Aquatic Invasions (2013) Volume 8, Issue 3: 271–280 doi: http://dx.doi.org/10.3391/ai.2013.8.3.03 Open Access

© 2013 The Author(s). Journal compilation © 2013 REABIC

Research Article

Photosymbiotic ascidians from oceanic islands in the tropical Pacific as candidates of long-dispersal species: morphological and genetic identification of the species

Mamiko Hirose1 and Euichi Hirose2* 1 Marine and Coastal Research Center, Ochanomizu University, Tateyama, Chiba 294-0301, Japan 2 Dept. of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan E-mail: [email protected] (MH), [email protected] (EH) *Corresponding author

Received: 13 April 2013 / Accepted: 20 August 2013 / Published online: 29 August 2013

Handling editor: John Mark Hanson

Abstract

Following the increase in seawater temperatures due to global warming, tropical species may expand their geographic ranges toward higher latitudes, and thus are noteworthy as potential introduced species. Among photosymbiotic ascidians, Trididemnum cyclops Michaelsen, 1921 and Diplosoma simile (Sluiter, 1909) have broad ranges in the tropical Pacific, including oceanic islands. Considering the taxonomic difficulties in identifying didemnid species, it is important to verify the species identification of specimens from oceanic islands and to examine genetic differences between them and conspecifics from continental islands. We examined zooid morphologies and partial sequences of the mitochondrial COI gene of T. cyclops and D. simile from oceanic islands in the Pacific (Bonin Islands and Hawai'i) to compare with those from the Ryukyu Archipelago (Japan), continental islands, and Caribbean Panama. The specimens from the oceanic islands were confirmed to be T. cyclops and D. simile. We assume that the colonies in oceanic islands were originally derived from the tropical western Pacific region, where the species richness is much higher than that in oceanic islands. The wide geographic range of these species suggests that T. cyclops and D. simile have long-range dispersal capabilities and broad environmental tolerances and may expand their distributions toward higher latitudes as seawater temperature increases due to global warming. Key words: COI; colonial tunicates; coral reef; cyanobacterial symbiosis; molecular phylogeny; oceanic islands

is reasonable to further examine photosymbiotic Introduction ascidians with large distribution ranges. In tropical and subtropical waters, about 30 Non-indigenous ascidians cause serious problems ascidian species are known to harbor cyanobacterial worldwide (Lambert 2007). Outbreaks of introduced symbionts such as Prochloron and Synechocystis ascidians often change native biota on large (Lewin and Cheng 1989; Hirose et al. 2009a). These scales and cause huge losses in aquaculture. are all colonial tunicates belonging to the family Following increases in seawater temperatures Didemnidae, which is the largest family in the due to global warming, tropical species could class Ascidiacea (Chordata: Tunicata) and probably expand their distribution ranges toward higher includes many undescribed species, particularly latitudes, and some species potentially become in tropical waters (Kott 2005). Microscopic exami- invasive in non-native regions. Among these, nation is often necessary for species identification photosymbiotic ascidians are high-risk candidates of didemnid species, because the zooids are very because their cyanobacterial symbionts are known small (sometimes measuring less than 1 mm in to produce various toxins (e.g., Donia et al. 2011), length) and simplified, resulting few morphological and the host ascidians are probably subjected to characters for species identification. Moreover, little or no predation. Additionally, species with the colonies often inhabit cryptic sites, such as large distribution ranges are supposed to have the undersides of, or crevasses in, coral limestones long-range dispersal capabilities and broad environ- or dead coral fragments. Accordingly, few mental tolerances, and thus would be powerful distribution records exist for many species (Kott candidates for becoming invasive. Therefore, it 2001; Monniot and Monniot 2001).

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The distribution ranges of photosymbiotic candidates expanding their distribution ranges ascidians differ from species to species. For toward higher latitudes following the global example, Didemnum molle (Herdmann, 1886) is warming of seawater. In the present study, we a conspicuous photosymbiotic species because of examined zooid morphologies and the partial its dome-shaped colony and its occurrence in sequences of the mitochondrial cytochrome oxidase exposed sites. This species is widely distributed subunit I gene of D. simile from oceanic islands from the western Indian Ocean to Fiji, and from in the Pacific (Bonin Islands and Hawai'i) and about 30ºN to more than 30ºS (Kott 2001), but compared them with those from Okinawa Island has never been recorded from some oceanic (Ryukyu Archipelago, Japan), a continental island, islands situated far from continents and other and from Caribbean Panama. island groups, e.g., French Polynesia (Monniot and Monniot 1987), the Bonin Islands (Hirose et al. 2007), and Hawai'i (Abbot et al. 1997). In Materials and methods contrast, Trididemnum cyclops Michaelsen, 1921 Animals and Diplosoma simile (Sluiter, 1909) have considerably broader distributions than D. molle The sampling of ascidian colonies was carried in the Pacific: the former species has been out by snorkeling in shallow subtidal areas (<3 recorded from French Polynesia and the Bonin m) in the Bonin Islands, Hawai’i, Taiwan, and Islands, and the latter has been recorded from Ryukyu Archipelago, from 2007 to 2012 (Figure 1). French Polynesia, the Bonin Islands, and Hawai'i Colonies of T. cyclops were collected from Ishijiro (Monniot and Monniot 1987; Abbot et al. 1997; Beach (26º38'00"N, 142º09'40"E) in Hahajima Hirose et al. 2007). Recently, colonies collected (Bonin Islands) on 14 May 2012. Trididemnum from Caribbean Panama were identified as D. simile miniatum colonies were collected from Bise based on their partial mitochondrial DNA (26º42'35"N, 127º52'47"E) off Okinawajima Island sequences as well as morphological features (Ryukyu Archipelago) on 15 June 2007, and T. (Hirose et al. 2012). These D. simile colonies may paracyclops colonies were collected from Agari- have been introduced from the Pacific. Vargas- hama (26º22'07"N, 127º09'00"E) in Tonakijima Ángel et al. (2009) reported a recent outbreak of Island (Ryukyu Archipelago) on 22 March 2012. D. simile in American Sãmoa, where overgrowth Diplosoma simile colonies were collected from by ascidian colonies threatens native scleractinian Ishijiro Beach (26º38'00"N, 142º9'40"E), Minami- corals. As mentioned above, species identification zaki (26º36'32"N, 142º10'35"E), and North Port of photosymbiotic didemnids is often problematic (26º41'55"N, 142º08'34"E) off Hahajima (Bonin due to the shortage of morphological characters for Islands) on 14 and 15 May 2012, Miyanohama species discrimination. For instance, we have (27º06'14"N, 142º11'40"E) and Kanameishi described six Diplosoma species as new species (27º04'58"N, 142º12'26"E) off Chichijima (Bonin from the Ryukyu Archipelago (Japan) since 2005, Islands) on 17 May 2012, and in the vicinity of using the number of stigmata of branchial sac as the Hawai'i Institute of Marine Biology (21º26'00" a novel character for taxonomy, and the recognition N, 157º47'12"E) off Coconut Island (Kaneohe, of the species was also supported by molecular Hawai'i) on 13 March 2012. To compare with phylogeny analyses (see Hirose and Hirose 2009). these specimens from oceanic islands, we used Considering the taxonomic difficulties, it is colonies collected from Crawl Key (9º15'47.78"N, important to verify whether the specimens from 82º07'51.64"W) and Isla Cristobal (9º16'56.4"N, oceanic islands are actually conspecific to those 82º15'25.7"W) off Bocas del Toro (Panama) on from continental islands, integrating morphological 22 and 27 June 2011 (Hirose et al. 2012) and from and molecular methods. Teniya (26º33'50"N, 128º8'30"E) off Okinawa In general, the species richness in tropical Island (Ryukyu Archipelago) on 13 September Indo-Pacific Oceans is highest in the Indonesian– 2007. Diplosoma ooru colonies were collected Philippine region and steadily decreases eastward from Nanwan (21º57'31"N, 120º45'57"E) in Kenting across the Pacific, consistent with dispersal from (Taiwan) on 23 March 2011, D. virens colonies a center of high diversity (e.g., Roberts et al. were collected from Yobuko-hama (26º21'31"N, 2002; Mora et al. 2003). This suggests that 127º08'31"E) in Tonakijima Island (Ryukyu T. cyclops and D. simile may have greater Archipelago) on 21 March 2012, and Uganzaki dispersal capabilities than other photosymbiotic (24º27'56"N, 127º07'22"E) in Ishigakijima Islands didemnid species, making them potential (Ryukyu Archipelago) on 13 September 2009,

272 Photosymbiotic didemnid ascidians from oceanic islands

Figure 1. Map of the North Pacific and Caribbean Sea indicating the regions of the collection sites listed in Table 1. Bn, Bonin Islands; Fu, Fukui; Hw, Hawai'i; Ka, Kagoshima; Ok, Okinawa; Pa, Caribbean Panama; Tw, Taiwan.

and D. watanabei colonies were collected from Hirose et al. (2009c). The PCR primer sequences Yobuko-hama in Tonakijima Island (Ryukyu were originally constructed by Yokobori (Hirose Archipelago) on 21 March 2012 (Figure 1). Some et al. 2009c) based on the sequence comparisons colonies were anesthetized with menthol and of tunicate COI genes, since amplification of the 0.37 M MgCl2 for approximately 2 h and then so-called 'Folmer’s region' of the COI gene (at fixed with 10% formalin–seawater for microscopic the 5' end of the cytochrome c oxidase subunit 1 observation. Some pieces from colonies were also region) was often unstable in our specimens. preserved in 99% ethanol for DNA extraction. Although the primers amplify a different region from the 'Folmer’s region' of the COI gene, more Microscopy sequence data were available in the public domain for the 'Yokobori's region' than for the Formalin-fixed specimens were dissected under a 'Folmer’s region' of photosymbiotic didemnid binocular stereomicroscope for species identifi- species. At least five clones were randomly selected cation. Zooids isolated from the fixed materials and sequenced for each sample. The sequences were observed under a light microscope equipped determined in this study have been deposited in with differential interference contrast (DIC) optics. the DNA databases of GenBank, European Several micrographs were combined to increase the Molecular Biology Laboratory (EMBL), and DNA depth of field using the post-processing image Data Bank of Japan (DDBJ) under the accession software Helicon Focus Pro 4.2.8 (Helicon Soft, numbers shown in Table 1. Ltd., Kharkov, Ukraine). Calcareous spicules in the tunics of T. cyclops colonies were photo- graphed under a light microscope and the diameters Phylogenetic analyses of 100 spicules were measured using ImageJ There were 16 partial COI gene sequences (three 1.44 (http://imagej/nih.gov/ij). Trididemnum sequences and 11 Diplosoma sequences) obtained in this study. Partial COI DNA extraction, amplification, and sequencing gene sequences data of six Trididemnum species Tissue samples were preserved in ethanol at – (eight sequences) and eight Diplosoma species 30°C. Genomic DNA was extracted from about (12 sequences) were retrieved from GenBank 30–40 zooids using a DNeasy Tissue Kit (Qiagen (Table 1). These were all available COI gene K.K., Tokyo, Japan) following the manufacturer’s sequence of the 'Yokobori's region' in Trididemnum protocol. Part of the mitochondrial COI gene was and Diplosoma. A total 36 COI sequences (11 amplified using UroCox1-244F (5′-CATTTWT Trididemnum sequences and 25 Diplosoma sequen- TTTGATTWTTTRGWCATCCNGA-3′) and Uro ces) were used for phylogenetic analyses, in which Cox1-387R (5′-GCWCYTATWSWWAAWACA Didemnum molle (AB433947), Lissoclinum TAATGAAARTG-3′), and the amplification and bistratum (AB433977) and L. punctatum (AB sequencing of the partial COI fragments followed 433976) were used as the outgroup (Table 1).

273 M. Hirose and E. Hirose

Table 1. List of Didemnidae species used for phylogenetic tree construction in this study.

Species Collection site, Regiona Abbreviation Acc. No. Reference

Diplosoma D. aggregatum Muiga, Miyakojima, Ok D. aggregatum_Miyako AB485997 Hirose and Hirose 2009 D. gumavirens Shinrihama, Kumejima, Ok D. gumavirens_Kume AB473642 Hirose et al. 2009b D. gumavirens Muiga, Miyakojima, Ok D. gumavirens_Miyako AB473643 Hirose et al. 2009b D. gumavirens Hirakubo, Ishigakijima, Ok D. gumavirens_Ishigaki AB485998 Hirose and Hirose 2009 D. ooru Shiraho, Ishigakijima, Ok D. ooru_Ishigaki AB485999 Hirose and Hirose 2009 D. ooru Nanwan, Kentign, Tw D. ooru_Taiwan AB813813 This study D. simile Teniya, Okinawa Is., Ok D. simile_Okinawa AB433975 Hirose et al. 2009c D. simile Crawl Key, Panama, Pa D. simile_PanamaCK AB813814 This study D. simile Isla Cristobal, Panama, Pa D. simile_PanamaIS AB813815 This study D. simile Coconuts Is., Kaneohe Bay, Hw D. simile_Hawai01 AB813816 This study D. simile Coconuts Is., Kaneohe Bay, Hw D. simile_Hawai02 AB813817 This study D. simile Ishjirou Beach, Hahajima, Bn D. simile_HahaIB AB813818 This study D. simile Minamizaki, Hahajima Is., Bn D. simile_HahaMZ AB813819 This study D. simile North port, Hahajima, Bn D. simile_HahaNP AB813820 This study D. simile Miyano-ura, Chichijima, Bn D. simile_ChichiMU AB813821 This study D. simile Kaname-ishi, Chichijima, Bn D. simile_ChichiKI AB813822 This study D. simileguwa Teniya, Okinawa Is., Ok D. simileguwa_Okinawa AB486000 Hirose and Hirose 2009 D. variostigmatum Teniya, Okinawa Is., Ok D. variostigmatum_Okinawa AB486001 Hirose and Hirose 2009 D. variostigmatum Hirakubo, Ishigakijima, Ok D. variostigmatum_Ishigaki AB486002 Hirose and Hirose 2009 D. virens Teniya, Okinawa Is., Ok D. virens_Okinawa AB433974 Hirose et al. 2009c D. virens Yobuko-hama, Tonakijima, Ok D. virens_Tonaki AB813823 This study D. virens Uganzaki, Ishigakijima, Ok D. virens_Ishigaki AB813824 This study D. watanabei Shimama, Tanegashima Is., Ka D. watanabei_Tanegashima AB473641 Hirose et al. 2009b D. watanabei Nagamahama, Kurimajima, Ok D. watanabe_Kurima AB479382 Hirose et al. 2009b D. watanabei Yobuko-hama, Tonakijima, Ok D. watanabei_Tonaki AB813825 This study Trididemnum T. clinides Bise, Okinawa Is., Ok T. clinides_Okinawa AB602782 Hirose and Hirose 2011 T. cyclops Hirakubo, Ishigakijima, Ok T. cyclops_Ishigaki AB433978 Hirose et al. 2009c T. cyclops Maeda, Okinawa Is., Ok T. cyclops_Okinawa AB499972 Hirose et al. 2010 T. cyclops Ishjirou Beach, Hahajima, Bn T. cyclops_HahaIB AB813826 This study T. miniatum Bise, Okinawa Is., Ok T. miniatum_Okinawa AB813827 This study T. nubilum Bise, Okinawa Is., Ok T. nubilum_Okinawa AB499975 Hirose et al. 2010 T. nubilum Ara Beach, Kumejima, Ok T. nubilum_Kume AB499976 Hirose et al. 2010 T. paracyclops Ara Beach, Kumejima, Ok T. paracyclops_Kume AB499973 Hirose et al. 2010 T. paracyclops Hirakubo, Ishigakijima, Ok T. paracyclops_Ishigaki AB499974 Hirose et al. 2010 T. paracyclops Agari-hama, Tonakijima, Ok T. paracyclops_Tonaki AB813828 This study T. savignii Nyu, Fu T. saviginii AB499977 Hirose et al. 2010 Didemnum molle Seragaki, Okinawa Is., Ok D. molle AB433947 Hirose et al. 2009c Lissoclimun L. bistratum Ara Beach, Kumejima, Ok L. bistratum AB433977 Hirose et al. 2009 c L. punctatum Ara Beach, Kumejima, Ok L. punctatum AB433976 Hirose et al. 2009c a Region: Bn, Bonin Islands; Fu, Fukui; Hw, Hawai'i; Ka, Kagoshima; Ok, Okinawa; Pa, Caribbean Panama; Tw, Taiwan.

Initial alignments were performed using (Jobb 2008); and phylogenetic analysis based on MUSCLE (Edgar 2004), and these were then Bayesian inference (BI) using MrBayes 3.12 inspected by eye and manually edited. Base (Ronquist and Huelsenbeck 2003). To select an frequencies, pairwise base differences, and genetic appropriate nucleotide substitution model, distances were calculated using PAUP* 4.0 Beta Modeltest ver. 3.7 (Posada and Crandall 1988) 10 (Swofford 2003). The following analyses were was used with PAUP. For data with all three performed on the aligned DNA sequences of the codon positions, the general time reversible partial COI gene: maximum likelihood (ML) using model with invariant sites and gamma distribution the October 2008 version of TREEFINDER (GTR+I+G) was selected as the best model using

274 Photosymbiotic didemnid ascidians from oceanic islands

Figure 2. Trididemnum cyclops from Hahajima (Bonin Islands, Japan). A, colonies in situ. B, thorax of a zooid (right side). C, tunic spicules. Arrows indicate three rows of stigmata. bs, branchial siphon; en, endostyle; es, esophagus (anterior part); pc, pigment cap; rt, retractor muscle. Scale bars: 0.1 mm in B; 20 µm in C.

Figure 3. Diplosoma simile from Teniya (Okinawa Island: A, E), Ishijiro Beach (Hahajima: B, F), Miyanohama (Chichijima: C, G), and Coconut Island (Kaneohe, Hawai'i: D, H). A–D, colonies in situ. E–H, thoraxes of zooids (right side) showing the stigma pattern; six stigmata in the first to third rows and five stigmata in the fourth row. Retractor muscle (rt) emerged from the bottom of the thorax. bs, branchial siphon; en, endostyle; es, esophagus; rc, rectum. Scale bars, 0.1 mm.

275 M. Hirose and E. Hirose

Akaike’s information criterion (AIC). For the BI analysis, a Markov chain Monte Carlo (MCMC) analysis was run for 1,000,000 generations, and trees were built at 100-generation intervals (burn-in = 2500). Statistical support for the ML tree was evaluated using a nonparametric bootstrap test with 1000 resampling events.

Results

Trididemnum cyclops from the Bonin Islands Small oval colonies were about 5 mm in diameter. Colonies were green due to Prochloron cells distributed in the peribranchial and cloacal cavities, while the colonial margin and bottom were white due to calcareous spicules in the tunic (Figure 2A). The thorax of zooids were strongly contracted in our specimens (Figure 2B). The thorax contained three rows of stigmata. The anterior end of the endostyle was always pigmented and referred as the “pigment cap.” Gonads were not found in the specimens. Stellate spicules were embedded in the tunic (Figure 2C), and their diameters were 29 µm on average (standard deviation = 6.0; maximum diameter = 48 µm).

Diplosoma simile from Okinawa Island, the Bonin Islands, and Hawai'i Colonies were thin sheets and entirely green due Figure 4. Bayesian inference (BI) consensus tree of Trididem- num spp. based on cytochrome oxidase subunit I (COI) gene to Prochloron cells distributed in the peribranchial sequences. The GTR+I+G model was used for the analysis. Bayes and cloacal cavities (Figure 3A–D). Colony posterior probability and bootstrap probabilities larger than 50% colors varied among colonies even within the for the maximum likelihood (ML) analyses are noted. New same collection site: some colonies inhabiting sequences from this study are in bold. shaded sites were bluish, while colonies from shallow, exposed sites often had white dots of pigment cells in the tunic. No spicules were present. Appearances of the thoraxes differed of all three codon positions from the eight somewhat among zooids depending on the extent haplotypes of Trididemnum specimens were of contraction in the fixatives (Figure 3E–H). 28.9% A, 9.7% C, 16.9% G, and 44.5% T, and The thorax had four rows of stigmata. Six the A+T composition frequency was 73.4%. The stigmata were in the first (top) to third rows and aligned COI sequences were 401 bp long, without five stigmata were in the fourth row (bottom). gaps (insertions/deletions). Figure 4 shows a BI The retractor muscle emerged from the underside consensus tree using the GTR+I+G substitution of the thorax. The thickness of the retractor model. The strict consensus ML under the muscle varied among specimens. There were no GTR+I+G substitution model is not shown. The spicules in the present species as well as the sequence of T. nubilum from Okinawa Island was other Diplosoma species. identical to that of the specimen from Kume Island. The sequences of T. paracyclops from Ishigaki and Kume islands were the same as the Phylogenetic analysis sequence of T. cyclops from Hahajima. The Eight haplotypes were obtained from the partial monophylies of T. cyclops and T. paracyclops COI gene sequences of 11 specimens from six individually were not supported, although the

Trididemnum species. The average base frequencies monophyly of T. cyclops + T. paracyclops was

276 Photosymbiotic didemnid ascidians from oceanic islands

Figure 5. Bayesian inference (BI) consensus tree of Diplosoma spp. based on cytochrome oxidase subunit I (COI) gene sequences. The GTR+I+G model was used for the analysis. Bayes posterior probability and bootstrap probabilities larger than 50% for the maximum likelihood (ML) analyses are noted. New sequences from this study are in bold.

supported by Bayesian posterior provability (BPP) were 401 bp long, without gaps (insertions/ and high bootstrap values. Monophyly of deletions). Sequence differences within each species Tridemnum was not supported. were lower (0–11.0%; 0–44/401 bp) than inter- Twenty-one haplotypes were obtained from specific sequence differences (10.2–22.4%; 41– the partial COI genes of 25 specimens from eight 90/401 bp). Figure 5 shows a BI consensus tree Diplosoma species. The average base frequencies using the GTR+I+G substitution model. The of all three codon positions from 21 haplotypes monophyly of each Diplosoma species was of Diplosoma specimens were 31.2% A, 10.5% supported by BPP and high bootstrap values, C, 16.7% G, and 41.7% T, and the A+T composition except D. simile in which monophyly was not frequency was 72.9%. The aligned COI sequences strongly supported (BPP = 0.63; 55% in ML).

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The monophyly of D. simile specimens from the basal position in the clade, another region Pacific Ocean (Okinawa Island, the Bonin (Folmer's) of the COI gene sequences from Islands, and Hawai'i) was supported by BPP and Caribbean specimens were the same as the high bootstrap values. sequences from Okinawa Island (Hirose et al. 2012). Here, the all specimens morphologically Discussion identified as D. simile were concluded to be conspecific. The photosymbiotic ascidians from oceanic The present results indicate that T. cyclops islands (i.e., the Bonin Islands and Hawai'i) were and D. simile probably have long-range dispersal verified as T. cyclops or D. simile, based on both capabilities, and we assume that the colonies morphological features and molecular phylogeny from oceanic islands were originally derived inferred from the partial sequences of mitochondrial from the tropical western Pacific region, where COI genes. According to Kott (1980), T. para- the species richness is much higher than that of cyclops Kott, 1980 is distinguished from T. cyclops the oceanic islands. Since didemnid ascidians are by differences in the colony, the spicules, the always viviparous, active dispersal is limited in proportion of zooids, the retractor muscles, the the larval period. However, didemnid larvae are course of the gut, and the number of vas deferens relatively large among ascidian species, and coils. However, Monniot and Monniot (1987) often settle and metamorphose into juvenile assigned both species to T. cyclops, considering colonies soon after their release from the mother these differences to represent intraspecific colony. Therefore, larval dispersal would be variation. Quantitative investigation showed that limited in didemnids and rafting on drifting the spicule sizes differed significantly between material in the sessile stage would be more the two species, but the monophyly of each species important for long-distance dispersal; as Jackson was not supported by molecular phylogenies (1986) stated that "Rafting is the only reasonable (Hirose et al. 2010). The sizes of colonies and explanation for the existence of the vast majority the diameters of the spicules in the present of clonal species on Indo-Pacific oceanic specimens from Hahajima were within the range islands." Worcester (1994) demonstrated that of those in T. cyclops. In the present phylogenetic rafting is a successful and important mode of trees based on partial COI gene sequences, dispersal in a botryllid ascidian, Botrylloides sp. T. cyclops and T. paracyclops formed a mono- It is possible that artificial transport via vessels phyletic clade, and these two species were mixed (on ship hulls and in ballast water), drifting up within the clade (Figure 4). This supports the buoys, and other artifacts are plausible vectors view of Monniot and Moniot (1987) regarding for the long-dispersal of didemnid species. T. paracyclops as a junior synonym of T. cyclops. In many photosymbiotic ascidians, algal In either case, the specimen from Hahajima was symbionts are transmitted from generation to identified as T. cyclops. generation (vertical transmission), whereby embryos The species of photosymbiotic Diplosoma can are known to acquire photosymbionts from their be discriminated through the liberating point of mother colony (Kojima and Hirose 2012 and the retractor muscle from the zooid and the references therein). In D. simile, pre-hatching number of stigmata in each row (Hirose and Su embryos collect Prochloron cells from the 2011), and the present specimens were consistent cloacal cavity of the mother colony using a without exception with the character states of specialized organ called the rastrum, and the D. simile (Figure 3E–H). Furthermore, partial COI rastrum, with numerous photosymbionts, is gene sequences distinguish many photosymbiotic folded in the posterior part of the larval trunk Diplosoma (Hirose and Hirose 2009). In the (Hirose 2000). Therefore, they enjoy the benefits phylogenetic tree, photosymbiotic Diplosoma of the symbionts from the beginning of their life, form two clades corresponding to the liberating and this may be an advantage for survival on the point of the retractor muscle (i.e., underside of drifting materials. It is uncertain why some the thorax and halfway down the esophagus), and species, such as T. cyclops and D. simile, have all the D. simile specimens examined here wide distribution ranges including oceanic formed a monophyletic clade supported by islands and other photosymbiotic species do not. moderate Bayesian posterior provability (BI) and A possible explanation is that T. cyclops and bootstrap value (ML) (Figure 5). While D. simile D. simile have broader environmental tolerances from Caribbean Panama branched at the most than other photosymbiotic species.

278 Photosymbiotic didemnid ascidians from oceanic islands

Will photosymbiotic ascidians expand their References geographic ranges toward higher latitudes following increases in seawater temperature? The Abbott DP, Newberry AT, Morris KM (1997) Reef and Shore Fauna of Hawaii, Section 6B: Ascidians (Urochordata). distribution ranges of photosymbiotic ascidians are Bishop Museum Press, Honolulu, HI, USA, 64 pp supposed to be limited by temperature minima Dionisio-Sese M, Maruyama T, Miyachi S (2001) Photosynthesis because their cyanobacterial photosymbionts are of Prochloron as affected by environmental factors. Marine susceptible to temperatures below 20ºC (Dionisio- Biotechnology 3: 74–79, http://dx.doi.org/10.1007/s10126000 0062 Sese et al. 2001), which is consistent with the Donia MS, Fricke WF, Partensky F, Cox J, Elshahawi SI, White distribution patterns of photosymbiotic ascidians JR, Phillippy AM, Schatz MC, Piel J, Haygood MG, Ravel J, in the Ryukyu Archipelago–Taiwan region. The Schmidt EW (2011) Complex microbiome underlying number of species gradually decreases toward secondary and primary metabolism in the tunicate— Prochloron symbiosis. Proceedings of the National Academy higher latitudes (Hirose 2013 and references of Sciences of the United States of America 108: E1423– therein), and 19 species have been so far E1432, http://dx.doi.org/10.1073/pnas.1111712108 recorded from the Yaeyama Islands (24–24º30'N; Edgar RC (2004) MUSCLE: multiple sequence alignment with the southernmost island group in the Ryukyus), high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797, http://dx.doi.org/10.1093/nar/gkh340 while only four species, including D. simile, Hirose E (2000) Plant rake and algal pouch of the larvae in the have been found in the Osumi Islands (ca. 30ºN; tropical ascidian Diplosoma similis: an adaptation for vertical the northernmost island group). In Taiwan, transmission of photosynthetic symbionts Prochloron. Zoological Science 17: 233–240, http://dx.doi.org/10.2108/zsj. photosymbiotic ascidians have been recorded 17.233 from Kenting (ca. 22ºN; South Taiwan) and Hirose E (2013) Didemnid ascidians harboring cyanobacteria Lyudao (22º40'N; island in southeast Taiwan) from Kumejima Island and Tonakijima Island, Ryukyu but not from Keelung (ca. 25ºN; the northern Archipelago, Japan. The Biological Magazine Okinawa 51: 41–49 coast of Taiwan; Hirose and Nozawa 2010; Hirose E, Hirose M (2011) A new didemnid ascidian Lissoclinum Hirose and Su 2011). The minimum winter water midui sp. nov. from Kumejima Island (Okinawa, Japan), with temperature at the sea surface is about 16ºC on remarks on the absence of a common cloacal system and the the northern and northwestern coasts of Taiwan presence of an unknown organ. 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The Biological Magazine Okinawa 45: 3–9 Their broad tolerance and wide dispersal abilities Hirose E, Neilan B, Schmidt E, Murakami A (2009a) Enigmatic suggest that they have the necessary attributes to life and evolution of Prochloron and related cyanobacteria expand their distribution range. It would be inhabiting colonial ascidians. In: Gault P, Marler H (eds), Handbook on Cyanobacteria. Nova Science Publishers, Inc., possible that they will become invasive in non- New York, NY, USA, pp 161–189 native regions, as reported for D. simile in Hirose E, Oka AT, Hirose M (2009b) Two new species of American Sãmoa (Vargas-Ángel et al. 2009). photosymbiotic ascidians in the genus Diplosoma from the Ryukyu Archipelago, with partial sequences of the COI gene. Zoological Science 26: 362–368, http://dx.doi.org/10.2108/zsj. Acknowledgements 26.362 Hirose E, Turon X, López-Legentil S, Erwin PM, Hirose M (2012) First records of didemnid ascidians harboring We thank Professor Robert A. Kinzie III (Hawai'i Institute of Prochloron from Caribbean Panama: genetic relationships Marine Biology) for his generous help for sampling and between Caribbean and Pacific photosymbionts and host hospitality in Hawai'i. Also thanks to the editor and the ascidians. Systematics and Biodiversity 10: 435–445, anonymous reviewers for their critical comments and productive http://dx.doi.org/10.1080/14772000.2012.735716 suggestions. The present study was partly supported by Grant-in- Hirose M, Hirose E (2009) DNA-barcoding in photosymbiotic Aid for Scientific Research (C) no. 23510296 from the Japan species of the genus Diplosoma (Ascidiacea: Didemnidae) Society for the Promotion of Science and by the International and a description of a new species from the southern Research Hub Project for Climate Change and Coral Reef/Island Ryukyus, Japan. 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