Ichthyol Res (2018) 65:92–100
https://doi.org/10.1007/s10228-017-0596-1
FULL PAPER
Cryptic genetic divergence in Scolopsis taenioptera (Perciformes:
Nemipteridae) in the western Pacific Ocean
Ryo Kakioka1,10 • Nozomu Muto1,11 • Hirohiko Takeshima1 • Arnold C. Gaje2,3 • Ramon S. Cruz2
Ulysses B. Alama2 • Armi May T. Guzman2 • Rex Ferdinand M. Traifalgar2 • Ricardo P. Babaran2
Osman Muda4 • Wahidah Mohd Arshaad4 • Sukchai Arnupapboon5 • Kamolrat Phuttharaksa6 •
Quan Van Nguyen7 • Thu The Pham7 • Hiroyuki Motomura8 • Fumihito Muto9 • Satoshi Ishikawa1
Received: 29 November 2016 / Revised: 20 July 2017 / Accepted: 20 July 2017 / Published online: 7 August 2017
Ó The Ichthyological Society of Japan 2017
Abstract We studied the phylogeny, population structure,
and demographic history of Scolopsis taenioptera in the
western Pacific Ocean. Using the 80 samples collected
from four locations, we obtained the nucleotide sequences
of mitochondrial cytochrome c oxidase subunit I and
cytochrome b genes. We identified two distinct lineages
showing a clear phylogeographic break that was possibly
due to the Pleistocene sea-level change. One lineage was
distributed in Iloilo (Philippines) and the other in Tereng-
ganu (Malaysia), Rayong (Thailand), and Ha Long Bay
(Vietnam). The Terengganu and Rayong populations
showed clear signs of demographic expansion; the Iloilo
and Ha Long Bay populations were relatively stable or
spatially expanded as geographically subdivided
populations.
Electronic supplementary material The online version of this
article (doi:10.1007/s10228-017-0596-1) contains supplementary
material, which is available to authorized users.
& Ryo Kakioka
kakiokar@gmail.com
1
Research Institute for Humanity and Nature, 457-4
Kamigamo-Motoyama, Kita ku, Kyoto 603-8047, Japan
2
College of Fisheries and Ocean Sciences, University of the
Philippines Visayas, Miagao, 5023 Iloilo, Philippines
3
College of Arts and Sciences, University of the Philippines
Visayas, Miagao, 5023 Iloilo, Philippines
4
Marine Fishery Resources Development and Management
Department, Southeast Asian Fisheries Development Center,
Taman Perikanan Chendering, 21080 Kuala Terengganu,
Malaysia
5 Present Address: School of Biological Sciences, Tokai
Training Department, Southeast Asian Fisheries
Development Center, P.O. Box 97, Phra Samut Chedi,
Samut Prakan 10290, Thailand
123
•
•
Keywords Marine biogeography  Coral Triangle 
Southeast Asia  Pleistocene sea-level changes  Sunda
Shelf
Introduction
The Indo-Pacific tropical waters support the greatest
diversity of marine organisms on Earth (Briggs 1999).
Species richness is concentrated particularly in the Coral
Triangle, the region encompassed by the Philippine
Archipelago, Malay Peninsula, New Guinea, and northern
Australia (Carpenter and Springer 2005). The exceptional
diversity of the Coral Triangle is attributable to the func-
tion of this region as the place of in situ divergences of
lineages, accumulation of lineages differentiated in
allopatry, and refugia, any of which are not mutually
exclusive (McManus 1985; Bellwood and Meyer 2009;
Cowman et al. 2013; Gaither and Rocha 2013). Vicariant
6
Eastern Marine Fisheries Research and Development Center,
2 Moo 2, Tambon Phe, Mueang Rayong, Rayong 21160,
Thailand
7
Institute of Marine Environment and Resources, Vietnam
Academy of Science and Technology, 18 Hoang Quoc Viet
Str., Cau Giay, Hanoi, Vietnam
8
The Kagoshima University Museum, 1-21-30 Korimoto,
Kagoshima 890-0065, Japan
9
School of Marine Science and Technology, Tokai University,
3-20-1 Orido, Shimizu ku, Shizuoka 424-8610, Japan
10
Present Address: Division of Ecological Genetics, National
Institute of Genetics, 1111 Yata, Mishima 411-8540, Japan
11
University, 1-1-1 Minamisawa-5-Jo, Minami ku,
Sapporo 005-8601, Japan
Genetic divergence in Scolopsis taenioptera
events in or around the Coral Triangle due to the glacial
environmental changes during the Plio-Pleistocene are
considered to have played a major role in generating the
diversity in any of the above processes (Bellwood and
Wainwright 2002). Many fish species in the Coral Triangle
indeed have geographically structured rather than pan-
mictic populations (Carpenter et al. 2011), despite the
absence of impermeable barriers at present. Therefore, the
elucidation of population genetic structures offers valuable
insights into the biological and physical processes that have
shaped the diversity of the Coral Triangle and Indo-Pacific.
To this end, complementary studies on species with dif-
ferent historical and ecological backgrounds are required to
generalise our understanding of the processes that are
shared by or are unique to the divergences of lineages
(Carpenter et al. 2011).
The lattice monocle bream Scolopsis taenioptera is a
coastal fish of the family Nemipteridae occurring on the
sandy and muddy bottoms at a depth up to 50 m. It is
distributed in the tropical and subtropical waters of the
western Pacific Ocean from Taiwan to New Caledonia and
eastern Indian Ocean from the Andaman Sea to north-
western Australia, and commonly marketed in Southeast
Asia (Carpenter and Niem 2001; Béarez 2003). This fish
can be a valuable system to study intraspecific divergences
in the Coral Triangle and Indo-Pacific, because it is widely
distributed in this region and it could have been subjected
to vicariance due to the extensive impacts of glacial
environmental changes on its habitats. Although allopatri-
cally distributed divergent lineages have been inferred in S.
taenioptera by Hung et al. (2017), our knowledge on the
population structure and demography of this species is still
limited because of the small number of samples per pop-
ulation used in the study.
The aim of this study was to examine the evolutionary
history of S. taenioptera in the western margin of the
Pacific Ocean. We used the molecular genetic approaches
based on the nucleotide sequences of two mitochondrial
genes. Phylogenetic and phylogeographic analyses were
conducted to reveal the possible genetic divergences and
regions of genetic breaks. We performed the historical
demographic analyses to elucidate the patterns of demo-
graphic changes of populations over time. Based on our
results, we discussed the potential biological and physical
processes that could have formed the spatial pattern of
genetic diversity.
Materials and methods
Specimen collection and molecular genetic experiments.
Eighty fish specimens were collected from four locations:
Iloilo, Philippines; Terengganu, Malaysia; Rayong,
93
Thailand; and Ha Long Bay, Vietnam (Fig. 1a, Table 1).
Genomic DNA was isolated using a Genomic DNA
Purification Kit (Promega, Madison, USA) from fin clips
preserved in 99% ethanol or dried muscle tissues that were
stored at –30 °C. Polymerase chain reaction (PCR) was
performed to amplify two mitochondrial genes: cyto-
chrome c oxidase subunit I (COI) and cytochrome b (cytb).
The COI gene was amplified using a primer cocktails
(‘COI-3’) by Ivanova et al. (2007): VF2_t1 (50 -TGT AAA
ACG ACG GCC AGT CAA CCA ACC ACA AAG ACA
TTG GCA C-30 ), FishF2_t1 (50 -TGT AAA ACG ACG
GCC AGT CGA CTA ATC ATA AAG ATA TCG GCA
C-30 ), FishR2_t1 (50 -CAG GAA ACA GCT ATG ACA
CTT CAG GGT GAC CGA AGA ATC AGA A-30 ) and
FR1d_t1 (50 -CAG GAA ACA GCT ATG ACA CCT CAG
GGT GTC CGA ARA AYC ARA A-30 ) (reaction at 94 °C
for 2 min, 35 cycles with 94 °C for 30 sec, 52 °C for
30 sec, 72 °C for 1 min, followed by a final extension at
72 °C for 7 min). Amplification of cytb was conducted
using one of three primer pairs: CbPercid_F (50 -CGG TAC
CCG GGG ATC ATG TGA CTT GAA AAA CCA CCG
TTG-30 ) and CbPercid_R (50 -CGA CTC TAG AGG ATC
CCC TCC ATC TCC GGT TTA CAA GAC-30 ) (94 °C for
1.5 min, 35 cycles with 94 °C for 42 sec, 47 °C for 45 sec,
72 °C for 1 min, followed by a final extension at 72 °C for
5 min) by Song et al. (1998) and Guo et al. (2007); Fish-
cytB-F (50 -CGG TAC CCG GGG ATC ATA CCA CCG
TTG TTA TTC AAC TAC AAG AAC-30 ) and THR-Fish2-
R (50 -CGA CTC TAG AGG ATC CCA ACC TCC GAC
ATC CGG CTT ACA AGA CCG-30 ) by Sevilla et al.
(2007) (94 °C for 1.5 min, 40 cycles with 94 °C for 30 sec,
50 °C for 35 sec, 72 °C for 1.5 min, followed by a final
extension at 72 °C for 5 min); and Cytb_UnvL2 (50 -CGG
TAC CCG GGG ATC ATC GAA CGT TGA TAT GAA
AAA CCA TCG T -30 ) by Sanciangco et al. (2011) and
CytbUnvH (50 -CGA CTC TAG AGG ATC CCA TCT
TCG GTT TAC AAG ACC GGT G-30 ) by Orrell et al.
(2002) and Sanciangco et al. (2011) (94 °C for 1.5 min,
35 cycles with 94 °C for 30 sec, 49 °C for 35 sec, 72 °C
for 1.5 min, followed by a final extension at 72 °C for
5 min). All the primers were tailed with M13 (M13F, 50 -
TGT AAA ACG ACG GCC AGT-30 or M13R, 50 -CAG
GAA ACA GCT ATG AC-30 ) by Messing (1983) to
facilitate sequencing (Ivanova et al. 2007). EmeraldAmp
PCR Master Mix (TaKaRa, Kusatsu, Japan) or Restorase
DNA Polymerase (Sigma-Aldrich, St. Louis, USA) was
used to conduct PCR in a 10-lL volume containing 0.5 lL
of each 5 lM primer, 1lL of DNA solution (*10–100 ng/
lL) messed up with distilled water on a Mastercycler ep
Gradient S thermal cycler (Eppendorf, Hamburg,
Germany).
PCR products were cleaned by ExoSAP-IT (Affymetrix,
Santa Clara, USA) at 37 °C. The PCR products were then
123
94 R. Kakioka et al.

Fig. 1 a Map of sampling

locations. Pie charts indicate
the occurrence of each lineage
at each sampling location; the
areas are proportional to sample
sizes. Dashed lines indicate
120-m isobaths based on
ETOPO1 bathymetric data
(Amante and Eakins 2009).
b Unrooted maximum
likelihood phylogenetic tree of
Scolopsis taenioptera based on
the concatenated sequences of
COI and cytb. Support values
(C75 % ML bootstrap
probability / C0.9 Bayesian
posterior probability) are
indicated along the branches.
Each node is labelled with a
haplotype name and the
occurring population with the
frequencies within the
parentheses

Table 1 Sampling locations, number of specimens (n), and genetic diversity indices of the examined populations of Scolopsis taenioptera
Location n k h ± SD p ± SD (9 103) D (P) FS (P) R2 (P)
Corrected Uncorrected

Iloilo 22 12 0.89 ± 0.05 4.31 ± 2.33 3.60 ± 1.97 –0.37 (0.40) –1.19 (0.31) 0.11 (0.34)
*
Terengganu 16 8 0.70 ± 0.13 0.98 ± 0.67 0.98 ± 0.67 –2.28 (\ 0.01) –3.36* (\ 0.01) 0.11 (0.13)
Rayong 28 17 0.82 ± 0.08 1.08 ± 0.70 1.07 ± 0.70 –2.58* (\ 0.01) –15.13* (\ 0.01) 0.04* (\ 0.01)
Ha Long Bay 14 6 0.77 ± 0.09 1.60 ± 1.01 1.60 ± 1.00 –0.05 (0.56) –0.02 (0.50) 0.14 (0.42)
k number of haplotypes; h haplotype diversity; SD standard deviation; p nucleotide diversity; Corrected/Uncorrected genetic distances were
corrected/uncorrected for substitution models; D Tajima’s D; FS Fu’s FS; R2 R2 statistics by Ramos-Onsins and Rozas. D, FS and R2 values
significantly depart from zero (D and R2, P \ 0.05; FS, P \ 0.02) are marked with *

sequenced using an Applied Biosystems 3500 Genetic
Analyzer (Life Technologies, Carlsbad, USA) with a Big-
Dye Terminator Cycle Sequencing Kit v3.1 (Life Tech-
nologies), M13 primers (Ivanova et al. 2007), and as
necessary, newly-designed internal sequencing primers
(StaeIntF1, 50 -ATT TAC ACG CAA ATG GAG CT-30 ;
StaeIntR1, 50 -CAC CTC CGA GTT TAT TTG GG-30 ) for
cytb. The obtained sequences of the 50 -end of COI (652 bp)
and complete cytb (1,141 bp) were deposited in DDBJ/ENA/
GenBank (accession numbers LC198257–LC198309).

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Genetic divergence in Scolopsis taenioptera
Molecular phylogenetic analyses. We conducted phy-
logenetic and population genetic analyses using concate-
nated sequences of COI and cytb, after checking with
Concaterpillar v1.7.2 (Leigh et al. 2008) that phylogenetic
congruence between the loci was not rejected (P = 0.35).
Maximum likelihood (ML) analyses were performed using
RAxML v8.2.4 (Stamatakis 2014). We conducted a rapid
bootstrap analysis with 1,000 replicates and a search for the
best-scoring ML tree in one single run. Optimal nucleotide
substitution models and partitioning were selected based on
corrected Akaike information criterion (AICc) using Par-
titionFinder v1.1.1 [Lanfear et al. 2012; Electronic Sup-
plementary Materials (ESM) Table S1], initially treating
each gene as a single locus partitioned by codon position.
The ‘greedy’ algorithm was used to search for the partition
scheme. Bayesian analyses were performed using MrBayes
v3.2.5 (Ronquist et al. 2012). Substitution models and
partition schemes were selected as above (ESM Table S1).
The MrBayes analysis was run for 107 generations with
two independent runs of four Markov chain Monte Carlo
(MCMC) chains and sampling every 1,000 generations.
After checking the trace files with Tracer v1.6 (Rambaut
et al. 2014) to ensure that the chains had reached conver-
gence, the first 25 % of the trees were discarded as burn-in.
The phylogenetic trees were visualised using FigTree
v1.4.2 (Rambaut 2014).
To assess the timing of the divergence of lineages, we
estimated the time to the most recent common ancestor
(TMRCA) using Bayesian inference implemented in
BEAST v2.4.3 (Bouckaert et al. 2014). We assumed
molecular clocks of 1, 5 and 10 % substitution per million
years (Ma), which are within the estimates for various fish
taxa (e.g. Lessios 2008). The substitution model HKY ? I
was selected based on AICc as implemented in jModelTest
v2.1.8 (Guindon and Gascuel 2003; Darriba et al. 2012),
treating each concatenated sequence as a single partition.
The tree topology was fixed to the one estimated using
RAxML. Midpoint rooting was used to assign the root
given a clear phylogenetic divergence (see Results and
Discussion; Sanderson and Shaffer 2002; Hess and Russo
2007). We performed four independent runs of MCMC
chains for 108 generations, sampling every 104 generations
following 107 generations of burn-in periods. After
checking for convergence using Tracer, the sampled trees
were summarised by TreeAnnotator of the BEAST package
and visualised using FigTree.
In addition, we constructed a neighbour-joining phylo-
genetic tree using MEGA v6.06 (Tamura et al. 2013) based
on the COI sequences of Scolopsis including those
retrieved from public databases (ESM Table S2): DDBJ/
ENA/GenBank, The Barcode of Life Data Systems
(BOLD; Ratnasingham and Hebert 2007) and Cryobanking
Program for Wildlife Genetic Material in Taiwan
95
(Academia Sinica 2008). After sequence alignment using
MUSCLE, the genetic distance was calculated under the
Kimura two-parameter model (Kimura 1980). Apparently
misidentified samples were excluded from the analysis.
The robustness of the tree was estimated using 1,000
bootstrappings. The specimen of S. taenioptera from the
Indian Ocean off northwestern Australia (DDBJ/EMBL/
GenBank accession number EF609455) has been deposited
at the Australian National Fish Collection of the Com-
monwealth Scientific and Industrial Research Organisation
in Hobart (Catalogue number H4029-01) and a photograph
of the specimen when fresh was reproduced in (Yearsley
et al. 1999:249). Identification of the specimen is herein
confirmed morphologically as S. taenioptera from the
photograph. Moreover, identification of several specimens
in the database records was revised according to Hung
et al. (2017; see ESM Table S2).
Population genetic analyses. The genetic diversity
indices, including the number of haplotypes (k), haplotype
diversity (h) and nucleotide diversity (p), were calculated
for each population using Arlequin v3.5.1.2 (Excoffier and
Lischer 2010). We estimated evolutionary distances that
were corrected for substitution models selected using
jModelTest and that were uncorrected (ESM Table S3). We
constructed statistically parsimonious networks using
PopART v1.7 (Leigh and Bryant 2015) to estimate rela-
tionships among the haplotypes. To study a geographic
population structure, we estimated the pairwise fixation
index (UST) to evaluate the genetic differentiation between
populations and genetic distance between populations (DA)
in Arlequin. The significance level of genetic differentia-
tion was adjusted by controlling the false discovery rate of
0.05 (Benjamini and Hochberg 1995). To test whether the
sequences had evolved under neutrality in each population,
Tajima’s D (Tajima 1989) and Fu’s FS (Fu 1997) were
estimated in Arlequin. A significant departure of these
indices from zero suggests a population expansion (\ 0) or
contraction ([ 0). Neutrality was also tested with R2
statistics (Ramos-Onsins and Rozas 2002) using ‘pegas’
v0.8-2 (Paradis 2010) in R v3.1.3 (R Core Team 2015).
The historical demographic expansion of each population
was also tested based on the distribution of pairwise
nucleotide differences (mismatch distribution analysis;
Rogers and Harpending 1992) in Arlequin. The fitness of
the observed data to the models of pure demographic
expansion in spatially unsubdivided populations (Rogers
and Harpending 1992) and spatial expansion in subdivided
populations (Ray et al. 2003; Excoffier 2004) was tested by
the sum of square deviations (SSD; Schneider and Excof-
fier 1999) and raggedness index (RI; Harpending 1994)
based on 104 parametric bootstrapping. Historical demo-
graphic changes of each population were estimated by
Bayesian skyline plot (BSP) analysis (Drummond et al.
123
96
2005) implemented in BEAST. The substitution model was
selected based on the AICc using jModelTest (ESM
Table S4), assuming each joined sequence as a single
partition. The molecular clocks of 1, 5 and 10 % Ma-1
were used. RAxML was used to estimate the tree topolo-
gies that were fixed throughout the BSP analyses. The most
frequent sequence of the Iloilo lineage (StaHap01; see
Fig. 1b and Table S5) was used to as an outgroup root the
tree of the Terengganu, Rayong and Ha Long Bay popu-
lations; the most frequent sequence of the Terengganu,
Rayong and Ha Long Bay (TRH) lineage (StaHap13; see
Fig. 1b and Table S5) was used to root the tree of the Iloilo
population. We performed four independent runs of
MCMC chains for 108 generations, sampling every 104
generations following 107 generations of burn-in periods.
The results with the stepwise (constant) model were sum-
marised using Tracer after checking for convergence.
Drawing maps. Maps were drawn using the R packages
maptools v0.9-1 (Bivand and Lewin-Koh 2017), marmap
v0.9.6 (Pante and Simon-Bouhet 2013) and PBSmapping
v2.69.76 (Schnute et al. 2015). The bathymetric data
derived from the ETOPO1 database (Amante and Eakins
2009).
Results and discussion
Genetic divergences. The ML and Bayesian phylogenetic
analyses using the concatenated sequences of COI and cytb
(Table 1, ESM Table S5) revealed two distinct genetic
lineages in Scolopsis taenioptera in the western Pacific
(ML bootstrap probability, 100 % and Bayesian posterior
probability, 1.0; Fig. 1b). The geographic distribution of
these lineages was separated: one was found in Tereng-
ganu, Rayong and Ha Long Bay (hereafter abbreviated as
TRH); the other originated exclusively from Iloilo
(Fig. 1a). The nucleotide sequences of the two lineages
Fig. 2 Statistically parsimonious network of Scolopsis taenioptera
based on the concatenated sequences of COI and cytb. Each disc
represents a unique haplotype, the area of which is proportional to
123
R. Kakioka et al.
were differentiated by 2.25 ± 0.15 % (Uncorrected p-dis-
tance; mean ± SD). The Bayesian divergence time analy-
sis estimated the TMRCA of the TRH and Iloilo lineages at
1.18 Ma before present (BP) (0.79–1.58 Ma BP, 95 %
highest posterior density; assuming the 1 % Ma-1 molec-
ular clock), 236 thousand years (ka) BP (159–318 ka BP; 5
% Ma-1), and 118 ka BP (80–160 ka BP; 10 % Ma-1), all
falling within the Pleistocene (ESM Fig. S1). The popu-
lation genetic analyses also supported the divergence,
wherein a significant genetic differentiation was found
between the TRH and Iloilo populations (0.88 B UST -
B 0.90; 1.97 B DA B 1.99; ESM Table S6). The Ha Long
Bay population showed moderate differentiation from the
other two TRH populations (UST = 0.28; DA = 0.05). The
haplotype network consisted of the TRH and Iloilo sub-
networks (Fig. 2).
Further divergent lineage was inferred by the phylo-
genetic analysis that included the COI sequences from
the existing studies (ESM Fig. S2). The haplotypes from
the TRH populations were included in a lineage that was
distributed on the Sunda Shelf (northern Malacca Strait,
northern Borneo, eastern Java and Bali), Lombok and
western Luzon (ESM Figs. S2, S3). Genetic divergence
among the haplotypes of the Sunda Shelf lineage was
generally small, although divergent haplotypes were
found in Bali and western Luzon. The Iloilo lineage was
distinct. Sequences from the Sahul Shelf (northwestern
and northeastern Australia and off western New Guinea)
was substantially differentiated from those of the Sunda
Shelf and Iloilo lineages (5.22 ± 0.35 %; p-distance,
mean ± SD). The Iloilo and Sunda Shelf lineages were
more closely related to each other (1.33 ± 0.17 %). In
contrast to the wide distribution range of the Sunda Shelf
and Sahul Shelf lineages, which have been detected in
Hung et al. (2017), the occurrence of the Iloilo lineage,
which we identified in the present study, was restricted
to Iloilo.
frequency; different colours correspond to the populations of origin.
Black dots represent unobserved hypothetical haplotypes. Each hatch
mark represents one mutational step
Genetic divergence in Scolopsis taenioptera
Our results show that S. taenioptera is differentiated into
at least three genetic lineages with a geographic structure
in the western Pacific. A common genetic break is known
in the Indo-Pacific, whereby one of the pairs of the sister
species or intraspecific lineages is distributed mainly in the
western Pacific and the other in the Indian Ocean (Bell-
wood and Wainwright 2002). The emergence of the Sunda
Shelf due to a glacial sea-level lowstand is considered as a
vicariant event that hinders gene flow between the oceans
by forming land barriers and by reducing sea currents
between the oceans; its postglacial submergence is sup-
posed to have allowed the colonisation and consequential
overlap of the diverged lineages in the Coral Triangle
(Gaither and Rocha 2013). The TRH–Sunda Shelf and
Iloilo lineages might represent an Indo–Pacific divergence
followed by interglacial dispersal (Lourie et al. 2005; Muto
et al. 2016). Older Indo–Pacific isolation is also one pos-
sible explanation for the divergence between the Sunda and
Sahul Shelf lineages that has been suggested to have
caused east–west phylogeographic breaks in the western
Pacific Ocean in other marine organisms (Barber et al.
2000; Lourie and Vincent 2004; Rohfritsch and Borsa
2005). The lowered water temperature of the seas between
the Sunda and Sahul Shelves due to the increased upwel-
ling during glacial periods (Fleminger 1986) can be another
barrier to the navigation of tropical organisms between the
two shelves. The restriction of migration of coastal species
between the Sunda and Sahul Shelves due to the existence
of intervening pelagic environment even during glacial
periods might also explain the divergence of the Sunda and
Sahul Shelf lineages and similar divergence in the con-
gener Scolopsis monogramma (Hung et al. 2017). The
glacial isolations of sea basins within the Coral Triangle
could have served as vicariant events (Carpenter and
Springer 2005). This has been suggested to have caused the
development of geographically isolated lineages in the
western Pacific Ocean (e.g. Ravago-Gotanco and Juinio-
Meñez 2010), causing in situ divergences of lineages. The
divergence of the Iloilo lineage could have been alterna-
tively caused by this process given its restricted range.
Although it is premature to draw a general conclusion
about the processes of divergence in S. taenioptera, our
results could infer that the glacial environmental changes
repeated during the Plio-Pleistocene have caused allopatric
divergence and lineage diversification in the Coral Triangle
and Indo-Pacific.
Genetic diversity and demographic histories of the
Iloilo and TRH populations. The Iloilo and Terengganu
population showed the highest and the lowest genetic
diversities, respectively (Table 1). The haplotypes from the
TRH population revealed a ‘star-like’ genealogy (Fig. 2).
The most frequent haplotype at the centre was shared by all
TRH populations. On the contrary, haplotypes were
97
sparsely distributed in the Iloilo subnetwork. The star-like
tree of the TRH subnetwork suggested a rapid population
expansion, whereas the sparse Iloilo subnetwork could
infer the prolonged maintenance of a mutation–drift equi-
librium (Slatkin and Hudson 1991).
The neutrality tests generally supported the expansion of
the Terengganu and Rayong populations, but not of the
Iloilo and Ha Long Bay populations (Table 1). The mis-
match distributions were consistent with the pure demo-
graphic and spatial expansion models, except for the
departures of the distributions of the Iloilo (P = 0.03;
RI = 0.097) and Terengganu (P = 0.02; SSD = 0.109)
populations from the pure demographic model (ESM
Table S7; ESM Fig S4). There was a clear distinction in the
estimated number of migrants exchanged between demes
under the spatial models between the Terengganu and
Rayong populations and the Iloilo and Ha Long Bay pop-
ulations, in that the former were infinite while the latter
were small (6.89 and 2.57, respectively). The BSP analyses
revealed the expansions of the Terengganu and Rayong
populations ranging from approximately 4 ka BP (assum-
ing the 10 % Ma-1 molecular clock) to 40 ka BP
(1 % Ma-1; Fig. 3), the ages of which were close to the
Last Glacial Maximum (LGM; *22–20 ka BP). By con-
trast, BSPs suggest the demographic stability or slight
expansion of the Ha Long Bay population, and the stability
of Iloilo population (note that apparent recent decrease may
reflect random sampling of simulated haplotype trees rather
than an actual change in population size; Grant 2015).
The historical demographic characteristics were distinct
between the populations on and away from the Sunda
Shelf. The Terengganu and Rayong populations, which are
located on the Sunda Shelf, consistently showed clear signs
of demographic expansion. This supports the growth of
these populations since the exposed Sunda Shelf began
submerging after LGM, when the sea level was 120 m
lower than the present (Hanebuth et al. 2000; refer to
120-m isobaths in Fig. 1a). It is possible that the Iloilo
population has been stable because the last glacial cycle
could have limited impacts on the coastal habitats in the
Philippine Archipelago compared to those on the Sunda
Shelf: the Philippine Archipelago has not had vast shelves,
but complex coastal habitats were available during both
glacial and interglacial periods (see ESM Fig. S3). On the
other hand, the Ha Long Bay population is not likely to
have been stable. First, the shelf habitats around Ha Long
Bay were diminished during the glacial sea-level lowstands
(see ESM Fig. S3). Further, although the annual mean sea-
surface temperature over the present distribution range of
S. taenioptera is not below 26 °C, the temperature around
Ha Long Bay dropped to 24–26 °C at LGM (Otto-Bliesner
et al. 2009). It is, thus, more likely that the Ha Long Bay
population has undergone range expansion after LGM from
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Fig. 3 Bayesian skyline plots
of the populations of Scolopsis
taenioptera based on the
concatenated sequences of COI
and cytb assuming mutation
rates of 1, 5 and 10 % Ma-1.
Solid lines show median;
shaded area represents 95 %
credibility intervals
the refugia (Ray et al. 2003; Excoffier 2004). The Iloilo
population could also have expanded from the glacial
refugia elsewhere, rather than a long-stable population at
the same place. These results support the idea that the
eustatic and environmental changes due to glacial cycles
have profound impacts on the demography of S. tae-
nioptera to varying degrees among regions. To further
reveal the location of glacial refugia and mode of post-
glacial expansion, extensive sampling over the distribution
range and further knowledge on the population structure
and demography would be necessary.
Inferences and future prospects. The present study
revealed a genetic break in S. taenioptera, supposing not
only that the lineages diversified allopatrically, but also
that the dispersal between regions has been limited. Given
that the benthic and reef fishes disperse mainly via pelagic
larvae (Sale 2004), S. taenioptera might have the larval
characteristics that hinder long-distance dispersal, such as
short pelagic larval stage, lack of rafting behaviour or
limited environmental tolerance (Luiz et al. 2012). More-
over, although sea currents interconnect the sea basins of
the western Pacific, the seasonal changes in direction and
velocity could restrict the route and extent of larval
transport at the spawning seasons (Ravago-Gotanco and
Juinio-Meñez 2010). The limited dispersal ability facili-
tates not only the allopatric divergence of lineages, but also
the extinction of local populations. Therefore, the conser-
vation of this species needs to incorporate the spatial
structure of populations for the establishment and man-
agement of conservation units.
Acknowledgements The authors thank Mahyam M. I., Raja Bidin R.
H., M. Katoh, O. Abe (Marine Fishery Resources Development and
123
R. Kakioka et al.
Management Department, Southeast Asian Fisheries Development
Center, Kuala Terengganu, Malaysia), Seah Y. G., Mazlan A. G.
(Universiti Malaysia Terengganu), Aziz A. (Universiti Putra Malay-
sia), V. Vilasri (Natural History Museum, National Science Museum,
Bangkok, Thailand), Chien P.V., Nhan D.V., Chien V.V. (Institute of
Marine Environment and Resources, Hai Phong, Vietnam), S.
Kimura, Y. Hibino, H. Suzuki (Mie University), M. Nakae, K. Mat-
suura (National Museum of Nature and Science, Tokyo, Japan), H.
Imamura (Hokkaido University), T. Yoshida, Jeong B., S. Tashiro
(Kagoshima University), A. Takagi, Yap M. and Y. Ogata (Research
Institute for Humanity and Nature, Kyoto, Japan; RIHN) for their help
in sampling and arrangements. This study was conducted under a
Memorandum of Agreement for joint research made by and among
the Department of Agriculture of the Republic of the Philippines
(DA), University of the Philippines Visayas (UPV), Kagoshima
University Museum, RIHN and Tokai University, facilitated by S.
L. Sanchez [Bureau of Fisheries and Aquatic Resources (BFAR),
DA]. P.J. Alcala (DA) provided a Prior Informed Consent Certificate,
and I.P. Cabacaba and S.M.S. Nolasco (BFAR, DA) provided a Fish
Specimen Export Certificate (no. 2016-39812). We thank the staff of
the Office of the Vice-Chancellor for Research and Extension, UPV,
and the UPV Museum of Natural Sciences, College of Fisheries,
UPV, including S.S. Garibay, V.G. Urbina, L.H. Mooc, C.J.N. Rubido
and E.P. Abunal, and graduate students of the College of Fisheries,
UPV for their support of this research collaboration. Malaysian
specimens were collected during the Japan Society for the Promotion
of Science (JSPS), Tokyo Asian Core Program, ‘Establishment of
Research and Education Network on Coastal Marine Science in
Southeast Asia’, supported by the Ministry of Higher Education,
Putrajaya, Universiti Putra Malaysia and Universiti Malaysia
Terengganu. The Thai specimens were collected with the supports of
the Training Department, Southeast Fisheries Development Center,
Bangkok, and the Eastern Marine and Research Center, Rayong. The
Vietnamese specimens were collected with the support of the Institute
of Marine Environment and Resources, and the Ha Long Bay Man-
agement Department and with a permission of use of the specimens
by the Biodiversity Conservation Agency, Ministry of Natural
Resources and Environment, Vietnam. This study was supported by
the projects ‘Coastal Area Capability Enhancement in Southeast Asia’
of RIHN (no. 14200061) and ‘Biological Properties of Biodiversity
Genetic divergence in Scolopsis taenioptera
Hotspots in Japan’ of the National Museum of Nature and Science,
the JSPS Asian Core Program ‘Establishment of Research and Edu-
cation Network on Coastal Marine Science in Southeast Asia’,
Grants-in-Aid for Scientific Research (C:26450265) and Young Sci-
entists (B:26840131) from JSPS, the fund for international collabo-
ration (NDT.16 TW/16; VAST04.08/17-18) from the Ministry of
Science and Technology, Hanoi, and Vietnam Academy of Science
and Technology Grant in Marine Sciences and Technology (VAST
06.04/18-19).
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