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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
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 environmental 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 examination 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).
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M. Hirose and E. Hirose
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. VargasÁngel 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
272
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 Agarihama (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), Minamizaki (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
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 identification. 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 photographed 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 sequences) 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).
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M. Hirose and E. Hirose
Table 1. List of Didemnidae species used for phylogenetic tree construction in this study.
Species
a
Collection site, Region a
Abbreviation
Acc. No.
Reference
Diplosoma
D. aggregatum
D. gumavirens
D. gumavirens
D. gumavirens
D. ooru
D. ooru
D. simile
D. simile
D. simile
D. simile
D. simile
D. simile
D. simile
D. simile
D. simile
D. simile
D. simileguwa
D. variostigmatum
D. variostigmatum
D. virens
D. virens
D. virens
D. watanabei
D. watanabei
D. watanabei
Muiga, Miyakojima, Ok
Shinrihama, Kumejima, Ok
Muiga, Miyakojima, Ok
Hirakubo, Ishigakijima, Ok
Shiraho, Ishigakijima, Ok
Nanwan, Kentign, Tw
Teniya, Okinawa Is., Ok
Crawl Key, Panama, Pa
Isla Cristobal, Panama, Pa
Coconuts Is., Kaneohe Bay, Hw
Coconuts Is., Kaneohe Bay, Hw
Ishjirou Beach, Hahajima, Bn
Minamizaki, Hahajima Is., Bn
North port, Hahajima, Bn
Miyano-ura, Chichijima, Bn
Kaname-ishi, Chichijima, Bn
Teniya, Okinawa Is., Ok
Teniya, Okinawa Is., Ok
Hirakubo, Ishigakijima, Ok
Teniya, Okinawa Is., Ok
Yobuko-hama, Tonakijima, Ok
Uganzaki, Ishigakijima, Ok
Shimama, Tanegashima Is., Ka
Nagamahama, Kurimajima, Ok
Yobuko-hama, Tonakijima, Ok
D. aggregatum_Miyako
D. gumavirens_Kume
D. gumavirens_Miyako
D. gumavirens_Ishigaki
D. ooru_Ishigaki
D. ooru_Taiwan
D. simile_Okinawa
D. simile_PanamaCK
D. simile_PanamaIS
D. simile_Hawai01
D. simile_Hawai02
D. simile_HahaIB
D. simile_HahaMZ
D. simile_HahaNP
D. simile_ChichiMU
D. simile_ChichiKI
D. simileguwa_Okinawa
D. variostigmatum_Okinawa
D. variostigmatum_Ishigaki
D. virens_Okinawa
D. virens_Tonaki
D. virens_Ishigaki
D. watanabei_Tanegashima
D. watanabe_Kurima
D. watanabei_Tonaki
AB485997
AB473642
AB473643
AB485998
AB485999
AB813813
AB433975
AB813814
AB813815
AB813816
AB813817
AB813818
AB813819
AB813820
AB813821
AB813822
AB486000
AB486001
AB486002
AB433974
AB813823
AB813824
AB473641
AB479382
AB813825
Hirose and Hirose 2009
Hirose et al. 2009b
Hirose et al. 2009b
Hirose and Hirose 2009
Hirose and Hirose 2009
This study
Hirose et al. 2009c
This study
This study
This study
This study
This study
This study
This study
This study
This study
Hirose and Hirose 2009
Hirose and Hirose 2009
Hirose and Hirose 2009
Hirose et al. 2009c
This study
This study
Hirose et al. 2009b
Hirose et al. 2009b
This study
Trididemnum
T. clinides
T. cyclops
T. cyclops
T. cyclops
T. miniatum
T. nubilum
T. nubilum
T. paracyclops
T. paracyclops
T. paracyclops
T. savignii
Bise, Okinawa Is., Ok
Hirakubo, Ishigakijima, Ok
Maeda, Okinawa Is., Ok
Ishjirou Beach, Hahajima, Bn
Bise, Okinawa Is., Ok
Bise, Okinawa Is., Ok
Ara Beach, Kumejima, Ok
Ara Beach, Kumejima, Ok
Hirakubo, Ishigakijima, Ok
Agari-hama, Tonakijima, Ok
Nyu, Fu
T. clinides_Okinawa
T. cyclops_Ishigaki
T. cyclops_Okinawa
T. cyclops_HahaIB
T. miniatum_Okinawa
T. nubilum_Okinawa
T. nubilum_Kume
T. paracyclops_Kume
T. paracyclops_Ishigaki
T. paracyclops_Tonaki
T. saviginii
AB602782
AB433978
AB499972
AB813826
AB813827
AB499975
AB499976
AB499973
AB499974
AB813828
AB499977
Hirose and Hirose 2011
Hirose et al. 2009c
Hirose et al. 2010
This study
This study
Hirose et al. 2010
Hirose et al. 2010
Hirose et al. 2010
Hirose et al. 2010
This study
Hirose et al. 2010
Didemnum molle
Seragaki, Okinawa Is., Ok
D. molle
AB433947
Hirose et al. 2009c
Lissoclimun
L. bistratum
L. punctatum
Ara Beach, Kumejima, Ok
Ara Beach, Kumejima, Ok
L. bistratum
L. punctatum
AB433977
AB433976
Hirose et al. 2009 c
Hirose et al. 2009c
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
274
(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
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.
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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
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
276
Figure 4. Bayesian inference (BI) consensus tree of Trididemnum 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
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)
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 interspecific 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).
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M. Hirose and E. Hirose
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. paracyclops 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 monophyletic 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
278
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.
Photosymbiotic didemnid ascidians from oceanic islands
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 (DionisioSese 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 nonnative 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-inAid 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.
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