Genetic population structure and low genetic diversity in the over-exploited sea cucumber Holothuria edulis Lesson, 1830 (Echinodermata: Holothuroidea) in Okinawa Island

Abstract

Understanding genetic connectivity is fundamental for ecosystem-based management of marine resources. Here we investigate the metapopulation structure of the edible sea cucumber Holothuria edulis Lesson, 1830 across Okinawa Island, Japan. This species is of economic and ecological importance and is distributed from the Red Sea to Hawai‘i. We examined sequence variation in fragments of mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S), and nuclear histone (H3) at six locations across Okinawa Island. We found higher haplotype diversity for mtDNA (COI: Hd = 0.69 and 16S: Hd = 0.67) and higher heterozygosity of nDNA (H3: H
E = 0.39) in populations from the west coast of Okinawa compared to individuals from populations on the east coast (COI: Hd = 0.40; 16S: Hd = 0.21; H3: H
E = 0.14). Overall population structure was significant (AMOVA results for COI: Φ
ST = 0.49, P < 0.0001; 16S: Φ
ST = 0.34, P < 0.0001; H3: Φ
ST = 0.12, P < 0.0001). One population in the east, Uruma, showed elevated pairwise Φ
ST values in comparisons with all other sites and a marked reduction of genetic diversity (COI: Hd = 0.25 and 16S: Hd = 0.24), possibly as a consequence of a shift to a more dominant asexual reproduction mode. Recent reports have indicated that coastal development in this area influences many marine organisms, and ecosystem degradation in this location could cause the observed decrease of genetic diversity and isolation of H. edulis in Uruma. Our study should provide valuable data to help with the urgently needed management of sea cucumber populations in Okinawa, and indicates particular attention needs to be paid to vulnerable locations.

Full text

1 23
Conservation Genetics
ISSN 1566-0621
Volume 17
Number 4
Conserv Genet (2016) 17:811-821
DOI 10.1007/s10592-016-0823-8
Genetic population structure and low
genetic diversity in the over-exploited
sea cucumber Holothuria edulis Lesson,
1830 (Echinodermata: Holothuroidea) in
Okinawa Island
Taha Soliman, Iria Fernandez-Silva &
James Davis Reimer

1 23
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RESEARCH ARTICLE
Genetic population structure and low genetic diversity in the
over-exploited sea cucumber Holothuria edulis Lesson, 1830
(Echinodermata: Holothuroidea) in Okinawa Island
Taha Soliman
1,2,3
•Iria Fernandez-Silva
1,4,5
•James Davis Reimer
1,6
Received: 24 July 2015 / Accepted: 10 February 2016 / Published online: 19 February 2016
ÓSpringer Science+Business Media Dordrecht 2016
Abstract Understanding genetic connectivity is funda-
mental for ecosystem-based management of marine resour-
ces. Here we investigate the metapopulation structure of the
edible sea cucumber Holothuria edulis Lesson, 1830 across
Okinawa Island, Japan. This species is of economic and
ecological importance and is distributed from the Red Sea to
Hawai‘i. We examined sequence variation in fragments of
mitochondrial cytochrome oxidase subunit I (COI) and 16S
ribosomal RNA (16S), and nuclear histone (H3) at six
locations across Okinawa Island. We found higher haplotype
diversity for mtDNA (COI: Hd =0.69 and 16S: Hd =0.67)
and higher heterozygosity of nDNA (H3: H
E
=0.39) in
populations from the west coast of Okinawa compared to
individuals from populations on the east coast (COI:
Hd =0.40; 16S: Hd =0.21; H3: H
E
=0.14). Overall
population structure was significant (AMOVA results
for COI: U
ST
=0.49, P\0.0001; 16S: U
ST
=0.34,
P\0.0001; H3: U
ST
=0.12, P\0.0001). One population
in the east, Uruma, showed elevated pairwise U
ST
values in
comparisons with all other sites and a marked reduction of
genetic diversity (COI: Hd =0.25 and 16S: Hd =0.24),
possibly as a consequence of a shift to a more dominant
asexual reproduction mode. Recent reports have indicated
that coastal development in this area influences many marine
organisms, and ecosystem degradation in this location could
cause the observed decrease of genetic diversity and isola-
tion of H. edulis in Uruma. Our study should provide valu-
able data to help with the urgently needed management of sea
cucumber populations in Okinawa, and indicates particular
attention needs to be paid to vulnerable locations.
Keywords Genetic diversity Coastal development 
Genetic structure mtDNA Okinawa Sea cucumber
Introduction
Okinawa Main Island is located in the central Ryukyu
Archipelago, a chain of islands along the eastern boundary
of the East China Sea and the western boundary of the
Philippine Sea. Located at 26°N, Okinawa Main Island
harbors some of the northernmost coral reefs in the world,
owing to the warm northward-flowing Kuroshio Current,
that through larval dispersal and nutrient transport main-
tains the populations of many coral reef organisms in this
unique region (Kamidaira et al. 2014).
Sea cucumbers play an important role in many marine
ecosystemsbecause of their influence on benthic communities
Electronic supplementary material The online version of this
article (doi:10.1007/s10592-016-0823-8) contains supplementary
material, which is available to authorized users.
&Taha Soliman
tahasoliman2000@yahoo.com
1
Molecular Invertebrate Systematics and Ecology Laboratory,
Graduate School of Engineering and Science, University of
the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213,
Japan
2
Aquaculture Department, National Institute of Oceanography
and Fisheries, Kayet-Bay, El-Anfoushy, Alexandria 21556,
Egypt
3
Okinawa Institute of Science and Technology Graduate
University, 1919-1 Tancha, Onna, Okinawa 904-0495, Japan
4
Section of Ichthyology, California Academy of Sciences, 55
Music Concourse Dr., Golden Gate Park, San Francisco,
CA 94118, USA
5
Department of Biochemistry, Genetics and Immunology,
University of Vigo, Vigo 36310, Spain
6
Tropical Biosphere Research Center, University of the
Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
123
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DOI 10.1007/s10592-016-0823-8
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(Power et al. 1996; Lessios et al. 2001; Uthicke et al. 2009).
These deposit feeders process carbonate sand and rubble
through their digestive tract and dissolve CaCO
3
as part of
their digestive process (Schneider et al. 2011). However, sea
cucumbers have recently come under increasingly strong
fishing pressure in many parts of the world (Toral-Granda
2008) due to rapidly rising consumer demand in East Asia
(Purcell et al. 2014a,b), and many populations have become
threatened or even locally extirpated (Friedman et al. 2011),
with extinction risks rising for more valuable species (Purcell
et al. 2014a,b). In Okinawa and the Ryukyu Archipelago sea
cucumber harvesting is a common practice (Fig. 1), but the
fishery has only recently (since 2013) become regulated.
Given the increase in demand and the ecological importance
of these ecosystem engineers, gaining a deeper understanding
of the population dynamics is needed to help inform man-
agement decisions. The study of genetic diversity and gene
flow is relevant to understanding the mechanisms by which
demographic exchange can overcome local extirpation. The
degree of interpopulation connectivity and the location of
genetic breaks shared among species in the community define
the spatial scale at which management is most effective in
ensuring population persistence (Toonen et al. 2011).
The edible sea cucumber, Holothuria edulis Lesson,
1830, is widely distributed in the Indo-Pacific region,
including the Ryukyu Archipelago, where it is known as
‘akamishikiri’. It inhabits a wide range of depths from 1 to
20 m and a broad variety of habitats, including coral reefs,
rocky reefs, and mudflats (Conand 2008; Purcell et al.
2009). As seen in many sea cucumbers, it reproduces both
sexually by pelagic spawning and asexually by fission, yet
the conditions that trigger each mode of reproduction are
not yet fully understood (Uthicke 1997). Holothuria edulis
is an edible species that is experiencing an increase in
demand following overexploitation of other, higher-value
sea cucumber species (Conand and Muthiga 2007; Choo
2008; Dissanayake and Stefansson 2010), as seen in Viet-
nam after the decline of Holothuria scabra (Choo 2008).
We have recently observed the harvesting of large quan-
tities of H. edulis (Fig. 1) around Okinawa Main Island,
where it is prepared and dried for food, medicine, or export
overseas. In the present study we have undertaken popu-
lation genetic analyses of the sea cucumber species H.
edulis to (i) investigate patterns of genetic diversity and
connectivity around Okinawa Main Island; and (ii) to infer
the demographic history of these populations. This is the
first investigation of population genetic structure of H.
edulis in Okinawa Main Island, and this study will con-
tribute useful information for the management of this
fishery in this region.
Methods
Sampling
Specimens of the sea cucumber H. edulis were collected by
snorkeling or SCUBA diving between April to October
Fig. 1 Observations of large quantities of dried sea cucumbers around Okinawa Main Island, (a,b) near Awase fishing port (taken by Taha
Soliman, 23 November 2014)
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2014 at six locations around Okinawa Main Island, Japan
(Table 1; Fig. 2), including four localities along the west
coast in the East China Sea [Motobu (HDM), Oyama
(HDO), Ryugu (HDR), and Sunabe (HDS)], and two on the
east coast facing the Philippine Sea [Uruma (HDU) and
Awase (HDW)]. We covered a large area for sampling at
each site (five people sampled H. edulis), and covered an
area of approximately 2000 m
2
to avoid any replicate
sampling. Samples of H. edulis were obtained non-lethally
through body wall-tissue biopsy and preserved in 99 %
ethanol in the field, and live animals were released back to
the location from where they were collected.
DNA extraction and PCR
Total genomic DNA was extracted from a small piece of
body wall tissue using a DNeasy Blood and Tissue
extraction kit (Qiagen, Tokyo, Japan) following the man-
ufacturer’s protocol. Polymerase chain reactions (PCR)
were used to amplify fragments of two mitochondrial DNA
markers; cytochrome oxidase subunit I (COI) and 16S
ribosomal RNA (16S), and one fragment of the nuclear
histone H3 (H3) gene, using the following primers: COIe-F
50-ATA ATG ATA GGA GGR TTT GG-30; COIe-R 50-
GCT CGT GTR TCT ACR TCC AT-30(Arndt et al. 1996),
16SA-R 50-CGC CTG TTT ATC AAA AAC AT-30; 16SB-
R5
0-GCC GGT CTG AAC TCA GAT CAC GT-30
(Palumbi et al. 1991), and H3a: 50-ATG GCT CGT ACC
AAG CAG ACV GC-30; H3b: 50-ATA TCC TTR GGC
ATR ATR GTG AC-30(Colgan et al. 1998), respectively.
PCR reactions were carried out in a 15 ll volume con-
taining 5–20 ng of template DNA, 0.5 lM of each primer,
and 10 ll of HotStarTaq
TM
Master Mix (Qiagen, Tokyo,
Japan), in deionized water. PCR reactions were performed
on Astec Thermocyclers (Astec Co., Ltd, Japan) with an
initial denaturation step at 95 °C during 15 min, 35 cycles
of denaturation at 94 °C for 1 min, annealing at 45 °C for
1 min, and extension at 72 °C for 1 min, and a final
extension step at 72 °C for 10 min. The amplification
products were purified with Exonuclease I and Alkaline
Phosphatase Shrimp (Takara) and incubated at 37 °C for
20 min, followed by deactivation at 83 °C for 30 min.
Purified PCR products were sequenced using an ABI Prism
automated sequencer at Fasmac Co., Kanagawa, Japan
(http://www.fasmac.co.jp/index.html), in both in forward
and reverse directions.
Table 1 Molecular diversity of populations of Holothuria edulis sampled across locations of the west and east coasts of Okinawa Main Island,
inferred from mtDNA and nDNA sequences
Location Populations (Code) Latitude and longitude N H Hd S pFu’s F
S
Fu’s F
S
(P-values)
COI
West coast Motobu (HDM) 26°40045.800 N 127°52055.400 E 19 5 0.65 6 0.0029 0.48 0.37
Oyama (HDO) 26°17001.200N 127°44020.500 E 30 6 0.77 8 0.0033 0.47 0.41
Ryugu (HDR) 26°31048.600N 127°55034.800 E 15 3 0.59 2 0.0015 0.11 0.39
Sunabe (HDS) 26°20026.200N 127°44039.000 E 24 5 0.74 7 0.0039 0.64 0.65
East coast Uruma (HDU) 26°19040.800N 127°56019.400 E 36 2 0.25 4 0.0022 3.50 0.93
Awase (HDW) 26°18027.100N 127°50010.300E 25 3 0.54 4 0.0034 2.72 0.92
16S rRNA
West coast Motobu (HDM) 26°40045.800 N 127°52055.400 E 21 6 0.65 5 0.0037 0.79 0.31
Oyama (HDO) 26°17001.200N 127°44020.500 E 22 6 0.68 5 0.0027 1.66 0.11
Ryugu (HDR) 26°31048.600N 127°55034.800 E 14 4 0.69 4 0.0041 0.85 0.73
Sunabe (HDS) 26°20026.200N 127°44039.000 E 20 4 0.66 2 0.0017 0.78 0.23
East coast Uruma (HDU) 26°19040.800N 127°56019.400 E 37 2 0.24 1 0.0006 0.48 0.37
Awase (HDW) 26°18027.100N 127°50010.300E 29 3 0.59 3 0.0029 2.24 0.88
H3 Na H
O
H
E
HWE
West coast Motobu (HDM) 26°40045.800 N 127°52055.400 E 24 7 0.12 0.35 0.0991 -1.75 0.18
Oyama (HDO) 26°17001.200N 127°44020.500 E 22 9 0.04 0.41 0.0004 0.32 0.68
Ryugu (HDR) 26°31048.600N 127°55034.800 E 14 9 0.02 0.29 0.0223 0.36 0.66
Sunabe (HDS) 26°20026.200N 127°44039.000 E 23 9 0.00 0.51 0.0000 5.71 1.00
East coast Uruma (HDU) 26°19040.800N 127°56019.400 E 40 10 0.02 0.16 0.1002 1.78 0.19
Awase (HDW) 26°18027.100N 127°50010.300E 23 10 0.03 0.41 0.1001 0.41 0.76
Nsample size, Hnumber of haplotypes, Hd haplotype diversity, Snumber of polymorphic sites, pnucleotide diversity, Na allele number, H
O
observed heterozygosity and H
E
Expected heterozygosity, HWE Hardy–Weinberg equilibrium
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Data analysis
Consensus sequences of COI, 16S, and H3 were aligned
and edited using Geneious v.8.1.3 (http://www.geneious.
com, Kearse et al. 2012), and MEGA v.6.0 (Tamura
et al. 2013). Numbers of haplotypes, haplotype diversity,
number of polymorphic sites and nucleotide diversity
were estimated with DNASP v.5.10.01 (Librado and
Rozas 2009). To investigate the demographic history of
H. edulis we conducted a neutrality test based on Fu’s
F
S
(Fu 1997) using Arlequin 3.5.1.2 (Excoffier and
Lischer 2010).
Fig. 2 a Map showing sampling sites of the sea cucumber Holothuria
edulis across Okinawa Island, Japan. Diagrams represent the distri-
bution of haplotypes (concatenated sequence of COI and 16S, as
defined by Fig. 4and Table 1), Chlorophyll a (b) and CDOM
(c) levels across Okinawa Island, Japan; averages from July 2002 to
January 2015. Chlorophyll a and CDOM data for 2002–2014, derived
from satellites, were downloaded from the National Aeronautic and
Space Administration Giovanni website (Acker and Leptoukh 2007),
developed and maintained by the NASA Goddard Earth Sciences
Data and Information Services Center. Yearly average chlorophyll a
and CDOM data and maps used in this study were derived from 9 and
4 km resolution data, respectively
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Allelic states of nuclear DNA (H3) sequences were
calculated using Phase 2.1 (Stephens and Donnelly 2003)
as implemented in DNASP v.5.10.01 (Librado and Rozas
2009). Three replicate phase runs were conducted using
100 burn-in steps and 1000 iterations. Phase results were
used to estimate observed heterozygosity (Ho), expected
heterozygosity (H
E
) and an exact test of Hardy–Weinberg
equilibrium (HWE) using 100,000 steps of Markov chain
in Arlequin 3.5.1.2 (Excoffier and Lischer 2010).
The best-fit model of DNA sequence evolution was
estimated using MEGA v.6.0 (Tamura et al. 2013). The
AIC (Akaike Information Criterion) indicated that Jukes-
Cantor (JC) for COI and 16S, and Tamura 3-parame-
ter ?Gamma (T92 ?G), with gamma value (G =0.05)
for H3 were the best-fit models. We computed pairwise U
ST
for all pairs of populations that incorporates a model of
sequence evolution (Weir and Cockerham 1984; Excoffier
et al. 1992). The significance of the genetic distances was
tested by permuting the haplotypes or individuals between
the populations (10,000 iterations) in Arlequin 3.5.1.2, and
simultaneous tests correction adjusted using the modified
false discovery rate (FDR) method (Narum 2006).
To test genetic partitioning among different populations
and population groups (shown in Table 3)ofH. edulis,we
tested the significance of the covariance components
associated with the different possible levels of genetic
structure (within individuals, within populations, within
groups of populations, among groups) using non-paramet-
ric permutation procedures as part of the AMOVA
framework implemented in Arlequin (Excoffier et al.
1992). In order to visualize the genetic relationships
between populations, GENALEX 6.5 (Peakall and Smouse
2012) was used to construct a principle coordinate analysis
(PCoA) of the pairwise U
ST
matrix. PopArt software
(http://popart.otago.ac.nz) was used for inferring and
visualizing genealogical relationships among populations
using median joining network approach (Bandelt et al.
1999). In order to estimate the isolation by distance, we
performed a Mantel test for each marker (COI, 16S and
H3). The significance of correlation between genetic dis-
tance (U
ST
, log) and geographic distance (log) were cal-
culated using IBDWS 3.23 (Jensen et al. 2005). The
geographic distances among sampling locations were
measured along Okinawa Main Island coast using Google
Maps (https://www.google.co.jp/maps).
Results
We sequenced a 545 bp fragment of the COI gene for 149
individuals and a 431 bp fragment of 16S for 143 indi-
viduals of H. edulis sampled across six locations of Oki-
nawa Main Island (Fig. 2; Table 1). Individual numbers
per population (N), number of haplotypes (H), haplotype
diversity (Hd), number of polymorphic sites (S), and
nucleotide diversity (p) for COI and 16S are listed in
Table 1. Overall, the numbers of haplotypes in COI and
16S were 8 and 9 respectively, and the corresponding
haplotype diversity was relatively similar in both COI
(Hd =0.84) and 16S (Hd =0.82). The average haplotype
diversity on the west coast (COI: Hd =0.69 and 16S:
Hd =0.67) was higher than that on the east coast (COI:
Hd =0.40 and 16S:Hd =0.21). We also sequenced a
327 bp of nDNA H3 for 146 specimens. Average values of
observed and expected heterozygosity in west coast pop-
ulations (Ho =0.05 and H
E
=0.39) were higher than that
in east coast population (Ho =0.03 and H
E
=0.29)
(Table 1). Non-significant Fu’s Fs tests provide no statis-
tical support for recent demographic changes in Okinawan
populations of H. edulis (Table 1).
The median-joining parsimony networks (Fig. 3a, b)
based on mtDNA haplotypes sampled across all Okinawan
populations of H. edulis revealed the presence of a mod-
erate number of mid-frequency haplotypes. Consistently,
the networks were not characterized by a starburst shape
(i.e. a few very common haplotypes and many singletons),
which is the most commonly observed pattern in popula-
tions of shallow coral reef organisms that has been attrib-
uted to population expansion following Pleistocene post-
glacial range expansion (Conroy and Cook 2000).
The networks also allow inferring trends of genetic
partitioning (Fig. 3). Seven COI haplotypes were sampled
in the east coast sites and four were detected in the west
(eight and four 16S haplotypes respectively). Although
most haplotypes were shared across sites, shifts in haplo-
type frequencies indicate a certain degree of genetic iso-
lation among sites (Fig. 2). The haplotype networks and
maps illustrate a decrease of genetic diversity and high
degree of isolation of the Uruma population (HDU) indi-
cated by the low number of shared haplotypes with other
sites. The parsimony networks constructed with H3 indi-
cated a majority of shared haplotypes with frequency shifts
among populations (Fig. 3c).
Pairwise comparisons among populations revealed a
high degree of genetic structuring of H. edulis across
Okinawa Island. Twelve of fifteen pairwise comparisons
had high and significant values of F
ST
(Appendix S1) and
U
ST
inferred from mitochondrial COI after correcting for
multiple comparisons (thirteen significant comparisons
inferred from 16S). Most comparisons between east and
west sites were significant (Table 2) and the degree of
genetic connectivity among sites at either side of the island
was also different. Whereas in the west, genetic cohesion
was indicated by lower pairwise U
ST
values and less sig-
nificant comparisons (Table 2), populations in the east
showed high and significant pairwise U
ST
values between
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Uruma and Awase (COI: U
ST
=0.75, P\0.0001; 16S:
U
ST
=0.49, P\0.0001; Table 2). Thus far, only these
sites on the eastern coast of the Island have been confirmed
to harbor H. edulis; during the course of this study we
searched many other locations and we did not find any
other H. edulis populations (T. Soliman, unpublished data).
The AMOVA indicated that overall population structure
was significant (COI: U
ST
=0.490, P\0.0001; 16S:
U
ST
=0.341, P\0.0001; H3: U
ST
=0.121, P\0.0001).
Although grouping populations into east and west coast sites
had no statistical support (COI: U
ST
=0.077, P\0.40;
16S: U
ST
=0.003, P=0.47; H3: U
ST
=–0.046, P=0.92;
Table 3), this was possibly driven by further genetic struc-
turing among sites within each coast as indicated by high and
significant U
SC
(COI: U
ST
=0.450, P\0.0001; 16S:
U
ST
=0.340, P\0.0001; H3: U
ST
=-0.157,
P\0.0001). However, additional subdivision into four
groups [(1) HDS ?HDO, (2) HDR ?HDM, (3) HDW, (4)
HDU] resulted in a marginally significant among groups
variance component (COI: U
CT
=0.517, P=0.02; 16S:
Fig. 3 Haplotype networks inferred from aCOI, b16S rRNA, and
cH3 sequences from Holothuria edulis across Okinawa Island, Japan.
Each circle represents a different haplotype, with size proportional to
its frequency. The colors represent the proportion of individuals from
each sampling site carrying a particular haplotype. Black circles are
unsampled haplotypes. HDM Motobu, HDO Oyama, HDR Ryugu,
HDS Sunabe, HDU Uruma, and HDW Awase
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U
CT
=0.241, P=0.11; H3: U
CT
=0.169, P=0.06;
Table 3). The Mantel test indicated significant correlations
between genetic distance and geographic distance among
populations for both COI (r=0.63, P=0.008) and 16S
(r=0.62, P=0.007), but this was not significant for H3
(r=–0.1688, P=0.67) (Fig. 4). Overall, the results of
mtDNA and nuclear DNA haplotype analyses of the sea
cucumber H. edulis showed a relatively clear geographical
pattern between east and west coast populations (Fig. 3).
The PCoA confirmed the genetic isolation of the Uruma
(HDU) based on mtDNA (COI and 16S), while H3 showed
the isolation of Uruma (HDU) and Sunabe (HDS) from the
other populations (Fig. 5). However, PCoA showed that
Motobou (HDM) and Oyama (HDO) were isolated from
other populations in the western coast of the Island.
Discussion
Our examination of mtDNA (COI and 16S) sequences
showed high levels of haplotype diversity and low levels of
nucleotide diversity among five populations of H. edulis
(HDM, HDO, HDS, HDR, HDW). Similar patterns have
previously been reported in other sea cucumber species
such as Holothuria mammata (Borrero-Pe
´rez et al. 2011);
H. polii (Vergara-Chen et al. 2010), H. arguinensis (Ro-
drigues et al. 2015), and H. atra and H. whitmaei (Skillings
et al. 2014). However, our study showed low haplotype
diversity and low nucleotide diversity in the Uruma (HDU)
population, and for nuclear DNA (H3) the HDU population
also had a low level of expected heterozygosity compared
to the other populations.
Our results indicate the genetic variation of H. edulis is
highly structured across Okinawa Main Island, and also
that there is a correlation between geographic distance and
genetic distance. Previously, Skillings et al. (2014)
observed a high degree of genetic structure over relatively
short geographic distances in H. atra in the Central Pacific,
and Vergara-Chen et al. (2010) observed genetic isolation
between sites inside and outside of a lagoon in Spain for H.
polii (but see Valente et al. 2014). Reduced gene flow in
sea cucumbers is counter-intuitive given the potential for
dispersal of the larval stages and the extended distribu-
tional ranges of some species (Skillings et al. 2014).
Although details of the larval biology of sea cucumbers are
largely unknown, the pelagic larval duration (PLD) has
been documented to be at least 20 days for some species
(Laxminarayana 2005), and sufficient for maintaining
species cohesion over the large geographic areas that H.
edulis,H. atra, and, to a lesser extent, H. polli are dis-
tributed in. In H. edulis, we observed a strong reduction of
gene flow over an unprecedentedly small geographic scale.
One possibility is that the biogeographic characteristics or
physical barriers of Okinawa Main Island promote such a
reduction of gene flow. White et al. (2015) recently
demonstrated that the amphipod species Leucothoe vul-
garis, which has limited larval dispersal, has different east
and west coast genotypes on Okinawa Main Island, and
speculated this was caused by Pleistocene sea level chan-
ges (Ni et al. 2014) and/or geographic discontinuity (Cas-
telin et al. 2012).
Another possibility to explain our observed results is
that the life history traits or reproduction patterns of H.
edulis favor low dispersal capability. Uthicke (1997)
revealed that *24 % of H. edulis undertake fission each
year and it has the same asexual reproduction pattern with
two other species of sea cucumber, H. atra and Stichopus
chloronotus. Uthicke’s estimate of annual fission rates
emphasizes that asexual reproduction is an important
method of population size maintenance for all holothurian
species on all reefs studied.
The most striking result of the present study was the
genetic break between the H. edulis population in Uruma
(HDU) and all other populations, which was supported by
genetic data from all three DNA markers examined.
Haplotype diversity was lower in the Uruma population
than in all the other populations, with low numbers of
haplotypes for the COI, 16S, and H3 markers, and with no
exclusive haplotypes located in this area. Strikingly, we
observed a high degree of genetic isolation between Uruma
and Awase (HDU and HDW), despite these sites being
very close to each other (*10 km apart).
Table 2 U
ST
pairwise values for Holothuria edulis across Okinawa
Main Island inferred from mtDNA and nDNA
Location HDM HDO HDR HDS HDU HDW
COI (above diagonal) and 16S (below diagonal)
HDM 0.14* -0.01 0.18* 0.64* 0.40*
HDO 0.37* 0.04 0.02 0.59* 0.35*
HDR 0.13 0.16 0.09 0.69* 0.38*
HDS 0.39* 0.01 0.07 0.61* 0.26*
HDU 0.27* 0.71* 0.47* 0.72* 0.75*
HDW 0.24* 0.11* 0.01 0.19* 0.49*
H3
HDM
HDO 0.17
HDR 0.41* 0.14
HDS 0.16* -0.23 0.23
HDU 0.50* 0.18* -0.11 -0.26
HDW -0.25 -0.68 0.56* 0.27 0.06
Sample sites are indicated in Table 1and Fig. 1.HDM Motobu, HDO
Oyama, HDR Ryugu, HDS Sunabe, HDU Uruma, and HDW Awase
* Significant values after correcting for multiple comparisons
(P\0.015)
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Table 3 Analysis of molecular variance (AMOVA) of Holothuria edulis for tests of different geographical groupings: A, B, C and D
Variation A B C D
Var. (%) Fixation indices Pvalue Var. (%) Fixation indices P value Var. (%) Fixation indices Pvalue Var. (%) Fixation indices Pvalue
COI
Among groups 7.73 U
CT
=0.077 0.40 53.37 U
CT
=0.533 0.06 53.04 U
CT
=0.530 0.17 51.67 U
CT
=0.517 0.02*
A. pop. within groups 41.48 U
SC
=0.449 \0.0001* 4.53 U
SC
=0.093 \0.0001* 11.12 U
SC
=0.237 \0.0001* 0.50 U
SC
=0.010 0.27
Within populations 50.78 U
ST
=0.492 \0.0001* 42.28 U
ST
=0.577 \0.0001* 35.84 U
ST
=0.642 \0.0001* 47.83 U
ST
=0.522 \0.0001*
16S rRNA
Among groups 0.27 U
CT
=0.003 0.47 8.75 U
CT
=0.088 0.33 21.25 U
CT
=0.213 0.15 24.09 U
CT
=0.241 0.11
A. pop. within groups 34.18 U
SC
=0.343 \0.0001* 27.25 U
SC
=0.299 \0.0001* 20.63 U
SC
=0.262 \0.0001* 11.78 U
SC
=0.155 0.03*
Within populations 65.55 U
ST
=0.344 \0.0001* 64.00 U
ST
=0.360 \0.0001* 58.12 U
ST
=0.419 \0.0001* 64.13 U
ST
=0.241 \0.0001*
H3
Among groups -4.58 U
CT
=-0.046 0.92 16.68 U
CT
=0.167 0.13 7.37 U
CT
=0.074 0.33 16.99 U
CT
=0.170 0.07
A. pop. within groups 16.42 U
SC
=-0.157 \0.0001* 7.02 U
SC
=0.084 \0.0001* 15.42 U
SC
=0.167 \0.0001* 4.21 U
SC
=0.051 0.04*
Within populations 88.16 U
ST
=0.118 \0.0001* 76.30 U
ST
=0.237 \0.0001* 77.21 U
ST
=0.228 \0.0001* 78.80 U
ST
=0.212 \0.0001*
U
CT
region variance component relative to total variance; U
SC
between population within region variance component divided by the sum of itself and within population variance; U
ST
sum of the
variance due to region and population within region divided by the total variance
AEast (g1) and West (g2), BWest (g1), BAwase (g2) and Uruma (g3), CWest & Awase (g1) and Uruma (g2) and D-g1(HDS&HDO), g2 (HDR&HDM), g3 HDW and g4 HDU
* Significant values (P\0.05)
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The observed reduction in genetic diversity and genetic
isolation at Uruma may be a consequence of a shift to
asexual reproduction. Previously, high rates of asexual
reproduction have been observed in Holothuria atra at
eutrified sites compared to pristine reefs (Conand 1996).
The Uruma population is located near the Kaichu-Doro
Causeway, the construction of which was recently shown
to have impacted much of the marine biota in the area,
although echinoderms were not examined in detail (Reimer
et al. 2015). As well, Kin Bay’s environment has much
higher chlorophyll a and CDOM levels than the sur-
rounding oligotrophic waters of Okinawa (Fig. 1), and
historical records indicate environmental degradation (red
tides, oil spills) occurring within Kin Bay from the 1970s
(Reimer et al. 2015). Such environmental differences and/
or degradation could at least partially explain the observed
differences in the Uruma population. However, much more
data are needed before such theories can be confirmed or
denied, and this is an area of research that should be
examined in more detail in future studies.
Fig. 4 MANTEL test for matrix correlation between log (genetic distance: U
ST
) and log (geographic distance) of COI, 16S and H3 among 6
populations of sea cucumber Holothuria edulis across Okinawa Island, Japan
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Friedman et al. (2011) noted that many of the Pacific’s
sea cucumber fisheries are under-performing due to over-
exploitation and ecological stress causing relatively large-
scale depletions of stock. Therefore, countries with rela-
tively less fisheries management have not been able to
preserve sea cucumber stocks to appropriate levels. Bio-
logical conservation of exploited species such as sea
cucumbers depends on local-level regulatory measures and
international organizations that regulate trade (Purcell et al.
2014a,b). Regardless of the causes of our observed results,
these results have important implications for the manage-
ment of H. edulis in Okinawa. The results highlight the
need to empirically investigate genetic connectivity, and
demonstrate the difficulty of making predictions for marine
resources without fine-scale data. The reduced gene flow
observed here at a reduced geographic scale (*10 km)
highlights the importance of local management of resour-
ces. Given the degree of genetic isolation, the Uruma
population may be demographically closed, implying the
risk of local extirpation is high. Therefore, studies on other
sea cucumber species in the Kaichu-Doro area and Oki-
nawa are needed to aid in the management this important
marine resource in Okinawa.
Acknowledgments The authors thank Dr. Franc¸ois Michonneau for
providing technical advice during the DNA extraction and T. Ohara,
O. Takama, K. Hamamoto, Dr. J. Montenegro, M. Mizuyama, and R.
Diaz for help with sample collection. T.S. was supported by Ministry
of Higher Education of the Egyptian Government during this study in
Japan. J.D.R. was funded by a Japan Society for the Promotion of
Science (JSPS) ‘Zuno-Junkan’ grant entitled ‘Studies on origin and
maintenance of marine biodiversity and systematic conservation
planning’. I.F.-S. was funded by a JSPS postdoctoral fellowship for
overseas researchers. We thank two anonymous reviewers for their
constructive comments, which improved the manuscript.
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