Two species of styelid colonial ascidians in the genus Syncarpa Redikorzev, 1913 are known from the northwest Pacific. The valid status of the lesser known species, Syncarpa composita (Tokioka, 1951) (type locality: Akkeshi, Japan), is assessed here. To assess the taxonomic identity of S. composita, we compared one of the syntypes and freshly collected topotypes of S. composita with a syntype of S. oviformis Redikorzev, 1913 (type locality: Ul’banskij Bay, Russia). Specimens of S. composita consistently differed from the syntype of S. oviformis in the number of oral tentacles, the number of size-classes of transverse vessels, and the number of anal lobes. In this paper, S. composita is redescribed as distinct from S. oviformis, and its phylogenetic position inferred within Styelidae based on the 18S rRNA and cytochrome c oxidase subunit I gene sequences. In our phylogenetic tree, Syncarpa formed a well-supported clade together with Dendrodoa MacLeay, 1824. In Syncarpa and Dendrodoa, a single gonad is situated on the right side of the body, which is unique among Styelidae, and thus can be a synapomorphy for this clade.

Chordata, COI, phylogeny, Sea of Okhotsk, taxonomy, Urochordata

Syncarpa Redikorzev, 1913 is a member of the ascidian family Styelidae and consists of two species, Syncarpa composita (Tokioka, 1951) and S. oviformis Redikorzev, 1913. The two nominal species S. corticiformis Beniaminson, 1975 and S. longicaudata Skalkin, 1957, all from the Northwest Pacific, have been synonymized with S. oviformis by Sanamyan (2000). This genus is defined by the following four characters: i) colonial, with zooids reproducing asexually, ii) a single, well-developed fold is present on each side of the pharynx, iii) a single gonad is situated on the right side of the body, and iv) the gonad has several branches. Syncarpa composita is only known by the original description based on material from Akkeshi, Japan (Tokioka 1951). It was originally placed in a new monotypic genus Syndendrodoa Tokioka, 1951, which has been synonymized with Syncarpa by Nishikawa (1995).

The phylogeny of ascidians including styelids has been investigated by Zeng et al. (2006), Pérez-Portela et al. (2009), Tsagkogeorga et al. (2009), Alié et al. (2018), and Delsuc et al. (2018). Among these, Alié et al.’s (2018) analysis was based on 4908 genes and included 16 OTUs from Styelidae. It recovered Styelidae as monophyletic with maximum branch-support values, which turned out to be sister to part of paraphyletic Pyuridae. Alié et al.’s (2018) phylogeny showed three major clades for Styelidae: i) Polyzoinae + Botryllinae, ii) Dendrodoa + Polycarpa + Polyandrocarpa zorritensis (Van Name, 1931), and iii) Astrocarpa + Styela. However, no member of Syncarpa has ever been placed on a phylogenetic context in any of the previous studies.

The aims of this study are to assess the taxonomic identity of S. composita based on type specimens and freshly collected topotypes andto infer the species’ phylogenetic position among Styelidae. In this paper, we redescribe the species and present the results of a multi-gene molecular analysis.

Eleven topotype colonies of S. composita were freshly collected by dredging, snorkeling, and SCUBA diving in the type locality, Akkeshi Bay, at depths of 3–5 m in June, August, and September 2017, and July 2018 (Table 1). One of the colonies was photographed underwater and in the laboratory with a Nikon COOLPIX AW130 digital camera. The live colonies were anesthetized with menthol; then a part of a zooid was cut off along with the tunic from each colony and preserved in 99% EtOH for DNA extraction. The colonies were preserved in 10% formalin-seawater for morphological observation; zooids were removed from the colonies and then dissected for morphological examination. Larvae for histological observation were dehydrated in an ethanol series, cleared in xylene, embedded in paraffin wax, sectioned at 5 µm thickness, and stained with hematoxylin and eosin. After sections were mounted on glass slides in Entellan New (Merck, Germany), they were observed under an Olympus BX51 compound microscope and photographed with a Nikon D5200 digital camera. These voucher specimens have been deposited in the Invertebrate Collection of the Hokkaido University Museum (ICHUM), Sapporo, Japan. For comparison, specimens deposited in the Seto Marine Biological Laboratory (SMBL), Shirahama, Japan, and the Zoological Institute of the Russian Academy of Sciences (ZIRAS), St. Petersburg, Russia, were also examined.

Total genomic DNA was extracted from a piece of the body wall tissue for eight specimens of S. composita as well as one specimen each of Botrylloides violaceus Oka, 1927, Pyura mirabilis (Drasche, 1884), Styela clava Herdman, 1881, and Styela plicata (Lesueur, 1823) (Table 1). The tissue was placed in a 1.5 mL tube after air-dried, then mixed with 180 µL of ATL buffer (Qiagen, Hilden, Germany) and 20 µL of proteinase K (>700 U/mL, Kanto Chemical, Tokyo, Japan), and incubated at 55 °C for ca. 10 h. To the lysis solution, 200 µL of AL buffer (Qiagen) was added and incubated at 70 °C for 10 min; then 210 µL of 99% EtOH was added. The rest of the DNA extraction was carried out following Boom et al.’s (1990) silica method.

Two gene markers were amplified from the genomic DNA by PCR. The nuclear 18S rRNA (18S) gene was amplified with the primer pair 1F/9R (Giribet et al. 1996). The mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the primers Sty_COI_F2 (5'-TTTGCCTTTAATAGTAAGAAGTCC-3') and Sty_COI_R1 (5'-CATCAAAACAGATGCTGATA-3') for S. composita and with the primer pair LCO1490/HCO2198 (Folmer et al. 1994) for the other ascidians. PCRs were performed in a 10-µL total reaction volume with 3 µL of each primer pair (10 µM), 0.5 µL of TaKaRa Ex Taq (TaKaRa, Kusatsu, Japan), 10 µL of 10 × Ex Taq Buffer (TaKaRa), 8 µL of dNTP mixture (TaKaRa), 1 µL of extracted DNA, and 68.5 µL of deionized water. Thermal cycling condition was 94 °C for 2 min; 35 cycles of 94 °C for 45 sec, 52 °C for 90 sec (for 18S) or 55 °C for 50 sec (for COI), and 72 °C for 55 sec; then 72 °C for 5 min. Amplification was verified by electrophoresis in 1% agarose gel. The PCR products were purified through enzymatic reaction with 24 mU/µL of Exonuclease I (TaKaRa) and 4.9 mU/µL of Shrimp Alkaline Phosphatase (TaKaRa). The purified PCR products were sequenced directly with a BigDye Terminator ver. 3.1 Cycle Sequence Kit (Applied Biosystem, Foster, CA, USA) and 3730 Genetic Analyzer (Applied Biosystems), using the same primer pairs for amplification, as well as the following internal primers for 18S: 3F, 5R (Giribet et al. 1996); and 2, bi (Whiting et al. 1997). Base calling was performed with GeneStudio Professional Edition ver. 2.2.0.0 (GeneStudio, Suwanee, GA, USA).

To infer the phylogenetic position of S. composita, 18S and COI sequences of 24 species of Styelidae were obtained from GenBank (Table 2). For 18S, alignment was carried out by MAFFT ver. 7 using the E-INS-i strategy (Katoh and Standley 2013); ambiguous sites were removed by using Gblocks ver. 0.91b (Castresana 2002). For COI, nucleotide sequences were manually edited by MEGA ver. 5.2.2 (Tamura et al. 2011) so that translated amino acid sequences were aligned straightforward without indels. 18S and COI sequences were concatenated by using MEGA ver. 5.2.2 (Tamura et al. 2011).

Bayesian inference (BI) was performed using MrBayes ver. 3.2.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). The best-fit substitution models selected by PartitionFinder ver. 2.1.1 (Lanfear et al. 2016) for BI were GTR+I+G for 18S and GTR+G for all the three codon positions of COI. Each Markov chain was initiated from a random tree and run for 5 × 10⁶ generations; trees were sampled every 100 generation from the chain. Burn-in fraction was set to be 0.25. A consensus of sampled trees was computed using the “sumt” command, and the posterior probability (PP) for each interior branch was obtained to assess the robustness of the inferred relationships. Values of run convergence indicated that sufficient amounts of trees and parameters were sampled (average standard deviation of split frequencies = 0.009823; average estimated sample size of tree lengths = 205.35; potential scale reduction factor of tree lengths = 1.005). Run convergence was also assessed with Tracer ver. 1.6 (Rambaut et al. 2014) to see if the effective sample size of each parameter exceeded 200. Maximum Likelihood (ML) analysis was performed by RAxML ver. 8.2.3 (Stamatakis 2014). One thousand fast-bootstrap replicates were conducted to evaluate nodal support.

Sincere thanks are offered to Ms Haruka Yamaguchi, Mr Hidenori Katsuragawa, Mr Shoichi Hamano (Akkeshi Marine Station, Hokkaido University); Mr Daiki Wakita, Dr Kevin Wakeman (Hokkaido University); Mr Hisanori Kohtsuka, Mr Mamoru Sekifuji, and Ms Michiyo Kawabata (Misaki Marine Biological Station, University of Tokyo) for their help in collecting the samples. We are grateful to Dr Yuko Takigawa (Kagawa University); Dr Shigeyuki Yamato (Seto Marine Biological Laboratory, Kyoto University); and Dr Igor Smirnov (Zoological Institute, Russian Academy of Sciences) for their help with specimen loans. Advice and comments given by Dr Teruaki Nishikawa (National Museum of Nature and Science) have been a great help in this study. This study was supported by Research Institute of Marine Invertebrates (FY 2018, No. 6). We thank Dr Keiichi Kakui (Hokkaido University), and the other members of Biodiversity 1 for their help.