 16S (highlighted in blue) markers.

ZMMU:Op-619 ZMMU:Op-620 ZMMU:Op-621 AC17-19 Japan Japan Russia Russia ZMMU:Op-619 Japan – 0 0.46 ± 0.32 0.69 ± 0.40 ZMMU:Op-620 Japan 0.32 ± 0.22 – 0.46 ± 0.32 0.69 ± 0.40 ZMMU:Op-621 Russia 0.32 ± 0.22 0.32 ± 0.22 – 0.23 ± 0.23 AC17-19 Russia 0.47 ± 0.27 0.47 ± 0.27 0.16 ± 0.16 – specimen is 48 × 0.1.0. The radular teeth are yellowish to light brownish. The central tooth is wide, with a low cusp (similar in length to adjacent lateral teeth or slightly longer, not retracted) and up to six (normally 4–5) large lateral denticles and a few intercalated smaller denticles (Figs 1D, E). Reproductive system: the ampulla is moderately short and swollen. The prostate is a slightly convoluted tube. The prostate transits to a penial sheath, which contains a conical penis with a long curved hollow chitinous stylet (Fig. 1H). A supplementary (“penial”) gland inserts into base of the penis. The seminal receptacle is long and narrow, with several swollen nodes. Habitat: Under stones in the intertidal in Peter the Great Bay, Russia; at a depth of 2–5 m on brown algae Sargassum spp. at Akkeshi Bay, Hokkaido, Japan. Reproduction: The egg mass was obtained in Spaseniya Bay, Peter the Great Bay on 10 September 2014: the egg mass is thick flattened spiral yellowish cord of 2.5 whorls. There are ca. 210 eggs (Fig. 2C). Distribution: Northern part of the Sea of Japan, Peter the Great Bay (Russia), and Pacific coast of Hokkaido (Japan). It is expected to occur in Japan at Hokkaido on the coast of the Sea of Japan and in northern Honshu.

Molecular analysis In the molecular study, two Trinchesia lenkae specimens from Japan and two from Russia, including one from GenBank publicly available, were used. A total of six sequences were generated in the present study and 16 sequences were extracted from GenBank (Table 1). The molecular study verifies that publicly available sequences from GenBank for specimen voucher number AC17-19 (Cella et al., 2016) corresponds to T. lenkae Martynov, 2002. Regarding the COI and 16S markers, the genetic distances amongst all the available data are 0–0.69 ± 0.40% (Table 2). COI p-distances within the T. lenkae population from Japan (0.3%), and within T. lenkae from Russia (0.16%) do not exceed 0.3%. The same result shows 16S p-distances within the T. lenkae population from Japan (0%), and within the T. lenkae specimens from Russia (0.23%). COI and 16S p-distances between the Japanese and Russian populations (0.4% and 0.57% respectively) are very small for interspecific distances. The minimum uncorrected p-distances for the COI fragments between T. lenkae and other species of the genus Trinchesia including the type species T. caerulea (Montagu, 1804), T. ornata (Baba, 1937), T. sibogae (Bergh, 1905), and T. speciosa (Macnae, 1954) were 17.32%, 12.98%, 18.72%, and 16.13% respectively, which significantly exceed COI p-distances between T. lenkae specimens (range 0.16–0.47%). Results obtained by PopART showed a network of haplotypes based on concatenated molecular data that clustered into two groups coincident with populations from the Japan (two specimens ZMMU:Op-619 and ZMMU:Op-620) and from Russia (two specimens ZMMU:Op-621 and AC17- 19). At the same time, network of haplotypes showing only three genetic mutations occurring between T. lenkae specimens from Japan and Russia populations that corresponds to intraspecific First Record of Trinchesia lenkae in Japan 25

Fig. 3. The haplotype network based on concatenated molecular data showing genetic mutations occurring within Trinchesia lenkae specimens from Japan and Russia.

Fig. 4. Phylogenetic tree of the representatives of the genus Trinchesia based on concatenated molecular data represented by Bayesian Inference (BI). Numbers above branches represent posterior probabilities from BI. Numbers below branches indicate bootstrap values for Maximum Likelihood (ML). differences (Fig. 3). The resulting concatenated tree provided better resolution than COI, and 16S trees separately (not shown). BI and ML methods were used to infer phylogenetic trees. The topology of the tree obtained by BI was the same as the one inferred by ML. All four T. lenkae specimens clustered in a highly-supported clade together (PP = 1, BS = 100) that is sister to the T. ornata clade (Fig. 4). Insignificant divergence within all genetic distances supports the considerable morphological similarity between specimens of T. lenkae from Japan and Russia (Figs 1–2, Table 2).

Discussion

Morphological and molecular analysis of the newly discovered specimens of T. lenkae from the Pacific side of Hokkaido reveals a high degree of similarity to the specimens from the type 26 T. Korshunova et al. locality in Peter the Great Bay, the Sea of Japan, in Russia. Diagnostic external and internal features, including 3-4 preanal ceratal rows, the presence of the peculiar tentacular processes of the anterior part of the foot, broad radular teeth with a low cusp and smaller intercalated denticles, a long receptaculum seminis and distinctly curved penial stylet are in total agreement between Japanese and Russian specimens (Figs 1, 2, respectively) and with the original description of this species (Martynov, 2002). The distinctive colour of the rhinophores (which are covered with orange pigment from half to 2/3 of their length) is also shared between Russian and Japanese specimens of T. lenkae. The colour of the ceratal digestive gland shows minor variations and includes brownish and reddish (Hokkaido) specimens and brownish reddish to reddish orange specimens (Peter the Great Bay). Also, in the original description “almost smooth” masticatory edges of the jaws were described (Martynov, 2002), whereas in this study, both for Russian and Japanese specimens, delicate denticles were observed (Figs 1G, 2G). This is explained because the jaws in the present study were investigated using SEM, whereas in the original description only light microscopy was available and thus the small delicate denticles were probably overlooked. The molecular analysis confirmed the high morphological similarity. Results obtained by PopART showed a network of haplotypes that clustered into two groups coincident with T. lenkae from Japanese and Russian localities (Fig. 3). Simultaneously, small genetic divergence between all investigated specimens supports the fact that the Japanese population belongs to T. lenkae, and this corresponds to the phylogenetic tree results (Fig. 4). Akkeshi Bay has a long history of opisthobranch investigations (Baba, 1935, 1957); however, species similar to T. lenkae were never reported either from vicinities of Akkeshi Marine Station or from other parts of Japan. The specimens of T. lenkae from the Russian part of the Sea of Japan come from Far-Eastern Natural Reserve, and thus represent a first record of this species for this protected area. Previously, with the first description of T. lenkae, the status of the genus Trinchesia was discussed and it was suggested to restore it and delineate it from the genus Cuthona (Martynov, 2002). Most recently, using an integrative molecular and morphological approach, the necessity of the restoration of the genus Trinchesia, together with some other genera within the family Trinchesiidae has also been confirmed (Korshunova et al., 2017a). The molecular and morphological data (presence of well-defined tentacular anterior processes of the foot, broad radular teeth with low but not retracted central cusp, and long curved penial stylet) of T. lenkae are concordant with the type species of the genus Trinchesia, T. caerulea, and therefore support the restriction of the genus Trinchesia s. str. (Korshunova et al., 2017a, b).

Acknowledgements

We give our special thanks to the Dr. Tomoko Fukuda (Mie University) for help with organizing the research trip to Hokkaido. Prof. Masahiro Nakaoka, the director of the Akkeshi Marine Station, is warmly thanked for permission to conduct field research at the station. The staff of the Akkeshi Marine Station and the crew of the research boats generously helped us during the field collecting. G. N. Davidovich and A. G. Bogdanov (Electron Microscopy Laboratory, Moscow State University) are gratefully acknowledged for support with electron microscopy. This work was supported by a research grant of MSU Zoological Museum (АААА-А16-116021660077-3, depository of specimens) and the Russian Science Foundation (grant 14-50- 00029, morphological & molecular study).

References

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(Received October 19, 2017 / Accepted January 26, 2018) 28 T. Korshunova et al.

Trinchesia lenkae Martynov, 2002 の日本からの新記録：ロシアのタイプ産地の 標本との形態的及び分子系統学的比較（裸鰓類：Trinchesiidae）

Tatiana Korshunova・藤田敏彦・Alexander Martynov

要 約

裸鰓類の一種である Trinchesia lenkae Martynov, 2002 が，日本国内から初めて厚岸湾から発見された。 新たに採集された日本産の個体は，タイプ産地である日本海北部ロシア極東地域のピョートル大帝湾の標 本と内・外部形態，および分子系統的に比較された。分子系統解析の結果，日本とロシアの個体群の間の 遺伝的均質性が高いことが示された。形態学的な近似性も含めると，日本の個体群はロシアの個体群と同 じ種に含まれることが明瞭に示された。