photosymbiotic ascidians from oceanic islands in the

Aquatic Invasions (2013) Volume 8, Issue 3: 271–280 doi: http://dx.doi.org/10.3391/ai.2013.8.3.03 © 2013 The Author(s). Journal compilation © 2013 REABIC

Open Access

271

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

Introduction

Non-indigenous ascidians cause serious problems worldwide (Lambert 2007). Outbreaks of introduced ascidians often change native biota on large scales and cause huge losses in aquaculture. Following increases in seawater temperatures due to global warming, tropical species could expand their distribution ranges toward higher latitudes, and some species potentially become invasive in non-native regions. Among these, photosymbiotic ascidians are high-risk candidates because their cyanobacterial symbionts are known to produce various toxins (e.g., Donia et al. 2011), and the host ascidians are probably subjected to little or no predation. Additionally, species with large distribution ranges are supposed to have long-range dispersal capabilities and broad environ-mental tolerances, and thus would be powerful candidates for becoming invasive. Therefore, it

is reasonable to further examine photosymbiotic ascidians with large distribution ranges.

In tropical and subtropical waters, about 30 ascidian species are known to harbor cyanobacterial symbionts such as Prochloron and Synechocystis (Lewin and Cheng 1989; Hirose et al. 2009a). These are all colonial tunicates belonging to the family Didemnidae, which is the largest family in the class Ascidiacea (Chordata: Tunicata) and probably includes many undescribed species, particularly in tropical waters (Kott 2005). Microscopic exami-nation is often necessary for species identification of didemnid species, because the zooids are very small (sometimes measuring less than 1 mm in length) and simplified, resulting few morphological characters for species identification. Moreover, the colonies often inhabit cryptic sites, such as the undersides of, or crevasses in, coral limestones or dead coral fragments. Accordingly, few distribution records exist for many species (Kott 2001; Monniot and Monniot 2001).

M. Hirose and E. Hirose

272

The distribution ranges of photosymbiotic ascidians differ from species to species. For example, Didemnum molle (Herdmann, 1886) is a conspicuous photosymbiotic species because of its dome-shaped colony and its occurrence in exposed sites. This species is widely distributed from the western Indian Ocean to Fiji, and from about 30ºN to more than 30ºS (Kott 2001), but has never been recorded from some oceanic islands situated far from continents and other 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 contrast, Trididemnum cyclops Michaelsen, 1921 and Diplosoma simile (Sluiter, 1909) have considerably broader distributions than D. molle in the Pacific: the former species has been recorded from French Polynesia and the Bonin Islands, and the latter has been recorded from French Polynesia, the Bonin Islands, and Hawai'i (Monniot and Monniot 1987; Abbot et al. 1997; Hirose et al. 2007). Recently, colonies collected from Caribbean Panama were identified as D. simile based on their partial mitochondrial DNA sequences as well as morphological features (Hirose et al. 2012). These D. simile colonies may have been introduced from the Pacific. Vargas-Ángel et al. (2009) reported a recent outbreak of D. simile in American Sãmoa, where overgrowth by ascidian colonies threatens native scleractinian corals. As mentioned above, species identification of photosymbiotic didemnids is often problematic due to the shortage of morphological characters for species discrimination. For instance, we have described six Diplosoma species as new species from the Ryukyu Archipelago (Japan) since 2005, using the number of stigmata of branchial sac as a novel character for taxonomy, and the recognition of the species was also supported by molecular phylogeny analyses (see Hirose and Hirose 2009). Considering the taxonomic difficulties, it is important to verify whether the specimens from oceanic islands are actually conspecific to those from continental islands, integrating morphological and molecular methods.

In general, the species richness in tropical Indo-Pacific Oceans is highest in the Indonesian–Philippine region and steadily decreases eastward across the Pacific, consistent with dispersal from a center of high diversity (e.g., Roberts et al. 2002; Mora et al. 2003). This suggests that T. cyclops and D. simile may have greater dispersal capabilities than other photosymbiotic didemnid species, making them potential

candidates expanding their distribution ranges toward higher latitudes following the global warming of seawater. In the present study, we examined zooid morphologies and the partial sequences of the mitochondrial cytochrome oxidase subunit I gene of D. simile from oceanic islands in the Pacific (Bonin Islands and Hawai'i) and compared them with those from Okinawa Island (Ryukyu Archipelago, Japan), a continental island, and from Caribbean Panama.

Materials and methods

Animals

The sampling of ascidian colonies was carried out by snorkeling in shallow subtidal areas (<3 m) in the Bonin Islands, Hawai’i, Taiwan, and Ryukyu Archipelago, from 2007 to 2012 (Figure 1). Colonies of T. cyclops were collected from Ishijiro Beach (26º38'00"N, 142º09'40"E) in Hahajima (Bonin Islands) on 14 May 2012. Trididemnum miniatum colonies were collected from Bise (26º42'35"N, 127º52'47"E) off Okinawajima Island (Ryukyu Archipelago) on 15 June 2007, and T. paracyclops colonies were collected from Agari-hama (26º22'07"N, 127º09'00"E) in Tonakijima Island (Ryukyu Archipelago) on 22 March 2012. Diplosoma simile colonies were collected from Ishijiro Beach (26º38'00"N, 142º9'40"E), Minami-zaki (26º36'32"N, 142º10'35"E), and North Port (26º41'55"N, 142º08'34"E) off Hahajima (Bonin Islands) on 14 and 15 May 2012, Miyanohama (27º06'14"N, 142º11'40"E) and Kanameishi (27º04'58"N, 142º12'26"E) off Chichijima (Bonin Islands) on 17 May 2012, and in the vicinity of the Hawai'i Institute of Marine Biology (21º26'00" N, 157º47'12"E) off Coconut Island (Kaneohe, Hawai'i) on 13 March 2012. To compare with these specimens from oceanic islands, we used colonies collected from Crawl Key (9º15'47.78"N, 82º07'51.64"W) and Isla Cristobal (9º16'56.4"N, 82º15'25.7"W) off Bocas del Toro (Panama) on 22 and 27 June 2011 (Hirose et al. 2012) and from Teniya (26º33'50"N, 128º8'30"E) off Okinawa Island (Ryukyu Archipelago) on 13 September 2007. Diplosoma ooru colonies were collected from Nanwan (21º57'31"N, 120º45'57"E) in Kenting (Taiwan) on 23 March 2011, D. virens colonies were collected from Yobuko-hama (26º21'31"N, 127º08'31"E) in Tonakijima Island (Ryukyu Archipelago) on 21 March 2012, and Uganzaki (24º27'56"N, 127º07'22"E) in Ishigakijima Islands (Ryukyu Archipelago) on 13 September 2009,

Photosymbiotic didemnid ascidians from oceanic islands

273

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 Yobuko-hama in Tonakijima Island (Ryukyu Archipelago) on 21 March 2012 (Figure 1). Some colonies were anesthetized with menthol and 0.37 M MgCl2 for approximately 2 h and then fixed with 10% formalin–seawater for microscopic observation. Some pieces from colonies were also preserved in 99% ethanol for DNA extraction.

Microscopy

Formalin-fixed specimens were dissected under a binocular stereomicroscope for species identifi-cation. Zooids isolated from the fixed materials were observed under a light microscope equipped with differential interference contrast (DIC) optics. Several micrographs 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). Calcareous spicules in the tunics of T. cyclops colonies were photo-graphed under a light microscope and the diameters of 100 spicules were measured using ImageJ 1.44 (http://imagej/nih.gov/ij).

DNA extraction, amplification, and sequencing

Tissue samples were preserved in ethanol at –30°C. Genomic DNA was extracted from about 30–40 zooids using a DNeasy Tissue Kit (Qiagen K.K., Tokyo, Japan) following the manufacturer’s protocol. Part of the mitochondrial COI gene was amplified using UroCox1-244F (5′-CATTTWT TTTGATTWTTTRGWCATCCNGA-3′) and Uro Cox1-387R (5′-GCWCYTATWSWWAAWACA TAATGAAARTG-3′), and the amplification and sequencing of the partial COI fragments followed

Hirose et al. (2009c). The PCR primer sequences were originally constructed by Yokobori (Hirose et al. 2009c) based on the sequence comparisons of tunicate COI genes, since amplification of the so-called 'Folmer’s region' of the COI gene (at the 5' end of the cytochrome c oxidase subunit 1 region) was often unstable in our specimens. Although the primers amplify a different region from the 'Folmer’s region' of the COI gene, more sequence data were available in the public domain for the 'Yokobori's region' than for the 'Folmer’s region' of photosymbiotic didemnid species. At least five clones were randomly selected and sequenced for each sample. The sequences determined in this study have been deposited in the DNA databases of GenBank, European Molecular Biology Laboratory (EMBL), and DNA Data Bank of Japan (DDBJ) under the accession numbers shown in Table 1.

Phylogenetic analyses

There were 16 partial COI gene sequences (three Trididemnum sequences and 11 Diplosoma sequences) obtained in this study. Partial COI gene sequences data of six Trididemnum species (eight sequences) and eight Diplosoma species (12 sequences) were retrieved from GenBank (Table 1). These were all available COI gene sequence of the 'Yokobori's region' in Trididemnum and Diplosoma. A total 36 COI sequences (11 Trididemnum sequences and 25 Diplosoma sequen-ces) were used for phylogenetic analyses, in which Didemnum molle (AB433947), Lissoclinum bistratum (AB433977) and L. punctatum (AB 433976) were used as the outgroup (Table 1).


274

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

MUSCLE (Edgar 2004), and these were then inspected by eye and manually edited. Base frequencies, pairwise base differences, and genetic distances were calculated using PAUP* 4.0 Beta 10 (Swofford 2003). The following analyses were performed on the aligned DNA sequences of the partial COI gene: maximum likelihood (ML) using the October 2008 version of TREEFINDER

(Jobb 2008); and phylogenetic analysis based on Bayesian inference (BI) using MrBayes 3.12 (Ronquist and Huelsenbeck 2003). To select an appropriate nucleotide substitution model, Modeltest ver. 3.7 (Posada and Crandall 1988) was used with PAUP. For data with all three codon positions, the general time reversible model with invariant sites and gamma distribution (GTR+I+G) was selected as the best model using


275

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.


276

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 to Prochloron cells distributed in the peribranchial and cloacal cavities (Figure 3A–D). Colony colors varied among colonies even within the same collection site: some colonies inhabiting 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 somewhat among zooids depending on the extent of contraction in the fixatives (Figure 3E–H). The thorax had four rows of stigmata. Six stigmata were in the first (top) to third rows and five stigmata were in the fourth row (bottom). The retractor muscle emerged from the underside of the thorax. The thickness of the retractor muscle varied among specimens. There were no spicules in the present species as well as the other Diplosoma species.

Phylogenetic analysis

Eight haplotypes were obtained from the partial COI gene sequences of 11 specimens from six Trididemnum species. The average base frequencies

Figure 4. Bayesian inference (BI) consensus tree of Trididem-num 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.

of all three codon positions from the eight haplotypes of Trididemnum specimens were 28.9% A, 9.7% C, 16.9% G, and 44.5% T, and the A+T composition frequency was 73.4%. The aligned COI sequences were 401 bp long, without gaps (insertions/deletions). Figure 4 shows a BI consensus tree using the GTR+I+G substitution model. The strict consensus ML under the GTR+I+G substitution model is not shown. The sequence of T. nubilum from Okinawa Island was identical to that of the specimen from Kume Island. The sequences of T. paracyclops from Ishigaki and Kume islands were the same as the sequence of T. cyclops from Hahajima. The monophylies of T. cyclops and T. paracyclops individually were not supported, although the monophyly of T. cyclops + T. paracyclops was


277

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) and high bootstrap values. Monophyly of Tridemnum was not supported.

Twenty-one haplotypes were obtained from the partial COI genes of 25 specimens from eight Diplosoma species. The average base frequencies of all three codon positions from 21 haplotypes of Diplosoma specimens were 31.2% A, 10.5% C, 16.7% G, and 41.7% T, and the A+T composition frequency was 72.9%. The aligned COI sequences

were 401 bp long, without gaps (insertions/ deletions). Sequence differences within each species were lower (0–11.0%; 0–44/401 bp) than inter-specific sequence differences (10.2–22.4%; 41–90/401 bp). Figure 5 shows a BI consensus tree using the GTR+I+G substitution model. The monophyly of each Diplosoma species was supported by BPP and high bootstrap values, except D. simile in which monophyly was not strongly supported (BPP = 0.63; 55% in ML).


278

The monophyly of D. simile specimens from the Pacific Ocean (Okinawa Island, the Bonin Islands, and Hawai'i) was supported by BPP and high bootstrap values.

Discussion

The photosymbiotic ascidians from oceanic islands (i.e., the Bonin Islands and Hawai'i) were verified as T. cyclops or D. simile, based on both morphological features and molecular phylogeny inferred from the partial sequences of mitochondrial COI genes. According to Kott (1980), T. para-cyclops Kott, 1980 is distinguished from T. cyclops by differences in the colony, the spicules, the proportion of zooids, the retractor muscles, the course of the gut, and the number of vas deferens coils. However, Monniot and Monniot (1987) assigned both species to T. cyclops, considering these differences to represent intraspecific variation. Quantitative investigation showed that the spicule sizes differed significantly between the two species, but the monophyly of each species was not supported by molecular phylogenies (Hirose et al. 2010). The sizes of colonies and the diameters of the spicules in the present specimens from Hahajima were within the range of those in T. cyclops. In the present phylogenetic trees based on partial COI gene sequences, T. cyclops and T. paracyclops formed a mono-phyletic clade, and these two species were mixed up within the clade (Figure 4). This supports the view of Monniot and Moniot (1987) regarding T. paracyclops as a junior synonym of T. cyclops. In either case, the specimen from Hahajima was identified as T. cyclops.

The species of photosymbiotic Diplosoma can be discriminated through the liberating point of the retractor muscle from the zooid and the number of stigmata in each row (Hirose and Su 2011), and the present specimens were consistent without exception with the character states of D. simile (Figure 3E–H). Furthermore, partial COI gene sequences distinguish many photosymbiotic Diplosoma (Hirose and Hirose 2009). In the phylogenetic tree, photosymbiotic Diplosoma form two clades corresponding to the liberating point of the retractor muscle (i.e., underside of the thorax and halfway down the esophagus), and all the D. simile specimens examined here formed a monophyletic clade supported by moderate Bayesian posterior provability (BI) and bootstrap value (ML) (Figure 5). While D. simile from Caribbean Panama branched at the most

basal position in the clade, another region (Folmer's) of the COI gene sequences from Caribbean specimens were the same as the sequences from Okinawa Island (Hirose et al. 2012). Here, the all specimens morphologically identified as D. simile were concluded to be conspecific.

The present results indicate that T. cyclops and D. simile probably have long-range dispersal capabilities, and we assume that the colonies from oceanic islands were originally derived from the tropical western Pacific region, where the species richness is much higher than that of the oceanic islands. Since didemnid ascidians are always viviparous, active dispersal is limited in the larval period. However, didemnid larvae are relatively large among ascidian species, and often settle and metamorphose into juvenile colonies soon after their release from the mother colony. Therefore, larval dispersal would be limited in didemnids and rafting on drifting material in the sessile stage would be more important for long-distance dispersal; as Jackson (1986) stated that "Rafting is the only reasonable explanation for the existence of the vast majority of clonal species on Indo-Pacific oceanic islands." Worcester (1994) demonstrated that rafting is a successful and important mode of dispersal in a botryllid ascidian, Botrylloides sp. It is possible that artificial transport via vessels (on ship hulls and in ballast water), drifting buoys, and other artifacts are plausible vectors for the long-dispersal of didemnid species.

In many photosymbiotic ascidians, algal symbionts are transmitted from generation to generation (vertical transmission), whereby embryos are known to acquire photosymbionts from their mother colony (Kojima and Hirose 2012 and references therein). In D. simile, pre-hatching embryos collect Prochloron cells from the cloacal cavity of the mother colony using a specialized organ called the rastrum, and the rastrum, with numerous photosymbionts, is folded in the posterior part of the larval trunk (Hirose 2000). Therefore, they enjoy the benefits of the symbionts from the beginning of their life, and this may be an advantage for survival on the drifting materials. It is uncertain why some species, such as T. cyclops and D. simile, have wide distribution ranges including oceanic islands and other photosymbiotic species do not. A possible explanation is that T. cyclops and D. simile have broader environmental tolerances than other photosymbiotic species.


279

Will photosymbiotic ascidians expand their geographic ranges toward higher latitudes following increases in seawater temperature? The distribution ranges of photosymbiotic ascidians are supposed to be limited by temperature minima because their cyanobacterial photosymbionts are susceptible to temperatures below 20ºC (Dionisio-Sese et al. 2001), which is consistent with the distribution patterns of photosymbiotic ascidians in the Ryukyu Archipelago–Taiwan region. The number of species gradually decreases toward higher latitudes (Hirose 2013 and references therein), and 19 species have been so far recorded from the Yaeyama Islands (24–24º30'N; the southernmost island group in the Ryukyus), while only four species, including D. simile, have been found in the Osumi Islands (ca. 30ºN; the northernmost island group). In Taiwan, photosymbiotic ascidians have been recorded from Kenting (ca. 22ºN; South Taiwan) and Lyudao (22º40'N; island in southeast Taiwan) but not from Keelung (ca. 25ºN; the northern coast of Taiwan; Hirose and Nozawa 2010; Hirose and Su 2011). The minimum winter water temperature at the sea surface is about 16ºC on the northern and northwestern coasts of Taiwan due to cold China coastal water, but is about 19ºC at Okinawa Island (26–27ºN). Note that D. simile has been widely recorded from the Ryukyus and Taiwan, as well as Indonesia, Singapore, the Great Barrier Reef, and various islands from the west and central Pacific. The distribution of T. cyclops ranges from the west Indian Ocean to the west and central Pacific and from the Ryukyus to the Great Barrier Reef. Their broad tolerance and wide dispersal abilities suggest that they have the necessary attributes to expand their distribution range. It would be possible that they will become invasive in non-native regions, as reported for D. simile in American Sãmoa (Vargas-Ángel et al. 2009).

Acknowledgements

We thank Professor Robert A. Kinzie III (Hawai'i Institute of Marine Biology) for his generous help for sampling and hospitality in Hawai'i. Also thanks to the editor and the anonymous reviewers for their critical comments and productive suggestions. The present study was partly supported by Grant-in-Aid for Scientific Research (C) no. 23510296 from the Japan Society for the Promotion of Science and by the International Research Hub Project for Climate Change and Coral Reef/Island Dynamics from University of the Ryukyus.

References

Abbott DP, Newberry AT, Morris KM (1997) Reef and Shore Fauna of Hawaii, Section 6B: Ascidians (Urochordata). Bishop Museum Press, Honolulu, HI, USA, 64 pp

Dionisio-Sese M, Maruyama T, Miyachi S (2001) Photosynthesis of Prochloron as affected by environmental factors. Marine Biotechnology 3: 74–79, http://dx.doi.org/10.1007/s10126000 0062

Donia MS, Fricke WF, Partensky F, Cox J, Elshahawi SI, White JR, Phillippy AM, Schatz MC, Piel J, Haygood MG, Ravel J, Schmidt EW (2011) Complex microbiome underlying secondary and primary metabolism in the tunicate—Prochloron symbiosis. Proceedings of the National Academy of Sciences of the United States of America 108: E1423–E1432, http://dx.doi.org/10.1073/pnas.1111712108

Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797, http://dx.doi.org/10.1093/nar/gkh340

Hirose E (2000) Plant rake and algal pouch of the larvae in the tropical ascidian Diplosoma similis: an adaptation for vertical transmission of photosynthetic symbionts Prochloron. Zoological Science 17: 233–240, http://dx.doi.org/10.2108/zsj. 17.233

Hirose E (2013) Didemnid ascidians harboring cyanobacteria from Kumejima Island and Tonakijima Island, Ryukyu Archipelago, Japan. The Biological Magazine Okinawa 51: 41–49

Hirose E, Hirose M (2011) A new didemnid ascidian Lissoclinum midui sp. nov. from Kumejima Island (Okinawa, Japan), with remarks on the absence of a common cloacal system and the presence of an unknown organ. Zoological Science 28: 462–468, http://dx.doi.org/10.2108/zsj.28.462

Hirose E, Nozawa Y (2010) Photosymbiotic ascidians from Kenting and Lyudao in Taiwan. Zoological Studies 49: 681–687

Hirose E, Su S-W (2011) A new record of a photosymbiotic ascidian from Kenting, Taiwan with a key to the photo-symbiotic species of the genus Diplosoma recorded in the Western Pacific. Collection and Research (Taichung) 24: 83–86

Hirose E, Kamijoh A, Oka AT (2007) Distribution of the photosymbiotic ascidians in Chichijima Island (Ogasawara Islands, Tokyo). The Biological Magazine Okinawa 45: 3–9

Hirose E, Neilan B, Schmidt E, Murakami A (2009a) Enigmatic life and evolution of Prochloron and related cyanobacteria inhabiting colonial ascidians. In: Gault P, Marler H (eds), Handbook on Cyanobacteria. Nova Science Publishers, Inc., New York, NY, USA, pp 161–189

Hirose E, Oka AT, Hirose M (2009b) Two new species of 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. 26.362

Hirose E, Turon X, López-Legentil S, Erwin PM, Hirose M (2012) First records of didemnid ascidians harboring Prochloron from Caribbean Panama: genetic relationships between Caribbean and Pacific photosymbionts and host ascidians. Systematics and Biodiversity 10: 435–445, http://dx.doi.org/10.1080/14772000.2012.735716

Hirose M, Hirose E (2009) DNA-barcoding in photosymbiotic species of the genus Diplosoma (Ascidiacea: Didemnidae) and a description of a new species from the southern Ryukyus, Japan. Zoological Science 26: 564–568, http://dx.doi.org/10.2108/zsj.26.564

Hirose M, Yokobori S, Hirose E (2009c) Potential speciation of morphotypes in the photosymbiotic ascidian Didemnum molle in the Ryukyu Archipelago, Japan. Coral Reefs 28: 119–126, http://dx.doi.org/10.1007/s00338-008-0425-0


280

Hirose M, Tochikubo T, Hirose E (2010) Taxonomic significance of tunic spicules in photosymbiotic ascidians: a quantitative and molecular evaluation. Journal of the Marine Biological Association of the United Kingdom 90: 1065–1071, http://dx.doi.org/10.1017/s0025315409991263

Jackson JBC (1986) Modes of dispersal of clonal benthic invertebrates: Consequences for species' distributions and genetic structure of local populations. Bulletin of Marine Science 39: 588–606

Jobb G (2008) TREEFINDER ver. Jan 2008. Munich, Germany. http://www.treefinder.de

Kojima A, Hirose E (2012) Transmission of cyanobacterial symbionts during embryogenesis in the coral reef ascidians Trididemnum nubilum and T. clinides (Didemnidae, Ascidiacea, Chordata). Biological Bulletin 222: 63–73

Kott P (1980) Algal-bearing didemnid ascidians in the Indo-West-Pacific. Memoirs of the Queensland Museum 20: 1–47

Kott P (2001) The Australian Ascidiacea, part 4, Aplousobranchia (3), Didemnidae. Memoirs of the Queensland Museum 47: 1–408

Kott P (2005) New and little-known species of Didemnidae (Ascidiacea, Tunicata) from Australia (Part 3). Journal of Natural History 39: 2409–2497, http://dx.doi.org/10.1080/00 222930500087077

Lambert G (2007) Invasive sea squirts: a growing global problem. Journal of Experimental Marine Biology and Ecology 342: 3–4, http://dx.doi.org/10.1016/j.jembe.2006.10.020

Lewin RA, Cheng L (eds) (1989) Prochloron, A Microbial Enigma. Chapman & Hall, Inc., New York, NY, USA, 129 pp, http://dx.doi.org/10.1007/978-1-4613-0853-9

Monniot C, Monniot F (1987) Les ascidies de Polynésie Française. Memoires du Museum National d'histoire Naturelle A 136: 1–155

Monniot F, Monniot C (2001) Ascidians from the tropical western Pacific. Zoosystema 23: 201–383

Mora C, Chittaro PM, Sale PF, Kritzer JP, Ludsin SA (2003) Patterns and processes in reef fish diversity. Nature 421: 933–936, http://dx.doi.org/10.1038/nature01393

Posada D, Crandall KA (1988) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 814–818, http://dx.doi.org/10.1093/bioinformatics/14.9.817

Roberts CM, McClean CJ, Veron JEN, Hawkins JP, Allen GR, McAllister DE, Mittermeier CG, Schueler FW, Spalding M, Wells F, Vynne C, Werner TB (2002) Marine biodiversity hotspots and conservation priorities for tropical reefs. Science 295: 1280–1284, http://dx.doi.org/10.1126/science.1067728

Ronquist F, Huelsenbeck J (2003) Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574, http://dx.doi.org/10.1093/bioinformatics/btg180

Swofford D (2003) PAUP* 4.0b7a, Phylogenetic Analysis Using Parsimony (*and Other Methods). Sunderland, MA, Sinauer Associates

Vargas-Ángel B, Asher J, Godwin LS, Brainard RE (2009) Invasive didemnid tunicate spreading across coral reefs at remote Swains Island, American Sãmoa. Coral Reefs 28: 53, http://dx.doi.org/10.1007/s00338-008-0428-x

Worcester SE (1994) Adult rafting versus larval swimming: dispersal and recruitment of a botryllid ascidian on eelgrass. Marine Biology 121: 309–317, http://dx.doi.org/10.1007/BF 00346739

photosymbiotic ascidians from oceanic islands in the

Documents