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RESEARCH ARTICLE
T. Kon
Æ
M. Nohara
Æ
M. Nishida
Æ
W. Sterrer
T. Nishikawa
Hidden ancient diversification in the circumtropical lancelet
Asymmetron lucayanum
complex
Received: 28 February 2005 / Accepted: 23 January 2006 / Published online: 25 February 2006
Springer-Verlag 2006
Abstract The tropical lancelet Asymmetron lucayanum
(= Epigonichthys lucayanus) is distributed from the
western Indian Ocean to the central Pacific Ocean, and
the western Atlantic Ocean. Molecular phylogenetic
analysis of mitochondrial cytochrome c ox idase sub-
unit I (COI) sequences (1,035 bp) of A. lucayanum (80
specimens from seven localities) showed clearly that
this species is genetically distinguished into three major
groups of geographical populations based on neighbor-
joining tree using maximum likelihood distance (HKY
model with invariable sites and gamma correction),
suggesting the existence of three cryptic species. Our
genetic data show that (1) inter-oceanic divergence
time between Clade B (the West-Centra l Pacific) and
Clade C (the Atlantic) (d = 6.6%, ca. 12 million years
ago) was smaller than intra-oceanic divergence time
between Clade A (the Indo-West Pacific) and Clade B
(d=39.5%, ca. 100 million years ago); (2) there are
two cryptic species in the West Pacific in sympatry;
and (3) high gene flow is implied between the Maldives
and the Ryukyus in Clade A (10,000 km distance), the
Philippines and Hawaii in Clade B (8,50 0 km distance),
and Barbados and Bermuda in Clade C (2,200 km
distance).
Introduction
The subphylum Cephalochordata (lancelets), the sister
group to the verteb rates (but also see Blair and Hedges
2005), has maintained its basic body organization for
several hundred million years (Holland and Chen 2001).
Lancelets are widely distributed in tropical and temper-
ate seas and consist of three genera (Branchiostoma,
Epigonichthys,andAsymmetron) with ca. 30 known living
species (Poss and Boschung 1996; Nishikawa and Nishida
1997; Nishikawa 2004). Adults of lancelets are benthic
inhabiting sandy and shell-sand bottoms, whereas larvae
are considered to be planktonic in both inshore and
offshore environments (Poss and Boschung 1996), having
a planktonic life stage of ca. 1.5–4 months (Wickstead
1975; Wu et al. 1994). Although most lancelet species
have rather restricted geographical distributions,
Asymmetron lucayanum (=Epigonichthys lucayanus)
is the only known species with wide circumtropical dis-
tribution, ranging from the western Indian Ocean to the
central Pacific Ocean, and the western Atlantic Ocean
(Nishikawa 1979; Tachikawa and Nishikawa 1979; Poss
and Boschung 1996; Nishikawa et al. 1997).
In the last decade, many studies of widespread species
with a pelagic larval phase have revealed the occurrence
of far more genetic structure than expected by their high
dispersal potential and present oceanographic condi-
tions (Palumbi et al. 1997; Benzie 1999). A number of
species traditionally seen as cosmopolitan are now rec-
ognized as assemblages of cryptic species with more
regionally restricted distribution (Palumbi 1992;
Knowlton 1993, 2000). Cryptic marine diversity has
been found acro ss a wide taxonomic range that includes
algae (Wright et al. 2000), sponges (Miller et al. 2001),
crustaceans (King and Hanner 1998; Kitaura et al. 2002;
Communicated by T. Ikeda, Hakodate
T. Kon (&) Æ M. Nishida
Department of Marine Bioscience, Ocean Research Institute,
The University of Tokyo, 164-8639, Tokyo, Japan
E-mail: kon@ori.u-tokyo.ac.jp
Tel.: +81-3-53516489
Fax: +81-3-53516488
M. Nohara
Yokohama R&D Center, HITEC Co., Ltd,
220–0005, Kanagawa, Japan
W. Sterrer
Bermuda Natural History Museum, PO Box FL 145,
Flatts FLBX, Bermuda
T. Nishikawa
The Nagoya University Museum,
Nagoya University, 464–8601, Aichi, Japan
Marine Biology (2006) 149: 875–883
DOI 10.1007/s00227-006-0271-y
Goetze 2003), cnidarians (Dawson and Jacobs 2001;
Holland et al. 2004), molluscs (Collin 2000; Kirkendale
and Meyer 2004), bryozoans (Hoare et al. 2001), asci-
dians (Tarjuelo et al. 2001), fishes (Miya and Nishida
1997; Colborn et al. 2001) and mammals (Wada et al.
2003). Lancelets, however, have remained little studied
in terms of evolutionary biology including phylogeny
and population genetics except for Nohara et al. (2004,
2005b).
The present study aims to resolve evolutionary rela-
tionships of the circumt ropical lancelet A. lucayanum,by
applying mitochondrial DNA (mtDNA) sequencing.
This species is somewhat unique morphologically among
the shallow-water lancelets by its possession of a long
urostyloid process and the peculiar morph ology of the
intertentacular membrane (Bigelow and Farfante 1948;
Nishikawa and Nishida 1997). Recent molecular phy-
logenetic studies indicate that the species is differentiated
remarkably from other extant lancelets (Nohara et al.
2004, 2005b).
Materials and methods
Sampling
A total of 84 individuals of A. lucayanum (Fig. 1) were
sampled from five Indo-West Pacific locations, Baa
Atoll in the Maldives (n=20), Negros Island in the
Philippines (n=5), Kuroshima Island in the Ryukyu
Islands (n=18), Lizard Island in the Great Barrier Reef
(GBR) (n=3), and Oahu Island in Hawaiian Islands
(n=13), and two Atlantic localities, Castle Harbour in
Bermuda (n=19) and Barbados (n=6) (Fig. 2). All
specimens were fixed and preserved in 99.5% ethanol
until use.
DNA preparation, amplification, and sequencing
The posterior pa rt of each specimen was digested for
12 h with proteinase K (10 mg/ml) in a lysis buffer
[10 mM Tris–HCl, pH 8.0; 2 mM EDTA; 1% SDS (w/
v)]. Total DNA of each lancelet was isolated from the
digested tissue solution using a standard phenol–chl o-
roform method and ethanol precipitation (Sambrook
and Russell 2001). The isolated DNA was resuspended
with TE buffer (10 mM Tris–HCl, pH 8.0; 2 mM
EDTA).
A fragment of the mtDNA that includes the cyto-
chrome c oxidase subunit I (COI) gene was amplified
using the AmphL109 (5¢–ATTCGNGCNGAAY
TNTCNCAGCC–3¢ ) and AmphH1325 (5¢–TCNGAA
TAYCGNCGWGGTATNCC–3¢) primers. The poly-
merase chain reaction (PCR) was carried out in a 15 ll
volume containing TaKaRa Ex Taq buffer (2 mM Tris–
HCl, pH 8.0; 2 mM MgCl
2
; 10 mM KCl
2
0.01 mM
EDTA; 0.1 mM DTT; 0.05% Tween 20; 0.05% Nonidet
P-40; 5% glycerol), 0.5 U TaKaRa Ex Taq polymerase,
2.5 mM each dNTP mixture, 0.5 lM each primer and
10–20 ng template DNA on a thermal cycler (GeneAmp
PCR System 9700, Applied Biosystems) for 30–35 cycles,
with the following profile: preheating at 96C for 2 min,
denaturing at 96C for 20–30 s; annealing at 45–60C
for 25–30 s and extension at 72C for 30 s.
Before sequencing the gene, the double-stranded
DNA obtained through PCR was purified with the
ExoSAP-IT (USB) composed of exonuclease I and
shrimp alkaline phosphatase. Direct sequencing of the
purified double-stranded DNA using the BigDye Ter-
minator Cycle Sequencing FS Ready Reaction Kit v.3.0
(Applied Biosystems) was performed on an ABI PRISM
3100 Genetic Analyzer (Applied Biosystems). All se-
quences were deposited in DDBJ/EMBL/GenBank
(accession numbers AB201315–AB201353 and
AB240554–AB240565).
Phylogenetic analysis
The DNA sequences were aligned using the multiple-
sequence alignment program CLUSTAL X, version 1.81
(Thompson et al. 1997). The sequences from the COI
gene were unambiguously aligned, allowing all sites to
be used in the analyses of that gene. The hierarchical
likelihood-ratio test approach (Huelsenbeck and Cra n-
dall 1997) was used to select the model of DNA evolu-
tion that best fitted the data, as implemented in the
program Modeltest 3.6 (Posada and Crandall 1998).
Modeltest was also used to estimate the parameters of
the model of evolution for input in PAUP*. The
appropriate model of nucleotide substitution was
HKY+I+C (Hasegawa et al. 1985). The base fre-
quencies were estimated to be A=0.2641, C=0.1848,
G=0.1820 and T=0.3691. Transition/transversion ratio
was 4.1122. Assumed proportion of invariable sires was
0.6053. Gamma distribution shape parameter was
35.6314.
In this study, we reconstructed phylogeny and esti-
mated divergence times between taxa of A. lucayanum
using the COI gene following Nohara et al. (2004).
Amplification of the ortholog ous mtDNA segment of
its congener, A. inferum , which should be the outgroup
for analysis, was unsuccessful, but our preliminary
analysis using the mitochondrial 16S rRNA gene re-
vealed that A. inferum is located far outside the cluster
of all the herein examined populations of the A. lu-
cayanum complex (T. Kon et al., unpublished data).
Fig. 1 One of the specimens of the Asymmetron lucayanum
complex from Chichijima Island, Ogasawara (Bonin) Islands,
Japan; 18.5 mm in body length. Photo by H. Tachikawa
876
Therefore, we used Branchiostoma belcheri (DDBJ/
EMBL/GenBank accession number: AB078191) to root
the trees. The large number of haplotypes required an
impractical length of time to search for the maximum
likelihood (ML) tree. Instead, the computationally
efficient, neighbor-joining (NJ) method (Saitou and Nei
1987) was used to construct a phylogenetic tree from
the ML distances estimated under the HKY+I+C,as
implemented in the program PAUP 4.0b (Swofford
2002). Support for internal branches within the NJ tree
was assessed using the bootstrap (Felsenstein 1985)
with 1,000 replicates.
Population structure analyses
A statistical parsimony network (SPN) in each clade of
NJ tree was constructed using the program TCS 1.18
(Clement et al. 2000), which employs the method of
Templeton et al. (1992). The SPN method builds a net-
work by connecting together sequences of one, and then
two, nucleotide differences, and so on, until all the se-
quences are included in a single network or the parsi-
mony connection limit is reached (Templeton et al.
1992). The parsimony connection limit is the maximum
number of nucleotide differences between the sequences
that could have been produced by single nucleotide
substitutions, with a statistical confidence of 95%. The
ancestral haplotype was also identified using the TCS by
the method of Castelloe and Templeton (1994), which
estimates ‘‘outgroup weights’’ based on haplotype fre-
quency and connectivity. The haplotype with the highest
outgroup weight is most likely the oldest. To test for
selective neutrality of COI sequences, Tajima’s (1989)
D-test was implemented for each clade. Population
genetics statistics were calculated using the program
Arlequin 2.000 (Schneider et al. 2000). Haplotype
diversity (h) and nucleotide diversity per site (p, based on
pairwise differences) values were estimated to quantify
genetic variation. Population structure was examined by
the analysis of molecular variance (AMOVA) (Excoffier
et al. 1992).
Divergence time estimation
We attempted to gain a rough estimation of divergence
time using COI variation based on Martin et al.’s (1992)
molecular clock for COI and cytochrome b (cytb) genes
in shark mtDNA (7.1·10
10
transversions/site · year)
following Nohara et al. (2004, 2005b), because a
molecular clock of lancelets was difficult to calibrate
using fossil data.
Results
Sequence divergence and phylogeny
DNA segments of 1,035 base pairs (bp) of the COI gene
were successfully sequenced for 80 of the 84 specimens.
A total of 265 nucleotide positions (25.6% in 1,035 bp)
varied in the ingroup, these variations defining 51
haplotypes (AL01–AL51), 44 of which were represented
in a single specimen. Base composition of the ingroup
(A: 25.1%, C: 20.7%, G: 18.7%, T: 35.5% on average)
was not remarkably biased and did not differ signi fi-
cantly from that of the outgroup (chi-square test,
P>0.1). The uncorrected average sequence divergence
(p-distance) between the ingroup and outgroup was
20.8% (range 19.9–21.9%), and the average ML dis-
tance based on HKY+I+C between them was 57.9%
(55.6–60.3%).
The NJ tree based on HKY+I+C model demon-
strated that the 51 haplotypes could be divided into three
major clades with high bootstrap values ranging from 98
to 100 % (Fig. 3a; Clade A, the Indo-West Pacific Ocean;
Clade B, the Central-West Pacific; Clade C, the Atlantic).
Clades A, B, and C consisted of 11 haplotypes (AL01–
AL11), 21 haplotypes (AL12–AL20, AL40–AL51) and
Fig. 2 The map of sampling localities and distribution of Asymmetron lucayanum modified from Nishikawa (1979), Tachikawa and
Nishikawa (1979), Poss and Boschung (1996) and Nishikawa et al. (1997). Localities are color coded: Maldives yellow, Ryukyus red, Great
Barrier Reef (GBR) purple, Bermuda green, Barbados light blue
877
19 haplotypes (AL21–AL39), respectively. Clade A was
sister group to Clades B and C. The average sequence
divergences (p-distance and ML distance) are summa-
rized in Table 1. The average p-distances were 18.9, 19.2
and 5.9% between Clades A and B, A and C, and B and
C, respectively. The average sequence divergences within
Clades A, B, and C were 0.5, 0.9, and 0.5%, respectively.
The average ML distances were 39.5, 41.9, and 6.6%
Fig. 3 a Neighbor-joining tree of 39 haplotypes (62 specimens) of
the lancelet Asymmetron lucayanum based on the cytochrome c
oxidase subunit I gene (1,035 bp), obtained using HKY+I+C
model. Numbers beside major internal branches indicate bootstrap
probabilities based on 1,000 replicates. b Statistical parsimony
network of three clades of the Asymmetron lucayanum, based on
1,035 bp of the COI gene. Networks were not joined if haplotypes
were separated by more than 14 mutations (95% probability
connection limit). Each circle represents one haplotype: size of
circles is proportional to haplotype frequency (1–20 specimens),
color codes follow those employed in Fig. 2. Circles within larger
circles (with dotted outline) represent haplotype sharing between
two populations. Small black dots represent putative (missing)
haplotypes that were not observed. Solid line circles indicate the
highest outgroup weight haplotype
878
between Clades A and B, Clades A and C, and Clades B
and C, respectively.
Population structure
Genetic diversity indices in the studied populations of
A. lucayanum are summarized in Table 2. Within pop-
ulations, we found 1–14 haplotypes, with low nucleotide
diversity of 0.08–0.85% in all populations, but the small
sample size precluded the GBR population from further
population analyses. Haplotype diversities were high
(h=0.52–1.00). Population structure, as defined by U
ST
values, was not significant in the Clades A and C,
whereas population structure in the Clade B was sig-
nificant betwe en Hawaii and the Ryukyus (U
ST
=0.21),
and Hawaii and the Philippines (U
ST
=0.22).
Three haplotype networks were recovered by TCS
based on COI sequences of 80 specimens from seven
populations (Fig. 3b). They were not connected with
each other due to the large genetic distance (>>14
mutations at 95% probability threshold) between their
haplotypes. The haplotype network of Clade A showed
a ‘‘star phylogeny’’ consisting of 31 specimens collected
from the Maldives, the Ryukyus and GBR. AL06 was
the most common haplotype shared by the Maldives
(n=14) and the Ryukyus (n=6), and the oldest as sug-
gested by the highest outgroup weigh t (0.81>0.02–0.03
in the others). Thre e haplotypes (AL01, 04 and 11) were
connected to AL06 by relatively long branches with five
or seven mutations. The Clade B network contained 28
specimens collected from the Ryukyus, the Philippines,
GBR, and Hawaii. It consisted of four subclades that
differed by three to 13 minimum mutations between
them. AL44 was the oldest as suggested by the highest
outgroup value (0.22 > 0.01–0.14 in the others). The
Clade C network also showed a ‘‘star phylogeny’’ con-
sisting of 21 specimens collected from Barbados and
Bermuda. AL21 was the most common haplotype
shared by both populations and the oldest haplotypes as
suggested by the highest outgroup weight
(0.40 > 0.03–0.11 in the others). Two haplotypes (AL24
and 30) were connected to AL21 by relativel y long
branches with five and seven mutations, respectively.
Divergence time
The estimated divergence times between the three clades
of A. lucayanum are summarized in Table 3. The diver-
gence times between Clades A and B, A and C, and B
and C were estimated as 100.6 million, 106.6 million and
12.6 million years ago (Mya), respectively.
Discussion
Discovery of cryptic species
Our molecular phylogenetic analysis of nucleotide se-
quences of mtDNA showed clearly that populations of
A. lucayanum collected from various areas are geneti-
cally clustered into three major clades A–C (Fig. 3a) .
Table 1 Matrix of genetic distances for the mitochondrial COI gene for three clades of Asymmetron lucayanum complex and Bran-
chiostoma belcheri (outgroup)
Clade Branchiostoma belcheri (outgroup) Asymmetron lucayanum complex
A (Indo-West Pacific Oceans) B (West Pacific Ocean) C (Atlantic Ocean)
B. belcheri 0.22 0.21 0.20
A. lucayanum
A 0.58 0.19 0.19
B 0.59 0.39 0.06
C 0.56 0.42 0.07
Values above the diagonal are uncorrected ‘‘p’’ distance; below diagonal are ML distance (HKY+I+C model)
Table 2 Genetic diversity within populations of three clades of Asymmetron lucayanum complex
Population N Number of haplotypes Haplotype diversity
h±SD
Nucleotide diversity
p±SD
A (Indo-West Pacific)
Maldives 20 7 0.5211±0.1346 0.000870±0.000705
Ryukyus 9 3 0.5556±0.1653 0.003167±0.002038
GBR 2 2 – –
B (West Pacific)
Ryukyus 9 7 0.9722±0.0640 0.008481±0.004905
Philippines 5 4 0.9000±0.1610 0.007923±0.005181
GBR 1 1 – –
Hawaii 13 9 0.9231±0.0572 0.006416±0.003642
C (Atlantic)
Bermuda 15 14 0.9905±0.0281 0.004454±0.002596
Barbados 6 6 1.0000±0.0962 0.003156±0.002178
879
It should be noted that the mtDNA COI sequence
divergence between Clade A and Clade B+C (p=18.6–
19.9% and ML d=38.3–47.0%) is similar to the inter-
specific value of orthologous mtDNA segment among
Branchiostoma species (p=16.9–20.8 %) (Nohara et al.
2004; 2005a). Whereas sequence divergence between
Clades B and C (p=5.8% and d=7.2%) is less than the
above values, this still falls within the range of inter-
specific values of other marine congeneric invertebrates
(d=4–31%) (Holland et al. 2004). In addition, inter-
clade values (p=5.6–19.2%) are much greater than in-
tra-clade values (p=0.4–0.9%). These extant clades
seem to have undergone relatively long and independent
lineage-sorting processes. Therefore, the present COI
sequence data suggests that ‘‘A. lucayanum’’ is an
assemblage of three cryptic species, two phylogenetic
extremities of which inhabit the Ryukyus and GBR in
the West Pacific Ocean in sympatry.
In view of nomenclature, the Atlantic Clade C is
referable to A. lucayanum Andrews, 1893, because the
type locality is the Bahamas in the Atlantic Ocean near
Barbados (Andrews 1893). On the other hand, nomen-
clatural problems still remain unsolved for the remain-
ing two cryptic species; A. lucayanum (sensu lato) has
a certain number of junior synonyms, established for
Pacific popul ations.
Population structure and suggested history
Haplotype networks and population statistics suggested
the following pop ulation history in the three clades
(Table 2 and Fig. 3b).
In Clade A, a recent decrease in population size can
be inferred from a combination of moderate haplotype
diversity (h=0.53) and small nucleotide diversity
(p=0.18%), which are classified as intermediate be-
tween Grant and Bowen’s (1998) first and second cat-
egories of population his tory, and significant negative
Tajima’s D -value (D=1.869, P=0.020). The popula-
tion size decrease in the Maldives may be due to
habitat reduction that the Maldivian atolls would have
suffered as steep cliff islands with rugged plateaux tops
during sea level drops at the glacia l maxima (Anderson
1998). AL06 is the most common haplotype and
detectable in the Ryukyus in the West Pacific and the
Maldives in the Indian Ocean across a distance of more
than 10,000 km by coastal routes. Despite our re-
stricted sampling at three sites in the Indo-West Pacific
Ocean, the existence of such a widespread haplotype, as
well as the absence of significant genetic differentiation
between the Maldives and the Ryukyus, suggests that
the frequent gene flow crossing the Sunda Shelf, known
as a distribution boundary for many marine animals
(Williams and Benzie 1998; marine Wallace’s line by
Barber et al. 2000), must have occurred recently in
evolutionary time, as postulated in the three-spot sea-
horse Hippocampus trimaculatus (Lourie and Vincent
2004).
In Clade B, haplotype diversity was large (h=0.97)
and nucleotide diversity was relatively large (p=0.8%),
suggesting that secondary contact has occurred among
differentiated lineages, three subclades in Hawaii (the
central P acific), three in the Ryukyus and two in the
Philippines (the western Pacific), by Grant and Bowen’s
(1998) criteria. Although population structure was sig-
nificant between the central and the western Pacific is-
lands, sharing haplotype AL50 by Hawaii and the
Philippines, and closely related haplotypes (AL50, 18
and 19) suggest that gene flow over the both Pacific is-
lands (8,500 km) must have occurred recently in evolu-
tionary time. Similarly, gene flow over the equa tor
between GBR, and the Ryukyus and Hawaii (5,000–
7,500 km) was suggested by the existence of closely re-
lated haplotypes (AL16, 17 and 44).
In the Atlantic Clade C, sharing of haplotype AL21
by Barbados and Bermuda and the lack of significant
genetic isolation among both localities (2,200 km dis-
tance) suggest that gene flow between populations in
the two Atlantic localities must have occurred recently
and is ongoing. The genetic continuity between Bar-
bados and Bermuda matche s that seen in the sea
urchin Lytechinus (Zigler and Lessios 2004), suggesting
the high gene flow caused by the Gulf Strea m. In
addition, the similarity of the fossil record between
Florida and Bermuda supports the genetic continuity
due to the Gulf Stream in the Last Interglacial period
(Muhs et al. 2002). Population statistics indicate a
combination of large haplotype diversity (h=0.99) and
small nucleotide diversity (p=0.41%), classifying into
Grant and Bowen’s (1998) second category, much like
coral reef fishes (Shulman and Bermingham 199 5) and
sea urchins (McCartney et al. 2000). Tajima’s D-test
was 2.21, being significantly negative (P=0.003).
Table 3 Matrix of average of transversions (above diagonal), K index (in parentheses, above diagonal), and estimated divergence time
(million years ago, below diagonal) among the three clades of Asymmetron lucayanum complex
Clade A (Indo-West Pacific) B (West Pacific) C (Atlantic)
A 68.9 (0.0714) 72.7 (0.0757)
B 100.6 9.2 (0.0090)
C 106.6 12.6
Divergence time estimated from Martin et al.’s (1992) molecular clock for cytochrome c oxidase subunit I and cytochrome b genes in
shark mtDNA (7.1·10
10
transversions/site · year). K indicates corrected proportion of transversions between compared sequences:
K=0.5 log
e
(1/(12Q)), where Q is an observed proportion of transversions
880
These imply that Clade C may have grown rapidly
after the population size reduction due to a decrease
in habitats of approximately 89% which occurred
during the glacial maxima (Bellwood and Wainwright
2002).
Speciation in Asymmetron lucayanum complex
In the present study, it should be noted that the inter-
oceanic divergence time between Clade B (the West-
Central Pacific) and Clade C (the Atlantic) is relatively
recent (11.7–13.0 Mya, the Middle Miocene), while the
intra-oceanic divergence time between Clade A (the
Indo-West Pacific) and Clade B is ancient (99.7–
101.2 Mya, the Cenomanian in the middle Cretaceous)
(Table 3 and Fig. 3). Divergence time between Asym-
metron and the remaining lancelet genera (Epigonich-
thys and Branchiostoma) was estimated at
approximately 162 Mya based on mitochondrial COI
and cytb genes (Nohara et al. 2005b). At that time (the
middle Jurassic), the initial breakup of Pangaea had
just begun (Smith et al. 1994), and therefore the
Atlantic Ocean had not yet appeared. It is thus rea-
sonable to assume that the Asymmetron lineage may
have originated in the east Tethys sea (representing the
origin of the Indian and the West Pacific Oceans).
Generally, it is difficult to estimate a dispersal route in
100 million years. If, however, separation between
Clades A and C (+B) occurred due to the Atlantic
expansion, as estimated in various marine animals
(Bellwood and Wainwright 2002), e.g. the Pacific spe-
cies of Branchio stoma (B. belcheri and B. malayanum)–
the Atlantic species (B. floridae and B. lanceolatum)
(Nohara et al. 2004 )andSepioteuthis lessoniana (living
in the Indo-West Pacific Ocean)–S. sepioidea (in the
Caribbean Sea) lineage (Anderson 2000), the west
Atlantic Clade C (called the species C) may have
moved via the Panamanian seaway by the middle
Miocene (ca. 13 Mya) into the Pacific Ocean, where the
species leading to Clade B (species B) was formed and
secondarily contacted Clade A (species A) (Fig. 4).
Whether or not Asymmetron dispersed in the above-
mentioned route, two sympatric morphologically iden-
tical species (A and B) have gone through separate
history for a long time. However, it is not plausible to
assume fundamental niche differences between these
sympatric species because they were simultaneously
collected from a sampling site in the Ryukyus for the
present study. Closer field or experimental studies are
necessary to solve the problem of how the two species
coexist and what reproductive isolating mechanism was
established.
Acknowledgements Our cordial thanks are due to Dr. G. Rouse
(University of Adelaide) for the material from the Great Barrier
Reef, to Drs. E. Cutler (Harvard University), E. Ruppert
(Clemson University), R. Langston (University of Hawaii), and
Ms. A. Fukunaga (University of Hawaii) for kind help and advice
for sampling, to Mr. Hassan Maaz Schareef of the Ministry of
Fisheries, Agriculture and Marine Resources, Republic of Mal-
dives for permission to collect the lancelets, to Mr. H. Tachikawa
(Natural Museum and Institute, Chiba) for the photo, and to
Drs. J.G. Inoue and Y. Yamanoue (ORI, University of Tokyo)
for their helpful discussion regarding phylogenetic analyses during
the preparation of this manuscript. This paper is Contribution
#91 of the Bermuda Biodiversity Project, Bermuda Aquarium,
Museum and Zoo. This study was financially supported by
Grants-in-Aid from JSPS (Nos. 13440253, 15380131, 16370044,
and 12NP0201), and NSF Grant DEB-0118804.
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