First record of a photosymbiotic ascidian Lissoclinum midui (Ascidiacea: Didemnidae) from Lyudao (Green Island), Taiwan euichi hirose 1 , yoko nozawa 2 and mamiko hirose 3 1 Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan, 2 Biodiversity Research Center, Academia Sinica, Nangkang, Taipei 115, Taiwan, 3 Marine and Coastal Research Center, Ochanomizu University, Tateyama, Chiba 294-0301, Japan Lissoclinum midui was collected from Lyudao (Green Island), Taiwan. This is the first record outside of the type locality, Kumejima Island (Ryukyu Archipelago, Japan), of this species. The colonies in Lyudao were found on non-geniculate coraline algae covering the coral limestones in the reef crest (0.5 – 1 m deep). They were not rare at the collection sites, and it is uncer- tain why this species has only been recorded in the two sites in Taiwan –Ryukyus. It may be that the shallow, wave-exposed reef crest is a poorly investigated habitat for small benthic invertebrates. Keywords: colonial ascidian, coral reefs, DNA-barcode, photosymbiosis, Prochloron, subtropical West Pacific Submitted 19 January 2014; accepted 26 January 2014 INTRODUCTION Lissoclinum midui Hirose & Hirose, 2011 is a colonial ascidian harbouring the cyanobacterial symbiont Prochloron in the tunic. The colonies investigated in the original description were found on non-geniculate coraline algae in the reef crest (0.5–1 m deep) off Shinri-hama (Kumejima Island, Ryukyu Archipelago, Japan) (Figure 1). This species has two character states that are unique among didemnid ascidians: a cloacal aperture in each zooid; and an unknown organ in the bottom wall of the thorax (Hirose & Hirose, 2011). Despite these unique features, L. midui has never been recorded from any site other than the type locality. In contrast, we have observed photosymbiotic ascidian fauna at various sites across Taiwan–Ryukyus (Hirose, 2013 and references therein). However, it is unlikely that the distribution range of this species is strictly limited to the type locality. We found multiple colonies of L. midui in the reef crest off Gongguan in Lyudao (Green Island), Tawain (Figure 1). A molecular phylogeny inferred from the cytochrome oxidase subunit I (COI) gene sequences supported the monophyly of this species as well as of the genus Lissoclinum. It is possible that the range of this species is broad but has merely not been recorded. MATERIALS AND METHODS Animals Colonies of Lissoclinum midui were collected from the reef crest off Gongguan (22838 ′ 42 ′′ N 121830 ′ 5 ′′ E, Lyudao, Taiwan) on 24 June 2013. The colonies were found as an aggregate, and those from the two aggregates were sampled for this study. The first author found four aggregates of colonies within a 1-hour survey by snorkelling. The colonies were photographed using a digital camera before they were scraped off the substrate using a steel knife. We fixed the colonies in 10% formalin – seawater after anaesthetizing them with menthol and 0.37 M MgCl 2 . Some specimens were deposited in the National Museum of Natural Science, Taichung, Taiwan (NMNS-7254-001). Some fresh colonies were preserved in 99% ethanol at 2308C for DNA analyses. The zooids were dissected from the fixed colonies and observed under a binocular stereomicroscope. In some photo- micrographs, several images were combined to increase the depth of field using the post-processing image software Helicon Focus Pro 4.2.8 (Helicon Soft, Ltd., Kharkov, Ukraine). DNA extraction, amplification and sequencing Tissue samples were preserved in ethanol at 2308C. Three preserved colonies from a colony aggregate were dissected under a stereo microscope, and genomic DNA was extracted from about 40 zooids using a DNeasy Tissue Kit (Qiagen K.K., Tokyo, Japan) following the manufacturer’s protocol. A segment of the mitochondrial COI gene was amplified using the primers UroCox1-244F (5 ′ –CATTTWTTTTGAT TWTTTRGWCATCCNGA – 3 ′ ) and UroCox1-387R (5 ′ – GCWCYTATWSWWAAWACATAATGAAARTG – 3 ′ ), fol- lowing the method of Hirose et al. (2009) for the amplifica- tion and sequencing of the partial COI fragments. Five clones were randomly selected and sequenced. Polymerase chain reaction products were treated with ExoSAP-IT (GE Healthcare) prior to the sequencing reactions. Sequencing Corresponding author: E. Hirose Email: euichi@sci.u-ryukyu.ac.jp 1 Marine Biodiversity Records, page 1 of 5. # Marine Biological Association of the United Kingdom, 2014 doi:10.1017/S175526721400013X; Vol. 7; e42; 2014 Published online