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ISSN: 1021-5506
Zoological Studies
An International Journal
Volume 51, Number 1
January, 2012
Published by Biodiversity Research Center
Academia Sinica, Taipei, Taiwan
Zoological Studies
CHIEF EDITOR
MANAGING EDITOR
LI, WEN-HSIUNG
LEE, SIN-CHE
Biodiversity Research Center,
Biodiversity Research Center,
ADVISORY BOARD
COLEMAN, DAVID C., USA
KNOWLTON, NANCY, USA
EDWARDS, JAMES, Denmark
,
O BRIEN, STEPHEN J., USA
WU, CHUNG-I, USA
EDITORIAL BOARD
AYALA, FRANCISCO J., USA
HWANG, PUNG-PUNG, Taiwan
TING, CHAU-TI, Taiwan
CHANG, CHING-FONG, Taiwan
LEE, LING-LING, Taiwan
TSO, I-MIN, Taiwan
CHANG, ERNEST S., USA
LOOF, ARNOLD DE, Belgium
WU, SHI-KUEI, USA
CHEN, CHAOLUN ALLEN, Taiwan
McCULLOUGH, DALE R., USA
XIA, XUHUA, Canada
CHIANG, TZEN-YUH, Taiwan
MOK, MICHAEL HIN-KIU, Taiwan
YEN, SHEN-HORN, Taiwan
DAI, CHANG-FENG, Taiwan
RANDALL, JOHN E., USA
YU, HON-TSEN, Taiwan
HUANG, RU-CHIH C., USA
SHAO, KWANG-TSAO, Taiwan
YU, SIMON S.J., USA
ASSISTANT EDITORS
CHEN, CHUN-CHIAO VANESSA, Biodiversity Research Center,
WU, CHIA-CHI KIKI, Biodiversity Research Center, Academia
Sinica, Taipei, Taiwan
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The publication of Zoological Studies, a bimonthly journal, is
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Pseudosiderastrea formosa sp. nov. (Cnidaria:
Anthozoa: Scleractinia) a New Coral Species
Endemic to Taiwan (photo by Y.Y. Chuang)
Zoological Studies 51(1): 1-11 (2012)
Prenatal Ethanol Exposure Increases Depressive-Like Behavior and
Central Estrogen Receptor α and Oxytocin Expressions in Adult Female
Mandarin Voles
Feng-Qin He1,*, Jie Zhang2, and Xiang Guo1
1
2
Institute of Brain and Behavioral Sciences, Department of Life Sciences, Xian Univ. of Arts and Science, Xian 710065, China
College of Life Sciences, Shaanxi Normal Univ., Xian 710062, China
(Accepted July 25, 2011)
Feng-Qin He, Jie Zhang, and Xiang Guo (2012) Prenatal ethanol exposure increases depressive-like behavior
and central estrogen receptor α and oxytocin expressions in adult female mandarin voles. Zoological Studies
51(1): 1-11. Prenatal exposure to ethanol is considered to increase risk of depression in offspring. Herein,
we tested the effect of prenatal ethanol exposure on adult female mandarin voles (Microtus mandarinus). We
verified depression-like behavior of female offspring exposed to ethanol prenatally (exposure group), and control
females (control group) during a forced swimming test. For the exposure group, ethanol doses (750 mg/kg body
weight in 0.9% saline, 10 ml/kg) were orally administered by clean drinking tubes to 5 pregnant females from
day 14 of their pregnancy until postnatal day 0. In the control group, 6 pregnant females received 0.9% saline (10
ml/kg) but no ethanol. At 90 d of age, the exposure and control groups were tested during a forced swimming,
and levels of serum estradiol (E2), estrogen receptor alpha-immunoreactive neurons (ERα-IRs) and oxytocinimmunoreactive neurons (OT-IRs) were measured. The exposure group exhibited significantly decreased
locomotion and increased immobility during the swim test. The level of serum E2 was higher in the exposure
group, and numbers of ERα-IRs in the bed nucleus of the stria terminalis (BNST), medial amygdaloid nucleus
(MeA), hypothalamic paraventricular nucleus (PVN), and supraoptic nucleus (SON) in the exposure group were
significantly lower. Numbers of OT-IRs in the hypothalamic PVN and SON of the exposure group were lower
than those of control animals. Our results suggest that prenatal ethanol exposure may lead to increases in
serum E2 levels and decreased ERα and OT in the central nervous system of adults and may be related to the
development of depression-like behaviors. http://zoolstud.sinica.edu.tw/Journals/51.1/1.pdf
Key words: Prenatal ethanol exposure, Forced swimming test, Estradiol, Estrogen receptor α, Oxytocin.
D
epression is a highly prevalent, chronic,
recurring, and potentially life-threatening mental
illness (Nestler et al. 2002, Berton and Nestler
2006), and prenatal ethanol exposure is associated
with an increased risk of depression in offspring
(Forrest et al. 1992, Larkby and Day 1997,
Mancinelli et al. 2007, Hellemans et al. 2010).
Both mouse and rat offspring, the mothers of which
had consumed moderate quantities of ethanol
throughout gestation, demonstrated dysregulation
of the hypothalamic-pituitary-adrenal (HPA) axis
which is common in depression and is primarily
reflected by increased HPA tone and activity
(Bale and Vale 2004, Hellemans et al. 2010).
Attention was recently focused on the role of
neurohypophyseal hormones such as oxytocin (OT)
on HPA axis activation in depression (Bao et al.
2008). In the central nervous system, the OT gene
is primarily expressed in magnocellular neurons in
the hypothalamic paraventricular nucleus (PVN)
and supraoptic nucleus (SON). Release of OT via
the neurohypophysis into the bloodstream and by
extrahypothalamic fibers projecting into the brain
exert a wide spectrum of central and peripheral
*To whom correspondence and reprint requests should be addressed. Tel: 86-29-88241902. Fax: 86-29-88253976.
E-mail:[email protected]
1
2
He et al. – Effect of Prenatal Ethanol in Adult Female Mandarin Voles
effects (Le Mevel et al. 1993). For example, in
addition to its regulatory role in social attachment,
OT was shown to have stress-protective effects
in animals, including humans (Heinrichs et al.
2003, Parker et al. 2005). One study showed that
peripheral OT levels are reduced in depression,
while another found no difference in OT plasma
levels between depressed and control patients
(van Londen et al. 1997, Slattery and Neumann
2010b). Although prenatal exposure to ethanol
(PAE) increases the HPA tone and results in HPA
dysregulation throughout life, paralleling many HPA
changes associated with depression (Hellemans
et al. 2010), neurobiological mechanisms of
depression need to be further researched.
Estrogen is a well-known regulator of mood,
and estradiol (E 2) treatment can result in both
depressant and antidepressant effects (Morrison
et al. 2006). The variable effects of estrogen on
the mood may be explained by opposing actions of
estrogen when mediated through estrogen receptor
alpha (ERα) or beta (ERβ). Research into this
area has yielded contrasting results. For example,
ERα was shown to play a role in the susceptibility
of females to major depressive disorders (Tsai
et al. 2003). However, ERα appears to be
unrelated to emotion in females (Malacara et al.
2004). Species differences in central (estrogen
receptor) ER expression may also contribute to
differences in the effects of E 2 on moods. For
example, ERα-positive cells are present in the
PVN of monogamous California mice, but not in
polygynous house mice, whereas ERα-positive
cells are present in the anterior hypothalamus
(AHA) of house mice but not in California mice
(Merchenthaler et al. 2004). Monogamous male
pine voles (Microtus pinetorum) express lower
levels of ERα-immunoreactive neurons (IRs) in the
medial amygdaloid nucleus (MeA) than polygynous
male montane (M. montanus) and meadow
voles (M. pennsylvanicus) (Cushing and WynneEdwards 2006). In addition, immunocytochemical
and in situ hybridization studies detected either low
levels or the absence of ERα-IRs in the PVN and
SON of rats (Hrabovszky et al. 1998).
Ethanol abuse during a woman’s pregnancy
produces a variety of deleterious effects in the
offspring (Hollstedt et al. 1977). However, the
mechanisms of those deleterious effects are
not entirely clear. Among the various different
animal models currently employed for screening
antidepressant compounds, the forced-swim test
(FST) is one of the most commonly used (Porsolt
et al. 1978, Wallace-Boone et al. 2008). Herein,
we utilized wild mandarin voles (M. mandarinus)
as animal models and explored the neurobiological
basis of depressive behavior. Mandarin voles are
ideal models for exploring the neurobiological basis
of various behaviors, which may provide abundant
neurobiological knowledge (Wang et al. 1997).
According to studies of mate preference and other
related characteristics, it was found that mandarin
voles have a monogamous mating system (Tai et
al. 2001, Guo et al. 2011). So far, the depressivelike behavior of the animal and relationships
among ERα, OT, and depressive-like behaviors
have not been reported. Some epidemiological
or experimental studies found that women have
a higher prevalence of mood disorders than men
(Hellemans et al. 2010), so we attempted to test
the hypothesis that prenatal exposure of female
pups to ethanol will affect the distribution of ERα
and OT in parts of the brain associated with
depressive-like behaviors.
MATERIALS AND METHODS
Subjects
Healthy adult female mandarin voles (n = 11,
weighing 30-36 g, 90 d old) were obtained from
an outbreed colony and reared in the Department
of Life Science, Xian Univ. of Arts and Science,
Xian, China. The colony of mandarin vole was
established in 1997 with wild-captured animals
(n = 300) from Lingbao City, Henan Province,
China. The animals were individually housed
in clear plastic cages (30 × 20 × 15 cm). The
voles were maintained under a 14: 10-h light:
dark photoperiod and at a temperature of 2426°C. Hardwood shavings and cotton batting were
provided as substrate and bedding. Rabbit chow
(Laboratory Animals Center, Xian Medical Univ.,
Xian, China), carrot, and malt were provided ad
libitum in this experiment. All methods for treating
voles were approved by the Institutional Animal
Care and Use Committee of Xian Univ. of Arts and
Science.
Female voles were brought into estrus with
estradiol benzoate (0.75 μg/g, 24 h before testing)
and progesterone (0.015 mg/g, 4-6 h before
testing), and the estrus state of the females was
monitored by taking vaginal smears (He et al.
2008). Each female in estrus was paired with an
adult male the bilateral or unilateral testes of which
had descended (n = 11, total = 11 pairs) until 2
ejaculations were observed (day 0 of pregnancy)
(Ward et al. 2002). These pregnant females were
allocated to either an ethanol or saline exposure
group. The gestation period in voles is only about
21 d long. In the ethanol exposure group, we
orally administered ethanol doses (750 mg/kg body
weight in 0.9% saline, 10 ml/kg, Sigma, St. Louis,
MO, USA) using clean drinking tubes to 5 pregnant
females from day 14 of the pregnancy until
postnatal day 0, so the animals were intubated
for 7 consecutive days. In the saline treatment
group, 6 pregnant females received 0.9% saline
(10 ml/kg) but no ethanol (Macenski and Shelton
2001, Erlwanger and Cooper 2008). The weight
of both ethanol- and saline-exposed pregnant
females 1 d before parturition was 55-65 g, the
number of live offspring per pair born was 1-5,
and the sex ratio was 1.87: 1 female: male (Tai
et al. 1999). Because the offspring were weaned
21 d after birth, all offspring were kept with their
mothers until postnatal day 21. Subsequently,
11 female offspring with similar weights (12-14 g)
were selected from the 11 pairs of vole individuals
and maintained in our laboratory under the same
conditions: 5 female offspring of 5 pregnant
females with ethanol exposure were the exposure
group, and 6 female offspring of 5 pregnant
females with saline treatment were the control
group. These female offspring were allowed to
grow up (weighing 30-36 g, 90 d old), and after the
behavioral testing, they were euthanized.
Forced swim test (FST)
From postnatal day 90, adult female offspring
were tested using the FST following Porsolt et
al. (1978), in which each animal was forced to
swim for 6 min in an open cylindrical container
(10 cm in diameter and 25 cm high) (Peng et al.
2007), containing 15 cm of water at 25 ± 1°C. The
water on an animal’s body was cleaned off with a
towel, and the body temperature was allowed to
return to normal next to a heater after each test.
The interval between each animal experiment
was 10 min. The investigator was blinded to the
treatment groups. The total duration of immobility
was scored following Zomkowski et al. (2004
2005). Each vole was judged to be immobile when
it ceased struggling and remained floating and
motionless in the water, making only movements
necessary to keep its head above water. A
decrease in the duration of immobility during the
FST was taken as a measure of antidepressant
activity.
3
Serum E2 quantification
Blood was taken from the retro-orbital
sinus at diestrus 2 d after each FST. All serum
samples were carefully collected and separated
from the blood by centrifugation, and serum
samples were stored at -80°C for 1 wk until being
assayed. Serum E 2 assays were performed
using commercial enzyme-linked immunosorbent
assay (ELISA) kits (R&D Systems, Minneapolis,
MN, USA). Serum samples were diluted 1: 20
to measure the optical density of E2 at 450 nm.
Enzyme-specific activities were determined using
a linear regression analysis (r > 0.95).
Tissue collection and ERα and OT immunocytochemistry
ERα and OT expressions were examined
1 h a f t e r b e h a v i o r a l t e s t i n g . Vo l e s w e r e
deeply anesthetized and perfused with a 0.1 M
phosphate-buffered solution (PBS, pH 7.4) and
4% paraformaldehyde in 0.1 M PBS. The brain
was removed within 3 min and placed in 4%
paraformaldehyde overnight. Prior to dissection,
brains were immersed in 30% sucrose until
saturated. Coronal sections (40 µm) were cut
on a cryostat, and consecutive sections were
collected in 2 vials containing 0.01 M PBS for 2
different immunohistochemical stainings. The
ERα antibody (sc-542; Santa Cruz Biotechnology,
Santa Cruz, CA, USA) is an affinity-purified rabbit
polyclonal antibody raised against a peptide
mapped at the C-terminus of ERα of mouse origin.
The OT antibody (AB911; Upstate Biotechnology,
Lake Placid, NY, USA) is also a rabbit polyclonal
antibody.
Floating sections were processed using the
primary antibody and a streptavidin/peroxidase
method (Bioss Co., Beijing, China). We incubated
1 vial per brain for 7 min with 3% H2O2 and then
washed them twice for 10 min each with distilled
water. We shrank the tissue in 0.01 M PBS.
Sections were preincubated for 90 min with normal
goat serum (SP-0023) and incubated at 4°C
overnight with the primary antibody solution (ERα
antibody 1: 100; OT antibody 1: 5000) diluted by
antibody diluent (0.01 M PBS containing 20%
bovine serum albumin and 1.7% Trition-X-100).
The next day, sections were washed 4 times
for 5 min each with 0.01 M PBS and incubated for
60 min in a 37°C water bath with a biotinylated
goat anti-rabbit antibody (SP-0023), followed
by another round of 4 washes for 5 min each
4
with 0.01 M PBS. After 60 min of incubation
with S-A/HRP and 4 washes for 10 min each
with 0.01 M PBS, sections were stained with
3, 3'-diaminobenzidine tetrahydrochloride to
visualize the immunoreactivity. Because ERα
was included in nuclei and OT was contained in
the cytoplasm, we counted stained nuclei and
cytoplasm using an Olympus microscope (Olympus
microscope is made in Tokyo, Japan). Slides
were randomized and coded for microscopic
analysis so that the counters were blinded to the
experimental treatment. Numbers of cells that
showed immunoreactivity were quantified by eye
per a standard area (200 × 200 μm) using a grid
sampling. We counted the number of ERα-IRs in
the anterior hypothalamus (AHA), bed nucleus of
the stria terminalis (BNST), lateral septum (LS),
medial amygdaloid nucleus (MeA), hypothalamic
paraventricular nucleus (PVN), and supraoptic
nucleus (SON); we selected these areas of the
brain because they are involved in emotion (Zhai et
al. 2008, Song et al. 2010). Different brain areas
were determined according to Nissl-stained brain
sections from mandarin voles and a stereotaxic
atlas of the rat brain.
For each brain nucleus, the criteria of 3
sections from anterior to posterior anatomically
matched between subjects were chosen and
counted to minimize variability. Individual means
for each animal were obtained by counting positive
neurons bilaterally in 3 sections from each nucleus.
Counts were separately performed for each
hemisphere, and results were averaged between
hemispheres. Numbers of OT-IR neurons were
quantified in 2 areas with a distinctive population of
OT-IR neurons: the PVN and SON. Sections were
chosen by their correspondence to the reference
control group
(A)
atlas plate and not by the level or intensity of ERαIR or OT-IR labeling. All immunohistochemical
procedures included negative controls (in which
the primary antibody was not added). For all
subjects, positive neurons were counted by a
trained experimenter blinded to the experimental
treatment. Sections were photographed with a
Nikon (Tokyo, Japan) camera attached to a Nikon
microscope.
Statistical analyses
All data were checked for normality using
a one-sample Kolmogorov-Smirnov test. The
majority of data were normally distributed and
analyzed using independent-sample t-tests. Data
are presented as the mean ± standard error (SE)
and alpha was set to p < 0.05. All statistical
analyses were conducted using SPSS 10.0 (SPSS,
Chicago, IL, USA).
RESULTS
The exposure group spent less time engaged
in locomotion (duration: F(1,9) = 13.1, p = 0.031;
frequency: F (1,9) = 5.015, p = 0.034) and more
time immobile (duration: F(1,9) = 13.204, p = 0.037;
frequency: F (1,9) = 6.620, p = 0.021) during the
FST than the control group (Fig. 1). The exposure
group had significantly higher serum E2 levels than
the control group (F(1, 9) = 6.667, p = 0.021) (Fig. 2).
We found a significant effect of ethanol on
the number of ERα-IRs in different brain regions
of adults. Numbers of ERα-IRs in the BNST
(F (1,9) = 2.295, p = 0.045), MeA (F (1,9) = 0.345,
p = 0.036), PVN (F(1,9) = 15.345, p = 0.001), and
ethanol exposing group
(B)
250
7
*
*
100
5
4
3
*
2
50
0
*
6
Frequency
Duration (s)
200
150
8
1
Locomotion
Immobility
0
Locomotion
Immobility
Fig. 1. Locomotion and immobility in the forced swimming test. (A) Durations of locomotion and immobility. (B) Frequencies of
locomotion and immobility. *Indicates a significant difference, p < 0.05. Values are the mean (± standard error).
SON (F(1,9) = 14.447, p = 0.002) of the exposure
group were significant lower than those of the
control group (Figs. 3, 4, 6), and those in the AHA
(F(1,9) = 0.271, p = 0.377) and LS (F(1,9) = 3.992,
p = 0.642) of the exposure group were slightly
lower than those of the control group (Figs. 3, 4).
We found reduced numbers of OT-IRs in the PVN
(F(1,9 ) = 2.379, p = 0.041) and SON (F(1,9) = 0.357,
p = 0.026) of the exposure group compared to the
control group (Figs. 5, 6).
DISCUSSION
Prenatal ethanol exposure increases depressive behavior in adult female offspring
Ethanol exposure resulted in a reduction in
locomotion time and an increase in immobility
during the FST for adult females, the mothers of
which were administered ethanol orally. Therefore,
prenatal ethanol exposure leads to increased
depressive-like behavior in adult offspring, and
results suggest that ethanol exposure during
brain growth spurts has long-term effects on
behavior, neurochemistry, and neuroendocrinology.
These findings confirm that alcohol increases
depressant-like behaviors via neurochemical
and neuroendocrine mechanisms. Although the
etiology of depressive disorders is at present
unknown, prenatal ethanol oral administration
during pregnancy can be considered in the
context of early life adversity or environments.
Programming of the fetal HPA axis by ethanol can
50
45
result in long-term alterations in the physiological
and behavioral profiles of offspring (Hellemans et
al. 2010), and we confirm results of much literature
that HPA programming ultimately confers increased
susceptibility to developing depression disorders if
stressors are encountered later in life (Macri et al.
2007), e.g., during the FST as employed herein.
Decreased expression of ERα-IRs may increase
depressive-like behavior
Our experiment is the 1st to show a positive
association between prenatal ethanol treatment
and serum estrogen levels. Previous studies
showed an increase in estrogenic levels after
ethanol treatment in women (Muti et al. 1998,
Juarez et al. 2002), men (Couwenbergs 1988),
and male rats (Esquifino et al. 1989). However,
why prenatal ethanol treatment increases serum
estrogen levels in adult female offspring is not yet
completely understood; it may due to increased
aromatization of E2 from testosterone, so ethanol
increases serum estrogen levels (Hilakivi-Clarke et
al. 1997).
Numbers of ERα-IRs in the BNST, MeA,
PVN, and SON significantly declined in females
exposed to ethanol in the present results, and the
BNST, MeA, PVN, and SON were demonstrated
to be involved in depression (Swaab et al. 2005,
Lee et al. 2009). Research from our own lab also
showed that these brain areas are correlated
with animal emotions (Zhai et al. 2008, Song et
al. 2010) and concurs with other studies which
showed that ERα is related to mood including
depression in females (Osterlund et al. 1999, Tsai
et al. 2003). In the present experiment, serum
*
90
Mean of expressing ERα-IRs
E2 concentration (ng/dl)
40
35
30
25
20
15
10
5
0
5
Control group
Ethanol exposing group
Fig. 2. Mean (± standard error) level of circulating serum
estradiol (E2) concentrations (ng/dl) according to an ELISA.
*Indicates a significant difference, p < 0.05.
control group
80
70
60
50
40
30
20
10
0
*
*
*
AHA
BNST
LS
MeA
PVN
*
SON
Fig. 3. Mean (± standard error) number of estrogen receptor
(ER)α immunoreactive cells following prenatal exposure to
ethanol in female voles. *Indicates a significant difference,
p < 0.05.
6
(A)
(B)
AHA
AHA
(C)
(D)
BNST
BNST
(E)
(F)
LS
LS
(G)
(H)
MeA
MeA
Fig. 4. Estrogen receptor (ER)α immunoreactivity in Microtus mandarinus females exposed to ethanol prenatally and a control group.
Anterior hypothalamus (AHA) of the control group (A) and ethanol-exposed group (B); bed nucleus of the stria terminalis (BNST) of the
control group (C) and ethanol-exposed group (D); lateral septum (LS) of the control group (E) and ethanol-exposed group (F); medial
amygdaloid nucleus (MeA) of the control group (G) and ethanol-exposed group (H). Scale bar = 200 µm.
E 2 levels of adult female offspring exposed to
prenatal ethanol treatment were higher than those
exposed to saline treatment, but numbers of ERαIRs in certain brain regions declined. E2 caused
the downregulation of ERα, which is consistent
with previous findings (Oliveira et al. 2004).
Species differences in central ER expression may
also contribute to differences in the effects of E2
on moods. For example, ERα-positive cells are
present in the PVN of monogamous California
mice, but not in polygynous house mice, whereas
ERα-positive cells are present in the AHA of house
mice but not in California mice (Merchenthaler
et al. 2004). Monogamous male pine voles (M.
pinetorum) express lower levels of ERα-IRs in
the MeA than polygynous male montane (M.
montanus) and meadow voles (M. pennsylvanicus)
(Cushing and Wynne-Edwards 2006). In addition,
immunocytochemical and in situ hybridization
studies detected either low levels or the absence of
ERα-IRs in the PVN and SON of rats (Hrabovszky
et al. 1998). Consequently, the varying effects of
ethanol on depression may result from different
properties of central ERα expression in different
species.
ERα is thought to modulate the activity of
corticotropin-releasing factor (CRF) neurons in
depression, and CRF expression is increased
in depression (Wang et al. 2008). Reduced
numbers of ERα-IRs in these brain regions raise
the possibility that a disturbed balance in the
production of the CRF may contribute to activation
of the HPA axis in depression. In contrast to
18
control group
Mean of expressing OT-IRs
16
14
12
10
*
8
*
6
4
2
0
PVN
SON
Fig. 5. Mean (± standard error) number of oxytocin (OT)immunoreactive cells following experimental treatment in adult
female mandarin voles. *Indicates a significant difference,
p < 0.05.
7
our study, another study found a significantly
increased expression of the ERα gene in the PVN
of depressed patients (Wang et al. 2008). This
discrepancy between studies may have been the
result of differences between expressions of the
ERα gene and protein between species.
Decreased expression of OT-IRs may lead to
increase depressive-like behavior
Numbers of OT-IRs in the ethanol-exposed
group were significantly lower in the PVN and
SON. Reduced numbers of OT-IRs in certain brain
regions may be one of the causes of depressivelike behavior. OT injected intraperitoneally
decreased immobility in rats undergoing the FST,
and this effect was even stronger following longterm treatment with OT (Arletti and Bertolini 1987).
An intracerebroventricular injection of OT did not
affect the behaviors of male and female rats during
an FST, even after a chronic treatment regimen
that did not affect depression behavior (Slattery
and Neumann 2010a). OT administered centrally
or systemically decreased the immobility time in
mice during an FST, but this effect was not blocked
by a non-peptide OTR antagonist (Ring et al.
2010). It is possible that results from one species
cannot be generalized to others (Insel et al. 1993).
Herein, prenatal ethanol exposure increased
serum estrogen levels, and OT significantly
decreased in the PVN and SON; but in rats, OT
binding in the brain showed a positive correlation
with serum estrogen (Larcher et al. 1995, Breton
and Zingg 1997). Again, these effects may be
species-specific (Insel et al. 1993).
Although in the present experiment, numbers
of cells that showed immunoreactivity were
quantified per a standard area (200 × 200 μm), a
limitation of this study is that the densities of ERα
and OT were measured and not total cell numbers,
and co-localization of ERα with OT occurred in the
hypothalamic PVN and SON in both the ethanolexposed and control groups. The central roles
of OT in behaviors and physiology are strongly
dependent on steroid hormones, and estrogen can
upregulate the production of OT messenger (m)
RNA (Caldwell et al. 1989) and the release of OT
(Johnson 1992). A membrane-bound receptor for
estrogen may regulate OT expression within the
PVN and SON (Sakamoto et al. 2007). Estrogeninduced OT binding in the brain is abolished in
ERα knockout mice (Young et al. 1998). Additional
evidence for OT regulation of E2 sensitivity comes
from studies of human breast cancer cell lines in
8
(B)
PVN
(A)
PVN
(D)
(C)
PVN
PVN
(F)
(E)
SON
SON
(H)
(G)
SON
SON
Fig. 6. Estrogen receptor (ER)α and oxytocin (OT) immunoreactivity in Microtus mandarinus females exposed to ethanol prenatally
and the control group. ERα-immunoreactive neurons (IRs) in the paraventricular hypothalamic nucleus (PVN) of the control group
(A) and ethanol-exposed group (B); OT-IRs in the paraventricular hypothalamic nucleus (PVN) of the control group (C) and ethanolexposed group (D); ERα-IRs in the supraoptic nucleus (SON) of the control group (E) and ethanol-exposed group (F); OT-IRs in the
supraoptic nucleus (SON) of the control group (G) and ethanol-exposed group (H). Scale bar = 200 µm.
which OT downregulates both ERα mRNA and
ERα protein expressions (Cassoni et al. 2002). It
is becoming increasingly clear that neuropeptides
interact with steroids to regulate moods (Cushing
et al. 2005). The fact that manipulations of
ethanol early in life alter ERα-IRs in adulthood
suggests that ethanol might not simply regulate
sensitivity to E 2 through activational effects,
but that organizational effects of OT produce
lasting changes in sensitivity to steroids. Our
data provide an additional mechanism by which
neuropeptides might act to alter or regulate sensitivity to steroids. This in turn has the potential
to alter depressive-like behaviors mediated by OT
and ERα. However, in the female rat, the PVN
contains only a negligible quantity of ERα, and
the estrogen-dependent regulation of oxytocin
synthesis in the PVN is mediated by ERβ (Patisaul
et al. 2003). Therefore, regulating the responses
to OT may be critical to ‘proper’ or species-specific
expression (Wang and Vries 1993, Winslow et al.
1993, Williams et al. 1994), and understanding the
relationship between the hormonal and peptide
regulation of depressive-like behavior and central
ERα and OT expression might differ in various
mammalian species (Wang et al. 1997).
Depression concerns hyperactivity of the
HPA axis, and this axis is driven by CRF release
by neurons located in the PVN, which causes
adrenocorticotropic hormone (ACTH) release at
the level of the pituitary (Meynen et al. 2007).
Because OT significantly inhibits the potentiating
effect of CRF-stimulated ACTH release (Suh et
al. 1986), OT seems to be able to stimulate and
inhibit activity within the HPA axis with short- and
long-term perspectives (Yegen 2010). In our
experiment, numbers of OT-IRs in the ethanolexposed group were significantly lower in the PVN
and SON, and reduced numbers of OT-IRs might
cause the release of CRF and ACTH, causing
hyperactivity of the HPA axis. Therefore, it is easy
to cause depressive-like behavior in these adult
female offspring prenatally exposed to ethanol.
Acknowledgments: We thank Prof. Benjamin
Bravery for valuable comments on the manuscript.
We also thank Prof. F.D. Tai of the College of
Life Science, Shaanxi Normal Univ., Xian, for
providing Mandarin voles from the experimental
outbreed colony. This research was supported
by the National Natural Science Foundation of
China (grant nos. 30970370 and 30670273) and
the Fundamental Research Fund for Central
Universities (GK200901011).
9
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Algal Symbionts Increase DNA Damage in Coral Planulae Exposed to
Sunlight
Badrun Nesa1, Andrew H Baird2, Saki Harii3, Irina Yakovleva4, and Michio Hidaka1,*
Department of Chemistry, Biology and Marine Science, Univ. of the Ryukyus, Okinawa 903-0213, Japan
ARC Centre of Excellence for Coral Reef Studies, James Cook Univ., Townsville, Queensland 4811, Australia
3
Sesoko Station, Tropical Biosphere Research Center, Univ. of the Ryukyus, Okinawa 905-0227, Japan
4
A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia
1
2
(Accepted August 24, 2011)
Badrun Nesa, Andrew H Baird, Saki Harii, Irina Yakovleva, and Michio Hidaka (2012) Algal symbionts
increase DNA damage in coral planulae exposed to sunlight. Zoological Studies 51(1): 12-17. To test the
hypothesis that algal symbionts make coral larvae more susceptible to high photosynthetically active radiation
(PAR) and ultraviolet radiation (UVR), symbiotic and non-symbiotic planulae of Acropora tenuis were exposed
to natural sunlight (high PAR and UVR) at an ambient temperature of approximately 27°C for 4 d. DNA damage
to host cells was detected using a comet assay (single-cell gel electrophoresis). Coral cells from symbiotic
planulae had longer comet tails than those from non-symbiotic planulae, indicating that cells in symbiotic larvae
had more DNA damage than those in non-symbiotic larvae. This result suggests that symbiotic algae are a
source of oxidative stress in larvae under conditions at the ocean surface.
http://zoolstud.sinica.edu.tw/Journals/51.1/12.pdf
Key words: Bleaching, Comet assay, Coral, Stress, Symbiosis, UVR.
R
eef-building corals contain endosymbiotic
dinoflagellates (zooxanthellae) within their
gastrodermal cells. Various types of stress
can cause corals to lose symbionts leading to
coral bleaching, which if prolonged can cause
the death of a colony (Brown 1997). While sea
surface temperature anomalies appear to be the
main cause of mass bleaching episodes, field
and laboratory studies have revealed that solar
radiation also plays an important role in coral
bleaching, either alone or in synergy with high
temperatures (see review by Baird et al. 2009a).
Planulae of broadcast-spawning corals have
an obligate planktonic duration of 2-4 d before
they are competent to settle (Fadlallah 1983),
and planulae of species with large eggs require
up to 48 h before they become motile (Babcock
and Heyward 1986). In addition, coral planulae
are positively buoyant due to the high proportion
of lipids in their tissues (Harii et al. 2007).
Consequently, some coral propagules may be
exposed to high solar radiation at the surface of the
ocean for at least a day following spawning.
Previous work demonstrated that planulae are
adversely affected by high temperatures (Edmunds
et al. 2001 2005). In particular, algal symbionts in
larvae exposed to high temperatures appear to be
a source of oxidative stress, inducing higher levels
of antioxidant activity and causing lipid breakdown
and increased mortality (Yakovleva et al. 2009).
Similarly, ultraviolet (UV) radiation (UVR) can have
a deleterious effect on coral larvae by reducing
survivorship (Gleason and Wellington 1995) and
rates of recruitment (Gleason et al. 2006). It is
possible that algal symbionts make coral larvae
more susceptible to high photosynthetically active
*To whom correspondence and reprint requests should be addressed. Tel: 81-98-895-8547. Fax: 81-98-895-8576.
12
Nesa et al. – DNA Damage in Coral Planulae
radiation (PAR) and UVR. Herein, we tested the
hypothesis that algal symbionts increase DNA
damage in planula larvae under conditions that
prevail on the ocean surface (high PAR and UVR)
using a single-cell gel electrophoresis assay known
as a comet assay.
Collection and maintenance of coral larvae
Colonies of Acropora tenuis were collected
from a reef at Ishigaki I., Okinawa, and transferred
to the Sesoko Station of the Tropical Biosphere
Research Center, Univ. of the Ryukyus, Japan
prior to spawning in May 2008. The corals were
maintained in running seawater prior to spawning.
Four colonies spawned on 25 May 2008. After
spawning, egg and sperm bundles from the 4
colonies were cultured following Babcock et
al. (2003) and kept in 2-L plastic containers in
0.22-μm-filtered seawater (FSW) at densities of
1 larva/ml. The water was replaced daily.
Inoculation of larvae with symbionts
Symbiodinium cells for use in inoculating
larvae were isolated from parent A. tenuis colonies.
Coral tissue was removed from the skeleton using
a WaterPik (Teledyne, WP-70J, Fort Collins, CO,
USA). Tissue suspensions were filtered through a
nylon mesh (180 μm) and then concentrated using
a centrifuge at 2400 rpm for 4 min. The algal
pellets were resuspended in FSW, filtered (40 μm),
and then rinsed twice with FSW using a centrifuge
at 1800-2000 rpm for 4 min. Densities of algal
cells were estimated using a hemocytometer.
To infect larvae of A. tenuis with the homologous symbionts, 3000 larvae (8 d old) were
transferred to each of 3 plastic containers
containing 400 ml of 0.2-μm-FSW with symbionts
at densities of 4.8 × 104 cells/ml and homogenized
Artemia sp. After 4 h, 1 L of FSW was added
to each of the containers to dilute the symbiont
concentration, and larvae were kept in this
condition overnight. The next day, larvae were
washed with FSW and placed in 400 ml of 0.2-μmFSW. Non-symbiotic larvae were treated similarly
but without inoculation. Both symbiotic and nonsymbiotic larvae were maintained in the laboratory
for 1 wk at room temperature (25-27°C). Water
was changed on days when larvae were sampled
to estimate the density of symbionts. Twelve to
13
17 larvae were sampled on days 1, 3, 5, and 7,
and the number of larvae with symbionts and the
number of symbionts per larvae were enumerated
under a fluorescence microscope (Nikon,
Microphot-FXA, Tokyo, Japan). The average
proportion of larvae infected over this period
(1-7 d after inoculation) was 96% ± 2.0% (mean
± standard error, n = 4), and the average number
of symbionts per larva on day 7 was 56.3 ± 10.3
(n = 11).
Exposure to solar radiation
Symbiotic and non-symbiotic larvae of A.
tenuis were exposed to the sun for 4 d on 9-12
June 2008. Six 2-L plastic containers each with
1 L of FSW were prepared. The containers were
square with a bottom area of 170 cm 2 and a
water depth of approximately 5.5 cm. Symbiotic
larvae were placed in each of 3 containers with
non-symbiotic larvae in each of the remaining 3
containers. The containers were held in an openair outdoor aquarium with running seawater to
maintain temperatures close to those on the ocean
surface (26-28°C). Daily changes in PAR (λ = 400700 nm), UV A radiation (UVAR, λ = 315-400 nm)
and UV B radiation (UVBR, λ = 280-315 nm) were
monitored in air every daylight hour throughout
the experiment using a light meter (LI-250A, LICOR, Lincoln, NE, USA) and a UV meter (UV203
Ultraviolet Radiometer, Macam Photometrics Ltd,
Livingston, Scotland). Mean (± standard error; SE)
daily unweighted doses of UVAR, UVBR, and PAR
during the 4-d experimental period were 374.1
± 124.7, 43.9 ± 14.6, and 28.8 ± 11.3 mol/m2/d,
respectively. The biologically effective UVBR
weighted by the DNA-damage action spectrum
(Setlow 1974) varied 0.41-0.98 kJ/m2/d. Mean
(± SE) daily temperatures in the aquaria ranged
from 26.44 ± 0.35°C on day 4 to 27.06 ± 1.82°C on
day 2.
Comet assay
A comet assay kit (Trevigen, Gaithersburg,
MD, USA) was used to detect possible DNA
damage in coral and symbiont cells. At the end
of the experimental period, two planulae from
each of the 3 replicate containers from each
treatment were placed into 1 ml of ice-cold 0.01 M
phosphate-buffered saline (PBS) containing
20 mM ethylenediaminetetraacetic acid (EDTA),
and cells were dissociated by pipetting for 2 min.
Dissociated cells were collected by centrifugation
14
(6000 rpm, 1 min) and mixed with 300 μl of
0.01 M PBS containing 20 mM EDTA. The cell
suspension (at 50 μl) was mixed with 500 μl of
low-melting-point agarose. This mixture (at 75 μl)
was applied to a comet slide and spread using a
pipette tip. Slides were placed in a refrigerator at
4°C for 20 min. Slides were then immersed in lysis
solution containing 1% sodium lauryl sarcosinate
for 30 min at 4°C. Slides were immersed in an
alkaline (pH 13) solution containing 200 mM
EDTA at room temperature for 20 min. Slides
were then placed on a horizontal electrophoresis
apparatus. TBE electrophoresis buffer was added
to the electrophoresis tray to cover the slides.
Electrophoresis was conducted at 20-22 V and
0.01 mA for 10 min at room temperature. After
that, slides were immersed in 70% ethanol for
5 min. DNA staining was performed by adding
50 μl diluted SYBR Green I to each circle of slides.
Samples were visualized and photographed
with a fluorescence microscope (OPTIPHOT-2,
Nikon, Tokyo, Japan) at 500x using a digital
camera (Nikon Digital Sight DS-LI). Tail lengths
of comets derived from coral cells were measured
using Image J (vers. 1.40) software (Research
Service Branch, National Institute of Health, USA).
The comet tail length is a good estimate of DNA
damage in cells (Lee and Steinert 2003). Numbers
of replicates were 3 and 2 for the aposymbiotic
and symbiotic planulae, respectively. At least 50
comets were measured in each of the replicate
assays.
DISCUSSION
The present results clearly show that
symbionts increased DNA damage in host cells
when exposed to environmental conditions that
prevail at the ocean surface. Rinkevich et al.
(2005) also demonstrated that isolated coral cells
containing algal symbionts suffer greater DNA
damage than coral cells without symbionts or algal
cells under UVR. Similarly, recent studies using
aggregates of dissociated coral cells also showed
that symbionts increase DNA damage and shorten
the survival time of coral cell aggregates under
thermal stress (Nesa and Hidaka 2009a b).
Levels of the DNA-weighted UVBR dose
(0.41-0.98 kJ/m 2/d) seen in the present study
can inhibit growth of some phytoplankton,
indicating UV-induced DNA damage (Buma et al.
1996 2000). It is likely that UVR is responsible
for directly damaging DNA and also causing
indirect damage to host tissues by increasing the
production of reactive oxygen species (ROS) and
oxidative stress (Lesser et al. 1990, Downs et al.
2002). DNA damage in symbiotic planulae might
be due to the production of ROS in coral cells with
algal symbionts (Yakovleva et al. 2009). Symbiotic
(A)
(B)
RESULTS
The comet assay revealed remarkable
differences in host cell nuclei between symbiotic
and non-symbiotic larvae. After 4 d of exposure
to solar radiation, host cell nuclei of non-symbiotic
planulae produced comets with no or only a short
tail. In contrast, host nuclei of symbiotic planulae
formed comets with long tails (Figs. 1, 2). The
average comet tail length was 15-times longer for
symbiotic planula samples than for non-symbiotic
ones (Fig. 3).
Comets derived from coral cells could be
distinguished from those derived from algal
symbionts. The symbionts on the comet slide
possessed intact cell walls in most cases and
did not show a typical comet shape (Fig. 4). We
could not detect DNA damage of algal symbionts
in planulae exposed to solar radiation for 4 d using
the present comet assay method.
Fig. 1. Fluorescence photomicrographs of nuclei of coral
cells from the comet assay of non-symbiotic (A) and symbiotic
planulae (B) of Acropora tenuis after 4 d of exposure to solar
radiation. Scale bar = 10 μm.
cnidarians generally have large quantities of O2
within their host tissues due to algal photosynthesis
(Dykens and Shick 1982), and UVR enhances
photosynthetically generated hyperoxia to produce
ROS in host tissues (Dykens et al. 1992). ROS
produced during algal photosynthesis diffuse to the
host cytosol (Downs et al. 2002, Tchernov et al.
2004).
1
In the present study, 8-d-old planulae were
used for inoculation of symbiotic algae and
were allowed to establish a stable symbiosis for
1 wk until being used in the stress experiment.
Thus the symbiotic planulae were 15 d old
when exposed to natural sunlight. The point in
coral life history at which the next generation
becomes infected by symbionts remains an open
question. Early reports suggested that infection
(B)
(A)
0.4
0.8
0.3
0.6
Frequency
Frequency
15
0.4
0.2
0.1
0.2
0
0
0
2 0
4 0
6 0
8 0
100
120
0
2 0
4 0
6 0
8 0
100
120
Comet tail length (µm)
Fig. 2. Histograms of comet tail lengths of coral cells in non-symbiotic (A) and symbiotic (B) planulae of Acropora tenuis exposed to
solar radiation for 4 d. Data from 3 (non-symbiotic) and 2 (symbiotic) replicated assays were separately pooled. Numbers of comets
measured were 150 and 100 for the non-symbiotic and symbiotic conditions, respectively.
(A)
80
(B)
(2)
60
40
20
(3)
0
Non-symbiotic
Symbiotic
Fig. 3. Comet tail length of coral cells of symbiotic and nonsymbiotic Acropora tenuis larvae exposed to solar radiation for
4 d. Mean ± S.D. The number in the parenthesis is the number
of replicated assays.
Fig. 4. Photomicrographs of zooxanthella cells on comet slides
of an Acropora tenuis larva exposed to solar radiation for 4 d.
(A) Fluorescence microscopic and (B) light microscopic images.
Scale bar = 10 μm.
16
occurred following metamorphosis (Babcock and
Heyward 1986); however, more-recent research
demonstrated that larvae can acquire symbionts.
For example, larvae of A. tenuis kept in 100-μmFSW contained zooxanthellae 37 days after
gamete release (Nishikawa et al. 2003). Similarly,
most planulae of A. monticulosa cultured in the
presence of sediment acquired zooxanthellae 9 d
after spawning (Adams et al. 2009). Inoculation
experiments with homologous symbionts showed
that A. digitifera and A. tenuis larvae first acquire
zooxanthellae 6 and 5 d, respectively, after
fertilization, and the number of zooxanthellae per
planula increased thereafter (Harii et al. 2009).
Therefore, our experimental conditions are not
unrealistic, and the infection of planulae with
symbionts is not in itself harmful.
The exact timing of acquisition of symbionts
by Acropora planulae in the field will have to
wait for further research, and it is not known
whether larvae regularly acquire zooxanthellae
during the planktonic phase. However, it is
likely that Acropora planulae can disperse
from the parent colony before they take up
symbionts. This strategy might render Acropora
corals a geologically widespread, dominant
reef builder. On the other hand, some corals
produce zooxanthellate eggs, and most brooder
corals release planulae containing zooxanthellae
(Harrison and Wallace 1990, Baird et al. 2009b).
How corals with a vertical mode of symbiont
transmission avoid UV damage or tolerate ROS
production during development and the planktonic
phase at the ocean surface is a challenging subject
for future research. Since concentrations of UVprotective mycosporine-like amino acids (MAAs)
are positively correlated with survival of planula
larvae (Gleason and Wellington 1995, Wellington
and Fitt 2003), MAAs might play an important role
in the survival of zooxanthellate planulae during
the planktonic phase.
The present experimental approach using
symbiotic and non-symbiotic Acropora planulae
demonstrated that symbiotic algae can be a burden
to coral planulae during the obligate planktonic
phase of propagules of many broadcast-spawning
species. We hypothesize that this may explain the
high proportion of broadcast spawning species that
lack symbionts in the eggs.
Acknowledgments: This work was supported
by a Grant-in-Aid for Scientific Research on
Innovative Areas “Coral reef science for symbiosis
and coexistence of human and ecosystem under
combined stresses” (no. 20121002) and a Grantin-Aid for Young Scientists (B) no. 20770017 (SH)
from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) and the 21st
century COE Program of the Univ. of the Ryukyus,
Japan. AHB thanks the Japanese Society for
the Promotion of Science and the Australian
Academy of Science for funding for a short-term
visit to Japan. We are grateful to T. Hayashibara,
Fisheries Research Agency, Ishigaki, Japan for
field assistance. The authors also thank the
staff of the Sesoko Station, Tropical Biosphere
Research Center, Univ. of the Ryukyus, Japan
where part of this study was conducted.
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Distributions of Testate Amoebae and Ciliates in Different Types of
Peatlands and Their Contributions to the Nutrient Supply
Tomasz Mieczan*
Department of Hydrobiology, Univ. of Life Sciences, Dobrzańskiego 37, Lublin 20-262, Poland
Tomasz Mieczan (2012) Distributions of testate amoebae and ciliates in different types of peatlands and their
contributions to the nutrient supply. Zoological Studies 51(1): 18-26. The influence of plant communities on the
structure, abundance, and biomass of testate amoebae and ciliates were investigated in bog and fens in eastern
Poland. Samples were collected in belts of Sphagnum, Phragmites, Carex, Utricularia, and Calliergonella.
Sampling was done on a monthly basis from Apr. to Nov. 2009. Comparisons of species numbers, abundances,
and biomass levels of testate amoebae and ciliates between Sphagnum mosses did not show statistically
significant differences. In carbonate fens, the average species numbers, abundances, and biomass levels
of testate amoebae and ciliates for Sphagnum, Calliergonella, and Utricularia were higher than those for
Phragmites and Carex. Based on differences in plant stem structure, 2 groups of habitats were distinguished.
The 1st group consisted of 2 vegetated zones with a sparse stem structure (Phragmites and Carex), while the
2nd group consisted of plant species with a decidedly more-complicated structure (Sphagnum, Calliergonella,
and Utricularia). The results demonstrated that water table depth, pH, and concentrations of total phosphorus
and total organic carbon strongly regulated the taxonomic composition and abundances of protozoa. Rates
of excretion of ammonia-nitrogen and phosphate-phosphorus proportionally decreased with an increase in
body weight. In experiments dominated by small protozoa, excreted amounts were significantly higher than in
experiments dominated by higher taxa. Average net excretion rates per protozoon of nitrogen ranged 1.0 × 10-5
- 3.72 × 10-5 µg/h and of phosphorus ranged 6.5 × 10-6 - 1.2 × 10-5 µg/h.
Key words: Wetlands, Protozoa, Nitrogen, Phosphorus, Excretion
P
eatlands are generally characterized
by rich biodiversity and also play key roles in
preserving the stability of ecological relationships
in particular regions (Flessa et al. 1998). At the
same time, they belong to the fastest disappearing
and most endangered ecosystems in Europe.
This is especially disquieting in combination with
progressive climate warming (Flessa et al. 1998,
Robson et al. 2005, Watters and Stanley 2006).
Although ecological research on carbon dynamics,
and plant and animal communities (e.g., copepods,
nematodes, and insect larvae) of peatlands is well
known (Walsh 1995, Wardle 2006, Watters and
Stanley 2006), in contrast, in the whole of Europe
and worldwide, very little is known about the
microorganisms and their roles in the functioning of
these ecosystems. Testate amoebae and ciliates
are good indicators of a variety of environmental
variables including the hydrology, pH, and nutrient
status (Mitchell et al. 2000 2004, Gilbert and
Mitchell 2006, Nguyen-Viet et al. 2007, Mieczan
2007 2009a b). Studies by many authors (Mitchell
et al. 2000, Mazei et al. 2007, Mieczan 2009a b)
reported significant relationships between numbers
of protozoan species and microhabitat types. In
hollows of raised-bogs, it was noted that there is a
decidedly higher species diversity and abundance
of protozoa, compared to hummocks. However,
*To whom correspondence and reprint requests should be addressed. Tel: 48-81-4610061 ext. 306. Fax: 48-81-4610061 ext. 304.
18
Mieczan – Testate Amoebae and Ciliates in Wetlands
research on the occurrence of protozoa (particularly
ciliates) in carbonate fens is lacking. Until recently,
only a few studies described the ecology of testate
amoebae in fens (Payne and Mitchell 2007, Jassey
et al. 2010, Lamentowicz et al. 2010, Payne
2011). On the other hand, studies concerning
ciliates in raised, ombrotrophic bogs suggest
an obvious qualitative and quantitative diversity
among individual plant species (Mieczan 2009a).
Thus, it seems that a similar differentiation should
be expected in the case of protozoa occurring in
others types of peatland microhabitats connected
with patches of different plant species
Wilkinson (2008) suggested that testate
amoebae, even if only a minor fraction of the total
microbial biomass, could be responsible for a
large proportion of nutrient recycling in peatland
communities. One role of protozoa is utilization of
organic particles and the regeneration of soluble
inorganic matter. A major portion of nutrients is
excreted within short time intervals due to rapid
growth rates of testate amoebae and ciliates
compared to larger zooplankton (Dolon 1997).
The role played by ciliates in removing nutrients is
relatively thoroughly studied in lake ecosystems
(Ejsmont-Karabin et al. 2004). However, research
on excretion of nitrogen and phosphorus by
testate amoebae and ciliates in peatlands is
lacking. Research by many authors showed that
nitrogen is a factor limiting production in peatbog
ecosystems (Watters and Stanley 2006, Kooijman
and Paulissen 2006). The fact that protozoan
distributions in peatland ecosystems seem to be
of an extraordinarily mosaic character, in terms
of both abundances and species structures,
suggests that a similar mosaic may be expected
in the case of nutrient dynamics. Since protozoa
may reach extremely high abundances in
peatbog ecosystems, they may have a significant
role in nutrient dynamics. Still, the question of
19
excretion rates by protozoa in peatlands remains
unanswered. Summing up, the current study was
designed to test the hypothesis that protozoan
communities in peatland ecosystems play major
roles in nutrient cycling, deficiencies of which can
be clearly observed, particularly in ombrotrophic
peatlands.
The present study had 3 aims: 1) to describe
testate amoeba and ciliate diversity; 2) to examine
relationships between environmental variables and
protozoa; and 3) to experimentally determine rates
of N and P excretion by protozoa in relation to their
body weights, the ambient temperature, and pH.
Study site
The study area comprised 3 peatlands: a
raised bog at Durne Bagno, a poor fen at JelinoKrugłe Bagno, and a rich carbonate fen at Bagno
Bubnów (Polesie National Park, eastern Poland,
51°N, 23°E). The peatlands selected for this
study represent various vegetation types. In
the raised bog and poor fen, the vegetation
is dominated by Eriophorum vaginatum (L.),
Carex acutiformis Ehrhart., Car. gracilis Curt.,
Sphagnum angustifolium (C.C.O. Jensen ex
Russow), S. cuspidatum Ehrh. ex Hoffm., and S.
magellanicum Bird. The carbonate fen is colonized
by Phragmites australis (Car.), Car. acutiformis
Ehrhart, Calliergonella cuspidata (Hedw.), and
Utricularia sp. (Table 1).
Field sampling and chemical analyses
Fieldwork was conducted monthly from Apr.
to Nov. 2009. Sampling sites were chosen to
achieve the highest diversity of microhabitats.
Table 1. Main characteristics of the peatland sites sampled in this study
Peatland
Durne Bagno
Krugłe Bagno/Jelino
Bagno Bubnów
Location
Area (ha)
Type of peatland
51°22.344'N, 23°12.303'E
213.2
Raised bog
51°24.099'N, 23°9.116'E
19.7
Poor fen
2308.6
Rich fen
51°22.364'N, 23°15.303'E
Dominant plants
Eriophorum vaginatum, Carex acutiformis,
Sphagnum angustifolium, S. cuspidatum, S.
magellanicum, S. palustre
S. magellanicum, S. angustifolium, Car.
acutiformis
Phragmites australis, Car. acutiformis,
Calliergonella cuspidata, Car. davalliana,
Comarum palustre, Utricularia sp.
20
The total dataset consisted of 168 samples from 6
sites. During each sampling occasion, 3 samples
were collected from each microhabitat. In the
raised bog and poor fen, microbial communities
were examined among different Sphagnum
species (SA, S. angustifolium; SC, S. cuspidatum;
and SM, S. magellanicum Bird) (with 72 total
samples). In the carbonate fen, testate amoebae
and ciliates were collected in belts of P. australis
(PH), Car. acutiformis (CR), Utricularia sp. (UT),
and Cal. cuspidata (CA) (with 96 total samples).
In each type of microhabitat, water was sampled
using a Plexiglas corer (1.0 m long, with an inside
diameter of 50 mm). Four subsamples, of about
0.5 L each, were pooled into a calibrated vessel
to form a composite sample (2 L), which was
concentrated using a 10-µm plankton net. The
1st sample was analyzed live. One liter of water
was immediately preserved with Lugols solution
(at a final concentration of 0.2%), allowed to settle
in a glass column for over 24 h in the laboratory,
and then concentrated to 30 ml. Finally, 0.1 ml
of the concentrated sample was counted using a
microscope at 400-1000x magnification. Abundances of testate amoebae and ciliates were
determined using the Utermöhl method (Utermöhl
1958). Morphological identification of testate
amoebae and ciliates was mainly based on works
by Foissner and Berger (1996), Charman et al.
(2000), and Clarke (2003). Biovolumes of testate
amoebae and ciliates were estimated by assuming
geometric shapes and converting to carbon using
the following conversion factor: 1 µm 3 = 1.1 ×
10-7 µg C (Gilbert et al. 1998).
In each plot, temperature, conductivity, pH,
dissolved oxygen (DO), total phosphorus (P tot),
total nitrogen (N tot ), and total organic carbon
(TOC) were measured. Physical and chemical
analyses were performed according to standard
methods for hydrochemical analyses (Golterman
1969). Temperature, conductivity, pH, and DO
were assessed at the sites with a multiparametric
probe (Hanna Instruments, Woonsocket, USA);
TOC was analyzed by a multiparametric UV
analyzer (Secomam, Ales Cedex, France); P tot
by a colorimetric method; and Ntot by the Kjeldahl
method.
Laboratory experiments
As a result of preliminary research carried
out in 2008, very high numbers of microorganisms
were found in peatbogs and fens, which made it
possible to immediately use them for laboratory
experiments, without needing to cultivate them
in order to acquire sufficient numbers. Surface
water samples were taken from individual
microenvironments in spring and summer 2009.
In an effort to define the rate of nutrient excretion
by protozoa, in the laboratory, microorganisms
were washed with deionized water and condensed
by filtration (through a 4-µm mesh size). Next,
protozoan (~6000) individuals were transferred to
a watch glass containing 100 ml of deionized water
(experiment no. 1), and then the watch glass was
filled with peatbog water previously filtered through
a 0.2-µm filter (experiment no. 2). Concentrations
o f N H 4+ a n d P O 43- w e r e a n a l y z e d u s i n g a
spectrophotometric method (APHA 1985) before
removing the microorganisms and again 5 h after
their removal. After 5 h, to determine the excretion
rate by protozoa, the water was filtered through a
4-µm-mesh filter, and the number of protozoa was
again counted with an inverted microscope. The
experiment was carried out at 3 pH values (of 4,
5, and 7) which had been observed in peatbog
ecosystems, and at a medium temperature of 1418°C noted during sample collection. Biovolumes
of microorganisms were estimated by multiplying
the numerical abundances by mean cell volume
measurements using appropriate geometric
formulae (Sherr et al. 1983). The experiment was
repeated twice during the vegetative season: in
spring when groups of protozoa were dominated
by small forms (< 60 µm), and in late summer
when larger species dominated (> 100 µm). Three
replicates were used for each pH level. Rates of
excretion were calculated as differences in P and
N concentrations between the samples containing
protozoa and the control. Protozoa were not fed
during these experiments, and nutrient excretion
rates of starved protozoa are ~30% lower than
those for fed ones (Taylor 1986, Dolon 1997).
Data analyses
The significance of differences between mean
density and biomass values of testate amoebae
and ciliates was verified by an analysis of variance
(ANOVA). Ordination methods were used to
examine the general structure of the protozoan
data and test links between the protozoa and
environmental data. A detrended correspondence
analysis (DCA), an unconstrained indirect method,
was used to measure and illustrate gradients
indicated by the protozoa. Because the length of
the gradient was > 2 standard deviations (SDs),
a canonical correspondence analysis (CCA),
a method which assumes unimodal speciesenvironmental relationships (Ter Braak 19881992), was used. A diversity analysis (i.e., the
Shannon-Wiener diversity index) was performed
using the Multivariate Statistical Package (MVSP
2002). Similarities of protozoa communities
among the peatlands were compared using the
Euclidean distance measure.
RESULTS
Environmental variables
The water table depth (DWT) was highly
variable among sites and samples, ranging 2055 cm (ANOVA, F = 26.5, p = 0.001). Statistically
s i g n i f i c a n t d i ff e r e n c e s a m o n g t h e s t u d i e d
peatlands were found in pH, conductivity, P tot,
Ntot, and TOC (ANOVA, F = 30.21-31.22, p = 0.001).
Among the studied peatlands, the highest average
pH value (pH 7.6) was noted in the rich fen with
the lowest in the bog and poor fen (pH 3.2-4.5).
TOC concentrations were highest in the bog and
poor fen; however the remaining parameters
(conductivity, P tot and N tot) were highest in the
carbonate rich fen. In the bog and poor fen,
chemical properties of the water were similar
between micro-sites (p > 0.05). In the rich fen,
chemical properties of the water significantly
differed between micro-habitats (ANOVA, F = 29.4,
p = 0.0012). The highest conductivity and
concentrations of Ptot, Ntot, and TOC were noted in
belts of Utricularia and Calliergonella (Table 2).
21
Protozoan diversity and density: general results
In total, 29 testate amoeba and 19 ciliate taxa
were identified. The highest numbers of testate
amoeba and ciliate taxa occurred in the bog and
poor fen (with respective totals of 23 and 15 taxa).
A lower number of taxa (16) was observed in
the rich fen. A comparison of species numbers,
abundances and biomass levels of testate
amoebae and ciliates among Sphagnum mosses
did not show significant differences (p = 0.560).
These differences were significant for microhabitats in the carbonate fen (ANOVA, F = 31.4,
p = 0.001). The highest species numbers (11-16)
were found in belts of Utricularia and Calliergonella,
and the lowest richness levels (6-9) were observed
in micro-habitats dominated by Typha, Phragmites,
and Carex. Samples were moderately diverse
with Shannon diversity ‘H’ values ranging from
3.2 in Sphagnum to 2.1 in Typha stands. In
the studied peatlands, numbers and biomass
levels of protozoa significantly differed among
the studied stands, with the lowest numbers in
Phragmites and Carex micro-habitats and the
highest numbers in Utricularia and Calliergonella.
In general, compositions of ciliates were similar
among Sphagnum mosses, Calliergonella, and
Utricularia. However testate amoeba communities
from the rich fen differed from the others (Figs. 1,
2). The most abundant testate amoeba taxa in the
mosses were Assulina muscorum and Euglypha
tuberculata type, and the most abundant ciliate
taxon was Chilodonella uncitata. In the carbonate,
rich fen, 2 groups of habitat were generally favored
by testate amoebae and ciliates. Plant beds
Table 2. Physical and chemical characteristics of water in the investigated peatlands (average values for
the period Apr.-Nov. 2009 ± standard deviation). SA, Sphagnum angustifolium; SC, S. cuspidatum; SM,
S. magellanicum; PH, Phragmites australis; CR, Carex acutiformis; UT, Utricularia sp.; CA, Calliergonella
cuspidata; DWT, depth to water table; TN, total nitrogen; TP, total phosphorus; TOC, total organic carbon
Microhabitat DWT (cm)
pH
Raised bog
SA
SC
SM
17 ± 5
15 ± 7
19 ± 6
3.3 ± 1
3.2 ± 1
3.6 ± 1.5
Poor fen
SA
SC
SM
9±4
11 ± 6
5±2
Rich fen
PH
CR
UT
CA
49 ± 3
41 ± 3
20 ± 5
22 ± 6
Dissolved oxygen (mg/L) Conductivity (µS/cm)
TN (mg/L)
TP (mg/L)
TOC (mg/L)
8.3 ± 3.3
10.1 ± 3.1
9.2 ± 3.1
29 ± 6.4
27 ± 8.2
32 ± 8.7
1.121 ± 0.02
1.13 ± 0.23
1.332 ± 0.25
0.222 ± 0.11
0.239 ± 0.11
0.251 ± 0.03
54 ± 11
56 ± 8
59 ± 7
4.5 ± 1
5.2 ± 2
4.6 ± 2
7.9 ± 3.3
8.9 ± 2.1
9.2 ± 2.1
48 ± 5.3
45 ± 4.5
48 ± 6.9
1.263 ± 0.06
1.53 ± 0.23
1.531 ± 0.28
0.241 ± 0.111
0.269 ± 0.06
0.275 ± 0.06
53 ± 13
49 ± 11
46 ± 15
7.9 ± 1
8.2 ± 1
7.2 ± 1
7.1 ± 1.5
8.3 ± 2.1
8.5 ± 2.3
6.9 ± 1.8
6.7 ± 1.8
321 ± 17
311 ± 23
421 ± 25
399 ± 31
2.111 ± 0.78
2.112 ± 0.98
1.563 ± 0.96
1.468 ± 0.48
0.311 ± 0.12
0.290 ± 0.16
0.368 ± 0.18
0.378 ± 0.13
15 ± 2
14 ± 4
23 ± 4
21 ± 6
The DCA showed that the species composition of protozoa clearly differed between the bog
and fen (Fig. 3). The 1st 2 DCA axes explained
20% of the total protozoan variability. Results
for all sites showed that axis 1 was significantly
correlated with DWT, Ptot, and TOC, whereas axis
2 was correlated with pH (p < 0.05). Sites were
(A) Testate amoebae
3.5
biomass
3
2.5
2
1.5
1
0.5
0
SA SC SM
Raised bog
SA SC SM
Poor fen
PH CR UT CA
Rich fen
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
(B) Ciliates
60
50
ind./ml
(A) Testate amoebae
RB-SA
RB-SC
RB-SM
PF-SC
PF-SA
PF-SM
RF-PH
RF-CR
RF-UT
RF-CA
density
40
30
20
10
0
0
20
40
60
80
100
120
140
µgC/ml
Correlations among testate amoebae, ciliates,
and environmental variables
separated into 2 main types of habitats: mossesSphagnum and vascular plants. In the canonical
correspondence analysis, all variables (DWT,
pH, and P tot and TOC concentrations) together
explained 40% of the variance (p < 0.001). The
CCA revealed that the proportion of testate
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
µgC/ml
with a “simple” structure (Phragmites and Carex)
were distinctly predominated by testate amoebae
(Hyalosphenia elegans) and ciliates (Strombidium
viride). Testate amoebae (Arcella discoides, Arc.
vulgaris, Centropyxis aculeata, and Cen. aerophila)
and small ciliates (of the Scuticociliatida) showed
significant connections with beds possessing
a decidedly complex structure (Utricularia and
Calliergonella).
ind × 102 ml
22
SA SC SM
Raised bog
SA SC SM
Poor fen
PH CR UT CA
Rich fen
Fig. 2. (A, B) Average (Apr.-Nov. 2009) density and biomass
of testate amoebae and ciliates within plant patches in the
investigated peatlands. SA, Sphagnum angustifolium; SC, S.
cuspidatum; SM, S. magellanicum; PH, Phragmites australis;
CR, Carex acutiformis; UT, Utricularia sp.; CA, Calliergonella
cuspidata.
(B) Ciliates
3.0
RB-SA
RF-UT
Rich fen
RF-CA
Poor fen
Raised bog
PF-SA
RB-SM
PF-SM
RB-SC
PF-SC
RF-PH
RF-CR
0
10
20
30
40
50
Fig. 1. (A, B) Similarity between testate amoeba and ciliate
communities in the investigated peatlands. RB, raised bog;
PF, poor fen; RF, rich fen; SA, Sphagnum angustifolium; SC, S.
cuspidatum; SM, S. magellanicum; PH, Phragmites australis;
CR, Carex acutiformis; UT, Utricularia sp.; CA, Calliergonella
cuspidata.
-0.9
Testate Amoebae
-1.0
Ciliates
4.0
Fig. 3. Detrended correspondence analyses (DCAs) of
protozoan samples (log-transformed data).
amoeba and ciliate data explained by each
explanatory variable and its significance strongly
varied among variables and between the bog
and fen. Microsites without Sphagnum were
usually characterized by a low water level, a low
pH, and a higher concentration of TOC. Moreabundant taxa in these habitats included Ass.
muscorum, Eug. tuberculata type, Nebela tincta,
Corythion-Trinema type, Chi. uncinata, Colpidium
colpoda, and Paramecium bursaria. The 2nd
group included species that were associated
23
with a higher water level and high pH (Archerella
flavum, Arc. wrightianum, Hyalosphenia elegans,
Neb. carinata, Cinetochilum margaritaceum,
and Codonella cratera). The 3rd group included
species associated with a high water level and
pH conditions and a higher concentration of Ptot
(Arc. vulgaris, Arc. discoides, Cen. aculeata,
Cen. aerophila, Colpoda steinii, Disematostoma
tetraedricum, Holosticha pullaster, Strombidium
viride, and the Stylonychia mytilus-complex) (Fig.
4).
(A) Testate amoebae
Axis 2
1.0
Cen pl.
pH
Plac spin.
Cen ac.
Ptot
Hya ele.
Hel pet
Neb boh.
Dif le.
Eug com.
Hya ov.
DWT
Arc cat.
Hel sph. Eug st.
Neb sp.
Neb car.
Arc sp.
Hya
pap.
Neb
col.
Arc dis.
Neb gris.
Cor-typ
Neb mil.
Arch fl. Dif le. Hya sub.
Eug cil.
Cor dub.
Dif gl.
Arc vul. Arc sp. Neb par.
Dif
sp.
-1.5
Cry ov.
Amph wr
Eug rot.
Ass sem.
Dif el.
Neb tin. Ass musc.
Trig arc.
Eug sp. Eug tub.
1.5
-1.0
Axis 1
(B) Ciliates
1.0
Aspid.
Loxodes
pH
Ptot
Stromb.
Eupl.
Leptoph. Halt.
Vortic.
TOC
P putr.
Col hirt. P burs.
C stein.
Cod.
Col spet.
Cinet.
Styl.
DWT
Uronema.
Holosticha. Chilod.
Axis 2
C Cuc. Paradil.
Kahl.
-1.5
Disemat.
1. 5
-1.0
Axis 1
Fig. 4. Biplots of the canonical correspondence analysis (CCA) of testate amoeba and ciliate data from investigated peatlands
with representation of environmental variables. Species data were log-transformed, and rare species were down-weighted. DWT,
depth of water table; Ptot, total phosphorus; pH, water reaction; TOC, total organic carbon. Testate amoebae: Amph wr., Amphitrema
wrightianum; Arc cat., Arcella catinus type; Arc dis., Arcella disoides type; Arc vul., Arcella vulgaris; Arc sp., Arcella sp.; Arch fl.,
Archerella flavum; Ass musc., Assulina muscorum; Ass sem., Assulina seminulum; Cen ac., Centropyxis aculeata type; Cen pl.,
Centropyxis platystoma type; Cor dub., Corythion dubium; Cor-typ, Corythion-Trinema type; Cry ov., Cryptodifflugia oviformis; Dif el.,
Difflugia elegans; Dif gl., Difflugia globulosa; Dif le., Difflugia leidyi; Dif sp., Difflugia sp.; Eug cil., Euglypha ciliata; Eug com., Euglypha
compressa; Eug rot., Euglypha rotunda type; Eug st., Euglypha strigosa; Eug tub., Euglypha tuberculata type; Eug sp., Euglypha sp.;
Hel sph., Heleoptera sphagnii; Hel pet., Heleoptera petricola; Hya ele., Hyalosphenia elegans; Hya ov., Hyalosphenia ovalis; Hya pap.,
Hyalosphenia papilio; Hya sub., Hyalosphenia subflava; Neb boh., Nebela bohemica; Neb car., Nebela carinata; Neb col., Nebela
collaris; Neb gris., Nebela griseola type; Neb mil., Nebela militaris; Neb tin., Nebela tincta; Neb sp., Nebela sp.; Plac spin., Placocista
spinosa type; Trig arc., Trigonopyxis arcula. Ciliates: Aspid., Aspidisca sp.; Chilod., Chilodonella uncinata, Cinet., Cinetochilum
margaritaceum, Cod., Codonella cratera; Col. hirt., Coleps hirtus; Col. spet., Coleps spetai; C. cuc., Colpoda cucullus; C. stein.,
Colpoda steinii; Disemat., Disematostoma tetraedricum; Eupl., Euplotes sp.; Halt., Halteria grandinella; Holosticha, Holosticha pullaster;
Kahl., Kahlilembus attenuotus; Leptoph., Leptopharynx costatus; Loxodes, Loxodes sp.; Oxytr., Oxytricha sp.; Paradil., Paradileptus
elephantinus; P. burs., Paramecium bursaria; P. putr., Paramecium putrinum; Stromb., Strombidium viride; Styl., Stylonychia mytiluscomplex; Uronema, Uronema sp.; Vortic., Vorticella companula.
24
Phosphorus and nitrogen excretion by
protozoa
DISCUSSION
The protozoa excreted measurable amounts
of ammonia-nitrogen (N-NH 4), and phosphatephosphorus (P-PO4), and there were no effects of
pH on excretion rates of ammonia or phosphate.
Concentrations of N-NH4 and P-PO4 were
significantly higher after a 5-h exposure, the same
as the control (ANOVA, F = 21.2, p = 0.0011).
Some significant additional data proving the
existence of dependence between individual size
classes of protozoa and the amount of excretion
were also ascertained. In experiments in which
small protozoa were dominant, amounts excreted
were significantly higher (ANOVA, F = 22.0,
p = 0.0012). Rates of excretion decreased proportionally to an increase in body weight. It was
also noted that in deionized water and prefiltered
peatbog water, amounts excreted were similar and
showed no statistically significant difference (Tables
3, 4).
Community structure in relation to environmental parameters
Numbers of identified taxa of testate
amoebae and ciliates were comparable to
other studies examining peatlands (Payne and
Mitchell 2007, Mieczan 2009a b, Jassey et al.
2010). In the present study, water levels, pH,
and TOC, were deciding factors constraining
communities of protozoa in peatlands. This
compares well to other studies (Tolonen et al.
1994, Velho et al. 2003, Payne and Mitchell 2007,
Mieczan 2009a b). There was also a significant
influence of total phosphorus on the occurrence
of protozoa. In previous research on testate
amoebae in relation to the chemical environment,
many of the significant explanatory variables
were nutrients (Mitchell 2004). Moreover, it was
demonstrated that the occurrence of testate
amoebae in minerotrophic fens in Greece was
significantly influenced by hydrological factors
(Payne and Mitchell 2007). The autecology of
Table 3. Excretion rates (µg protozoa × h) of ammonia and phosphate in laboratory experiments (average
value ± S.D.)
Protozoa/ size
Dry weight × cell
(µg)
Filtered peatbog water
< 60 µm
15,221
> 100 µm
40,150
Deionized water
< 60 µm
> 100 µm
Excretion rates
Excretion rate
(µg N-NH4 × cell × h) (µg P-PO4 × cell × h)
Control
Protozoa (after 5 h)
N-NH4 (mg/L) P-PO4 (mg/L)
N-NH4 (mg/L) P-PO4 (mg/L)
1.211 ± 0.211 0.265 ± 0.026
2.061 ± 0.238 0.455 ± 0.112
2.8 × 10-5
6.5 × 10-6
1.200 ± 0.217 0.260 ± 0.056
1.600 ± 0.212 0.650 ± 0.026
-5
1.3 × 10
1.3 × 10-5
16,221
0
0
0.720 ± 0.243 0.111 ± 0.043
3.72 × 10-5
2.4 × 10-6
43,200
0
0
0.300 ± 0.111 0.360 ± 0.111
1.0 × 10-5
1.2 × 10-5
Table 4. Relationships (Pearson’s correlations coefficients) of the rate of excretion with individual body
weights of protozoa, pH, and temperature
Experiments
Filtered peatbog water
Body weight
pH
Temperature
Deionized water
Body weight
pH
Temperature
ns, not significant.
r
p
r
p
-0.58
ns
0.43
0.01
ns
0.05
-0.62
ns
0.41
0.01
ns
0.05
-0.63
ns
0.41
0.01
ns
0.05
-0.64
ns
0.38
0.01
ns
0.05
many species living in investigated peatlands
corresponds well to published data (Mitchell et
al. 2000, Opravilová and Hájek 2006, Jassey et
al. 2010). In the wettest microhabitats with lowpH species such as Arcella vulgaris, Archerella
flavum, Arch. wrightianum, Hyalosphenia elegans,
Neb. carinata, Cinetochilum margaritaceum, and
Codonella cratera were present, and in the driest
ones are species such as Assulina muscorum, the
Corythion-Trinema type, the Euglypha tuberculata
type, Neb. tincta, Chilodonella uncinata, Colpidium
colpoda, and Paramecium sp. The genus
Centropyxis is often reported as characteristic
of high-pH habitats, i.e., calcium-rich fens. The
results are in keeping with the recognized moisture
preferences of these species. Opravilová and
Hájek (2006) reported that species compositions
of both the vegetation, involving vascular plants
and bryophytes, and moss samples characterize
testacean assemblages better than even long-term
measured water-chemistry data. In the present
study, species richness levels and abundances
of protozoa were similar between different
species of mosses. The lack of any statistically
significant difference in protozoan abundances
may be related to the fact that all moss species
were situated in sphagnum hollows with waters of
similar physical and chemical properties. On the
other hand, in a rich fen, both the abundance and
species diversity among the protozoa clearly varied
among individual plant species. It was observed
in the present study that species diversity and
abundance, and the biomass of protozoa increased
in the most architecturally complex habitats of
Utricularia and Calliergonella beds. According
to Mieczan (2008), more-structurally complex
plants provide a more-attractive environmental for
protozoa by better providing food and refuge.
Nutrient excretion
Significant differences between protozoan
size and excretion intensity suggest their
particularly vital role in bogs. For an average
population of 1000 protozoa in 1 ml of water
in peatbogs, the average net excretion rate
of nitrogen was 0.58 µg (as N-NH 4 )/d and of
phosphorus was 0.22 µg (as P-PO 4 )/d (data
presented in Table 3). The obvious prevalence of
small forms in such peatbogs means that during
the vegetative period (from Apr. to Nov.), these
microorganisms can supply ca. 139 µg N-NH 4
and 53 µg P-PO 4 .μg/d Obviously, these are
minimum excretion volumes compared to those
25
that may be noted in field conditions where the
abundance of food is relatively high. A significant
effect of a cell’s size on the rate of nitrogen and
phosphorus excretion was also observed by
other authors. However, their works examined
lake and sea ecosystems (Taylor 1986, Dolan
1997). No significant relationship between the
excretion volume and pH was detected. Similar
observations were noted by Dolon (1997). On the
other hand, studies carried out by Liu et al. (2007)
revealed an increase in phosphate excretion at pH
6.8-8. Ciliates are considered major consumers
of bacterial production in aquatic ecosystems.
However, recent studies showed that testate
amoebae are also able to consume a large fraction
of bacterial populations in peatbogs (Mieczan
2007). The consumption of bacteria constitutes a
major portion of nutrient regeneration in peatbogs.
Due to their abundance and relatively high weightspecific excretion rates, protists probably account
for a large portion of nutrient regeneration in a
variety of peatbog ecosystems (hypothesis 2).
These results show that vascular plant, moss,
testate amoeba, and ciliate communities respond
differently to ecological gradients. Factors which
most highly affect their occurrences are probably
water depth, pH, Ptot, and TOC. In accordance with
the 1st hypothesis, factors limiting the occurrence
of these microorganisms are the complexity of
the plant cover, groundwater level, and trophic
parameters. Statistically significant differences
between the size of the protozoa and the intensity
of excretion suggest that they have a key role in
nutrient cycling in bogs, which confirms the 2nd
hypothesis.
Acknowledgments: This work was partially
financed by project N N304 209837 from the
Ministry of Science and Higher Education,
Warsaw, Poland. I would like to thank L. Błędzki
for methodical comments on the laboratory
experiments. I am also grateful to M. Marzec
and M. Niedźwiecki for conducting the chemical
analyses.
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An Experimental Study of the Response of the Gorgonian Coral
Subergorgia suberosa to Polluted Seawater from a Former Coastal
Mining Site in Taiwan
Isani Chan1, Li-Chun Tseng1, Samba Kâ1, Ching-Fong Chang2,3, and Jiang-Shiou Hwang1,3,*
Institute of Marine Biology, National Taiwan Ocean Univ., 2 Pei-Ning Road, Keelung 202, Taiwan
Institute of Aquaculture, National Taiwan Ocean Univ., 2 Pei-Ning Road, Keelung 202, Taiwan
3
Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean Univ., 2 Pei-Ning Road, Keelung 202,
Taiwan
1
2
(Accepted September 6, 2011)
Isani Chan, Li-Chun Tseng, Samba Kâ, Ching-Fong Chang, and Jiang-Shiou Hwang (2012) An
experimental study of the response of the gorgonian coral Subergorgia suberosa to polluted seawater from a
former coastal mining site in Taiwan. Zoological Studies 51(1): 27-37. The response of the gorgonian coral
Subergorgia suberosa to heavy metal-contaminated seawater in the Yin-Yang Sea of Liang-Dong Bay, a former
mining site in northeastern Taiwan, was investigated. Subergorgia suberosa bioaccumulation and tissue injury
were recorded and examined throughout the study period. Heavy-metal concentrations in tissues of the corals
showed a significantly increasing trend with incubation time. Cu, Zn, and Cd each showed characteristic
bioaccumulation in this soft coral. Metallic Zn accumulated, but rapidly dissipated. In contrast, Cu easily
accumulated, but was slow to dissipate, and Cd was only slowly absorbed and dissipated. Our results indicate
that significant bioaccumulation of heavy metals occurred in S. suberosa coral. Histopathological and scanning
electron microscopic results identified polyp necrosis, mucus secretion, tissue expansion, and increased
mortality in S. suberosa corals exposed to water polluted with heavy metals.
Key words: Subergorgia suberosa, Heavy metals, Bioaccumulation, Histological examination.
C
oral reefs play vital roles in marine ecosystems, by serving as home to 25% of all marine
fish species and other fauna important to the
marine food chain (Bryant et al. 1998, Selkoe et al.
2008, Almany et al. 2009). Worldwide, coral reef
ecosystems have recently experienced increasing
direct and indirect threats from anthropogenic
pressures, including pollutants (Bruno et al. 2007,
Jones et al. 2009, Haas et al. 2010, Mendonca
et al. 2010, Niggl et al. 2010, Tseng et al. 2011).
Consequently, coral reefs are being degraded and
damaged by land- and sea-based human activities
within the Pacific region (Bryant et al. 1998,
Chanmethakul et al. 2010, Tseng et al. 2011).
Previous studies showed that environmental
pollutants can accumulate in marine organisms,
such as polynuclear aromatic hydrocarbons
(PAHs) in copepods and polychaetes (Chandler
et al. 1997, Cailleaud et al. 2007a), tributyltin
(TBT) in bivalve mussels (Huang and Wang 1995),
polychlorinated biphenyls (PCBs) in copepods,
corals, fish, crabs, and lobsters (Miao et al. 2000,
Cailleaud et al. 2007a, Chang et al. 2008), and
nonylphenols (NPs) Polyethoxylates (NPEs) in
copepods (Cailleaud et al. 2007b). However,
there are few reports of corals being used as
marine bioindicators of environmental pollution to
monitor the presence of trace metals (Howard and
*To whom correspondence and reprint requests should be addressed. Tel: 886-935289642. Fax: 886-2-24629464.
27
28
Chan et al. – Subergorgia suberosa in Polluted Seawater
Brown 1986, Brown 1987, Shen and Boyle 1987).
Corals can incorporate trace metals by a variety
of mechanisms: dissolved trace metal species
can migrate into crystal lattices, and trace metal
particulates may become trapped within skeletal
cavities by the uptake of trace metals from coral
tissue or through feeding (Howard and Brown
1984). Different coral species were shown to be
good tracers of pollutants in marine environments
(Pai et al. 1990a, Guzman and Jimenez 1992,
Bastidas and Garcia 1999). Hence, continual
pollution inputs can result in the accumulation
of trace metals and other pollutants in marine
organisms (Reichelt-Brushett and Harrison 1998).
Coral reefs are widely used as environmental
indicators, because they are known to experience
such threats (Guzman and Jimenez 1992,
Reichelt-Brushett and Harrison 2000, Guzman and
Garcia 2002, Kawahata et al. 2004, Yap 2007).
However, the accumulation of heavy metals in soft
corals has rarely been discussed.
In Liang-Dong Bay, off the northeastern
coast of Taiwan, there is a mixture of blue normal
and brown abnormal seawater locally known as
the Yin-Yang Sea (YYS). The body of water is
considered polluted as a result of copper refining
by the Taiwan Metal Mining Corporation (TMMC)
(Yang and Yeh 1990). Although the factory
was closed in 1987, the YYS still exists in the
estuary of Liang-Dong Creek where abandoned
pits are located. It was suggested that the iron
and aluminum dissolved in the effluent of the
abandoned pits form flakes of ferric and aluminous
hydroxides when they come into contact with the
near-neutral pH waters of the estuary (Pai et al.
1990b, Yang and Yeh 1990). These flakes adsorb
heavy metals from suspended particles of clay and
are transported by estuarine waters to form the
YYS (Yang and Yeh 1990).
In this study, we investigated responses of
the soft coral Subergorgia suberosa to exposure
to this polluted seawater from the YYS. The
aims of the present study were (i) to examine the
bioaccumulation of trace metals by the soft coral
species S. suberosa, and (ii) to determine the
extent and form of tissue damage resulting from
exposure to the heavy metal-polluted water of the
YYS.
Coral colonies and water collection
Subergorgia suberosa coral samples were
collected from the coastal area of Heping I. (Fig. 1)
by scuba divers at a depth of 26 m. Coral colonies
were cut into 17-cm lengths using scissors, and
specimens were rapidly placed in an icebox
containing clean ozone-treated seawater, and
supplied with air bubbles during transportation
to the laboratory. Experimental water for the
bioaccumulation study was collected from the
YYS at Liang-Dong Bay (Fig. 1), and water for the
control group experiments was collected from the
Aquaculture Department of National Taiwan Ocean
Univ., Keelung, Taiwan.
Experimental procedures
Eighteen S. suberosa colonies were
distributed equally among 6 tanks, and these
were divided into 3 treatments containing water
as a control (control group), and 3 treatments
containing YYS sample water (treatment group).
The YYS treatment water was not renewed during
the experimentation. At weeks 1, 4, 5, 6, and 7
of the experiment, 5-cm lengths of coral (skeleton
with tissue attached) were collected from the
control and experimental tanks for trace metal
analyses. The water tanks were maintained within
a salinity range of 34.5-35.0 psu at 24-26°C, with
a seawater flow rate of 7-9 cm/s. The water tanks
were illuminated from 06:00 to 18:00 with natural
daylight, and were kept in darkness in the period
18:00 to 06:00. Fresh Artemia franciscana nauplii
were provided twice daily as food, during the
morning at 10:00 and in the evening at 23:00.
Analysis of water and coral metal contents
Concentrations of Cu, Zn, and Cd in water
samples taken from the YYS and in tissues
of coral samples taken from Heping I. were
determined by atomic absorption spectroscopy
(AAS) using a Perkin-Elmer atomic absorption
spectrometer (Perkin Elmer 2000 DV ICP, 710
Bridgeport Avenue Shelton, Connecticut, USA).
Water samples were stored at 4°C until the
AAS analysis was conducted. Coral samples
(a combination of tissues and skeleton) were
placed into vials, sealed, and stored in a freezer
at -40°C for 24 h. Samples were uncapped and
dried in a hot-air oven (model EYELA natural
oven NDO-450 OND, Tokyo, Japan) at 50°C for
48 h. Samples were pulverized and digested in
aqua regia solution (3 ml HNO3 and 1 ml HCl) for
24 h. Digested samples were then heated to 60°C
until approximately 1 mL of the digested solution
remained. Milli-Q water was added to bring the
solution to 25 ml (Millipore, Pocklington, York,
England).
Histological observations
At weeks 1 and 7, 5-cm samples of S.
suberosa were cut from the experimental and
control tanks. The samples were fixed with
neutral buffered formalin (10%) and embedded in
paraffin of similar density to that of the S. suberosa
sample. Wax sections of 3-10 and 6-8 µm were
removed, and then dehydrated using 70% alcohol.
Xylene was used as the cleaning agent to remove
alcohol trapped within the wax medium. After
cleaning, samples were embedded in paraffin
wax, sectioned with a microtome, and placed on
microscope slides. The glass slides were then
placed in a warm oven for approximately 15 min
to allow the section to adhere to the slide. The
29
embedding paraffin wax covering the tissue was
removed by washing with a xylene solvent to allow
the water-soluble dye to penetrate the sections.
The tissue was then stained with hematein, and
a glass cover slip was placed over the specimen
for protection. The stained slides were repeatedly
immersed in an alcohol solution to remove any
water, and then in xylene, until a permanent
resinous substance had formed beneath the glass
coverslip covering the section (Howard and Smith
1983). Sections were studied using an Olympus
SZX10 microscope and images were recorded with
an Olympus Imaging Corp. model NO.E-3 camera.
The software program, ACD See Pro 2.5 was used
to edit the resulting photographs.
Scanning electron microscopic (SEM) observations
Subergorgia suberosa SEM samples were
oven-dried for 24 h, and then attached to SEM
stubs using a liquid colloidal silver paste. Samples
were then sputter-coated with gold (Jeol JFC
1100 E ion-sputtering system, Tokyo, Japan) and
observed with an SEM (Hitachi S-4700, Tokyo,
East China Sea
East China Sea
25°19'34"
25°40'
25°14'44"
Yin-Yang Sea
Latitude (°N)
Taiwan Strait
Heping I.
25°09'55"
Pacific Ocean
121°33'53"
22°55'
South China Sea
20°10'
119°10'
121°00'
Longitude (°E)
122°50'
Fig. 1. Location of Heping I. and Liang-Dong Bay (Yin-Yang Sea) in northern Taiwan.
121°43'32"
121°53'11"
30
Japan) (Humphreys et al. 1974).
140
Data analysis
120
T h e e ff e c t o f t h e t r e a t m e n t t y p e a n d
period of exposure on variations in heavy metal
concentrations in the corals were analyzed using a
two-way analysis of variance (ANOVA). Statistical
analyses were carried out using SAS software
(Cary, NC, USA). Pearson’s correlations were
used to correlate heavy-metal accumulation in
coral tissues with time.
100
(A) Cu
Control
Experimental
80
60
40
20
0
80
(B) Zn
Metal levels in the water
Variations in the collected water sample
metal concentrations are shown in table 1. High
metal concentrations occurred in the LiangDong Bay (YYS) sample, with 13.67 µg/L Cu,
2.26 µg/L Zn, and 12.72 µg/L Cd. In contrast,
metal concentrations were lower in Heping I. water
samples, at 0.260 µg/L Cu, 0.450 µg/L Zn, and
0.124 µg/L Cd.
Concentration (mg Kg-1)
RESULTS
60
40
20
0
1.4
(C) Cd
1.2
Metal levels in the coral
1.0
Variations in S. suberosa metal concentrations
are shown in figure 2. Prior to the start of the
experiment (week 1), corals collected from Heping
I. showed high zinc concentrations (37.98 mg/kg),
in contrast to Cu (5.64 mg/kg) and Cd (0.51 mg/kg)
levels.
After 4 weeks of exposure to the YYS water
sample, Cu concentrations in the corals were
significantly greater than Zn and Cd concentrations.
Metal concentrations significantly increased at
1-7 wk of the experimental treatment, showing
increases of 5.64 to 95.42 mg/kg for Cu, 38.76 to
63.43 mg/kg for Zn, and 0.67 to 0.91 mg/kg for Cd.
Concentrations of Cu and Zn in coral tissues were
0.8
0.6
0.4
0.2
0.0
week 1 week 4 week 5 week 6 week 7
Time
Fig. 2. Variations in the concentration of Cu, Zn, and Cd within
Subergorgia suberosa from the control and experimental
treatments during the experiment. Results are given as the
mean ± standard deviation of 3 replicates.
Table 1. Concentrations of heavy metal in samples from Heping I. and the Yin-Yang Sea compared to
ANZECC and ARMCANZ standards (2000). Concentrations are expressed in µg/L
Water
ANZECC and ARMCANZ
Yin-Yang Sea, Liang-Dong Bay
Heping I.
Cu
0.003-0.370
13.67
0.260
Zn
0.100-15.000
2.26
0.450
Cd
0.001-1.1
12.72
0.124
several orders of magnitude greater than in YYS
water samples from the surrounding area. The
bioaccumulation of Cu and Zn in the coral, after
7 wk of exposure to YYS water, was significantly
greater than that for Cd.
Metal concentrations in the control treatment
( n o r m a l s e a w a t e r ) s h o w e d a d i ff e r e n t y e t
significant trend. Concentrations of Cu in S.
suberosa tissues were stable in weeks 4-7, during
which time, concentrations varied 4.28-5.64 mg/kg
(Fig. 2A). However, Zn concentrations dramatically
decreased from weeks 1 (37.98 mg/kg) to
4 (6.76 mg/kg), then remained at a constant
value in weeks 4-7 (6.57 mg/kg) (Fig. 2B). Cd
concentrations in tissues of S. suberosa coral
slightly decreased from weeks 4 to 7 from 0.51 to
0.45 mg/kg (Fig. 2C).
Results of a two-way ANOVA showed that the
3 heavy metals significantly impacted S. suberosa
tissues (p < 0.001, Table 2) for all treatment
groups. Zn showed significant changes in tissue
concentrations following the 7-wk exposure to
YYS water (p < 0.001, two-way ANOVA) and at
week 4 (p < 0.001, two-way ANOVA). Interactions
between S. suberosa and the heavy metals, Cu
and Cd, were not significant for the treatment
(p = 0.118) or period (p = 0.125). Coral tissue
heavy-metal bioaccumulation showed a significant
increasing trend with exposure time for Cu
(r = 0.623, p < 0.030, Pearson’s correlation), Zn
(r = 0.827, p < 0.001, Pearson’s correlation), and
Cd (r = 0.592, p < 0.042, Pearson’s correlation)
(Fig. 3).
Histological observations
In the control tanks, coral cells remained
intact with no damage to the tentacles, polyps, or
outer epidermal cells (Fig. 5A, B). However, coral
in the experimental tanks containing water from
the YYS exhibited damaged tentacles, malformed
polyps and outer epidermal cells, and increased
mucus secretion, tissue expansion, and mortality
of juvenile corals (Fig. 5C, D).
SEM observations
Intact tissues of S. suberosa were identified
in the control tank (Fig. 6A). Magnification of S.
suberosa surface tissues in the experimental tanks
revealed mucus secretion, tissue necrosis, and
1.2
Variable
Zn, y = -22.7 + 11.6x, Pearson’s correlation, r = 0.827, p = 0.001
d.f.
F
Significance
1
3
3
34.796
2.129
2.286
< 0.001**
0.137
0.118
1
3
3
180.059
9.771
10.194
< 0.001**
0.001**
0.001**
1
3
3
5.966
1.491
2.225
100
0.9
80
60
0.6
Cd (mg Kg-1)
Cu and Zn (mg Kg-1)
Few polyp extensions were observed in
the experimental tanks compared to those in the
control tanks (Fig. 4A, B). Following experimental
treatments (YYS water), corals exhibited mucus
secretion and advanced stages of tissue necrosis
(Fig. 4C, D). Fewer polyps were present in the
experimental treatment tanks than were present
in the controls (Fig. 4C). Corals in the YYS water
exhibited a greater magnitude of tissue expansion
than that of control colonies. For some colonies,
tissue expansion was only observed in parts of the
colony.
1.5
Cd, y = -0.3 + 0.18x, Pearson’s correlation, r = 0.592, p = 0.042
120
Polyp extension, mucus secretion, and tissue
necrosis
Table 2. Results of ANOVA testing: the effects of
heavy metals on S. suberosa. Significance level =
p < 0.05
Cu, y = -56.2 + 22.3x, Pearson’s correlation, r = 0.623, p = 0.030
140
31
40
0.3
20
0
4
5
6
Time (wk)
7
8
0.0
Fig. 3. Pearson’s correlations between concentrations of 3
heavy metals in coral tissues and the experimental period of
weeks 4-7 in the present study.
Cu
Treatment
Period
Treatment × period
Zn
Treatment
Period
Treatment × period
Cd
Treatment
Period
Treatment × period
0.027*
0.255
0.125
32
abnormal spicule appearance, with scales ranging
from 20 µm to 1 mm wide (Fig. 6B-F).
DISCUSSION
As expected, metal pollution was higher in
the waters of Liang-Dong Bay (YYS) than it was at
Heping I. High concentrations of metals in LiangDong Bay are in agreement with Chu et al.’s (1995)
results for this site. According to Australian and
New Zealand standards for fresh and marine water
quality (ANZECC and ARMCANZ 2000, Hickey
et al. 2001), levels of metal pollution in LiangDong Bay and Heping I. are relatively high and at
noxious levels. However, Cd concentrations in the
southern coastal areas of Taiwan are low (Lee et
al. 1998). Our data indicate a large dispersion of
(A)
cadmium and copper (Fig. 3). We propose that this
situation was caused by irregular volumes of mine
water discharged into the coastal area. Seawater
samples from the coastal area were collected
for this study. Thus, concentrations of heavy
metals were diluted by mixing with seawater from
offshore areas (natural seawater) or precipitation
runoff from streams. Samples for this study were
collected from the field, and some results showing
high concentration of contaminants.
Subergorgia suberosa was exposed to water
from Liang-Dong Bay for a 7-wk period. Heavy
metal concentrations increased in coral tissues
and reached high levels over the study period. S.
suberosa can accumulate high concentrations
of trace metals over time, and there are reports
of corals’ ability to accumulate metals in their
tissues (Esslemont 2000, Esselmont et al. 2000,
(B)
P
P
(C)
(D)
P
M
Fig. 4. Pictures of Subergorgia suberosa showing (A, B) polyp expansion in the control treatments (arrow); (C) mucus secretion in the
experimental tanks; (D) tissue necrosis (arrowhead). P, polyp; M, Mucus.
Rainbow 2002, and Mitchelmore et al. 2007). In
contrast, some coral species do not accumulate
metals (Brown and Holley 1982). Esselmont et al.
(2000) reported that varying metal concentrations
in tissues might result from specific selectivity for
a given metal by a coral. This may explain our
observations of differences in concentrations of
Cu, Zn, and Cd in S. suberosa. However, the
differences may also be linked to the concentrations of these metals (Cu, Zn, and Cd) in the
YYS water.
The average concentrations obtained for
trace metals in S. suberosa tissue samples in our
study exceeded previous metal concentrations
reported for coral tissues and skeletons.
However, our observations concur with results of
Glynn et al. (1989), who also noted high metal
concentrations when analyzing coral tissue and
(A)
33
skeletal materials. In this study, coral metal
concentrations were significantly affected by
the exposure time and treatment. Zn has an
important role in coral biological function. At low
concentrations, Zn is actively taken up by coral
to meet its metabolic needs (Ferrier-Pagès et
al. 2005). The extent of Zn bioaccumulation is
dependent upon the duration of exposure and the
seawater concentration. One group also reported
a decrease in Zn in a coral skeleton resulting
from tissue detoxification (Ferrier-Pagès et al.
2005). Decreases in Zn concentrations seen for
coral in the control group can be explained by
the slow release of bioaccumulated Zn, probably
from tissues (Ferrier-Pagès et al. 2005). The
low Zn concentration, compared to that of Cu,
occurs by a variety of processes: substitution of
dissolved metals species into the crystal lattice of
(B)
T
T
OE
P
P
OE
2.0 mm
2.0 mm
(C)
(D)
T
T
P
OE
P
2.0 mm
OE
2.0 mm
Fig. 5. Histology of S. suberosa corals in the control tank (A, B) showing that the tentacles (T), polyps (P), and outer epidermal cells
(OE) are intact. Experimental tanks C and D show that the water with heavy-metal concentrations damaged the tentacles (T), polyps (P),
and outer epidermal cells (OE) of S. suberosa corals.
34
the mineral skeleton, substitution of metals into
coral tissues, trapping of particulate matter within
skeletal cavities, and coral feeding (Howard and
Brown 1984, Guzman and Jimenez 1992, Bastidas
and Garcia 1999). Heavy-metal intake can occur
during feeding by the tentacular food capture of
zooplankton, which may be rich in heavy metals
(Howard and Brown 1984, Anthony 2000). It was
reported that metal accumulation can occur via
food and water intake, with differences depending
on the species, metal, and food source (Wang and
Fisher 1999). Metal uptake from water and protein
(A)
sources is an important route for bioaccumulation
(Howard and Brown 1984). Our study produced
similar results to those of previous studies into
ingested food pathways, including Zn uptake via
phytoplankton (Weeks and Rainbow 1993) and the
uptake of Cd via zooplankton (Munger and Hare
1997) and sediments (Selck et al. 1998).
Cailleaud et al. (2007a b) showed that hydrophobic organic compounds accumulating in
copepods display seasonal variations. Copepods
are the major taxon of zooplankton in marine
ecosystems (Hwang et al. 2004, Soussi et al. 2007,
(B)
MS
P
x 50
0000
15 KV
1 mm
(C)
x 50
0000
15 KV
1 mm
(D)
TN
x 100
TN
0000
15 KV 500 µm
(E)
Sp
x 200
0000
15 KV 200 µm
(F)
TN
TN
x 500
0000
15 KV 100 µm
x 2.0 K
0000
15 KV 20 µm
Fig. 6. Scanning electron microscopic microphotographs of S. suberosa corals in the control tank (A) showing intact tissues and polyps
(P) induced by water with heavy-metal concentrations (B), and mucus secretion (MS) (C-F). Magnification of S. suberosa also shows
tissue necrosis (TN) and the appearance of spicules (Sp).
Hwang and Martens 2011) and therefore play an
important role in transferring materials to higher
consumer levels in the ecosystem (Tseng et al.
2008 2009). Several previous studies showed that
the bioaccumulation of heavy metals in copepods
is diverse and significant (Fang et al. 2006, Hsiao
et al. 2006 2010 2011). Our results suggest that
Cu, Cd, and Zn can be transferred from the coral’s
prey, Artemia, via feeding behavior. Future studies
might consider the quantity of heavy metals that
are transferred from copepods to corals through
the complex marine food web.
Our results confirm that benthic bioaccumulation from ambient water is progressive.
Subergorgia suberosa exhibited mucus secretion
and tissue necrosis in response to extended
exposure to water samples from the YYS. This
suggests that metal pollution results in an increase
in mucus secretion, a stress defense mechanism
and a response to environmental changes by
corals (Hayes and Bush 1990, Brown and Bythell
2005). We also found that corals under stress
may show symptoms of necrosis. Necrosis in
corals is not widely reported. McClanahan et al.
(2004) suggested that necrosis may be due to a
breakdown of the function of the immune system
as a consequence of external stress or disease.
High concentrations of Cu and Zn present in the
YYS samples may have caused the observed
reduction in polyp extensions. Indeed, ReicheltBrushett and Harrison (2005) reported that metals
present in a coral reef impair the success of
fertilization. Thus, metals are highly associated
with the reduced numbers of polyp extensions of
these corals.
The gorgonian species in this study was
exposed to maximal concentrations of 13.67 µg/L
Cu, 2.26 µg/L Zn, and 12.72 µg/L Cd. Mitchelmore et al. (2003) found that corals exhibited
bleaching after 48 h of exposure to Cu and Cd at
concentrations of 10-40 µg/L, and death without
prior bleaching with exposure to 50 µg/L. Thus,
corals used in this study were not exposed to
high constant concentrations, and so did not
completely die out; furthermore, concentrations
of metal ions in the experimental tank may have
decreased during the experimental period.
Thus, several colonies of corals were kept in the
experimental treatment tanks, and an individual
organism’s metabolism determined how the metals
accumulated. This provides an explanation for
the reduced effects exhibited by the colonies
seen in our study, because surviving colonies can
regenerate tissues, in contrast to observations
35
made by Mitchelmore et al. (2003 2007).
Our results indicate that the bioaccumulation
of heavy metals was significantly and positively
correlated with the exposure period. Wang and
Fisher (1999) suggested that the efficiency of
metal assimilation is dependent on environmental
conditions. Our results also confirmed that temporal factors affected the bioaccumulation of
metals in this soft coral.
This study demonstrated that despite closure
of the TMMC, pollution is still significant and
harmful to the development of marine organisms,
and particularly to corals in the YYS located in
Liang-Dong Bay. High concentrations of trace
metals at Heping I. gave corals from these areas
a distinct signature. The high concentrations
of trace metals found in Subergorgia suberosa
coral probably arose as a result of contaminants,
which were incorporated into tissue and skeletal
materials during juvenile formation of the corals,
and continued to accumulate over time. In
summary, this study provides new insights into the
bioaccumulation of trace metals by the soft coral
species S. suberosa. Heavy-metal-contaminated
water produced negative effects on S. suberosa
corals by causing tissue necrosis, mucus secretion,
tissue expansion, and increased mortality of
juvenile corals. The results of this study are useful
for coral reef management, with respect to marine
pollution.
Ackowledgments: We thank the Center of
Excellence for Marine Bioenvironment and
Biotechnology (99529001A) of National Taiwan
Ocean Univ. and Taiwan International Corporation
Development Funds (ICDF) for financial support.
We are grateful for grants (NSC97-2611-M-019-004
and NSC99-2611-M-019-009) from the National
Science Council of Taiwan. We acknowledge the
constructive suggestions and help from Prof. C.F.
Dai, Dr. M. Sha, and Dr. H.-U. Dahms.
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Lipid Contents and Fatty Acid Compositions of Idotea baltica and
Sphaeroma serratum (Crustacea: Isopoda) as Indicators of Food
Sources
Ermelinda Prato1,*, Antonio Danieli2, Michele Maffia2, and Francesca Biandolino1
1
2
CNR – Institute for Coastal Marine Environment, Section of Taranto, Via Roma 3, Taranto 74100, Italy
Laboratory of General Physiology, Department of Biological and Environmental Science and Technology. Univ. of Lecce, Monteroni,
Lecce I-73100, Italy
(Accepted June 30, 2011)
Ermelinda Prato, Antonio Danieli, Michele Maffia, and Francesca Biandolino (2012) Lipid contents and fatty
acid compositions of Idotea baltica and Sphaeroma serratum (Crustacea: Isopoda) as indicators of food sources.
Zoological Studies 51(1): 38-50. The lipid and fatty acid (FA) compositions of Idotea baltica and Sphaeroma
serratum, from Mar Piccolo basin at Taranto (Ionian Sea), Italy, were analyzed during winter and summer to
assess their feeding habits. The 2 isopods showed strong similarities in total lipid contents. Phospholipids
(PLs) were the major lipid class in both species, followed by triacylglycerols (TAGs). A low proportion of energystorage lipids suggested a regular food supply. Twenty-seven fatty acids were identified in the species studied.
Unsaturated FAs (UFAs) represented the predominant proportion in both species in the seasons studied.
Among them, monounsaturated FAs (MUFAs) showed higher levels. Regarding FAs corresponding to the
potential food of the 2 isopods studied, I. baltica and S. serratum displayed different FA profiles. Large amounts
of 18:2n-6 and18:3n-3 were found, especially in S. serratum suggesting a specific selection of phytodetritus
from green algae or terrestrial material of neighboring vegetation. The FA marker for diatoms of I. baltica
differed from that of S. serratum, although both species showed major consumption of diatoms during summer.
Idotea baltica showed higher levels of 22:6n-3 and 20:4n-6 in winter suggesting a preference for dinoflagellates
and macroalgae in this period. High levels of the carnivorous marker (the 18:1n-9/18:1n-7 ratio) reflected
consumption of animal materials, especially in winter. Examination of trophic markers indicated that I. baltica
and S. serratum consumed a mixed diet, showing that they have the ability to choose among available food
sources. http://zoolstud.sinica.edu.tw/Journals/51.1/38.pdf
Key words: Idotea baltica, Sphaeroma serratum, Lipids, Fatty acids, Trophic markers.
P
redicting food web patterns is fundamental
to understanding the processes involved in
estuarine ecosystems. Food webs in estuaries are
often complex, largely due to the high diversity of
both producers and consumers inhabiting these
ecosystems, as well as relatively extreme and
variable environmental conditions.
Among major approaches, the use of fatty acid
(FA) biomarkers to establish trophic interactions
within estuarine ecosystems is widely used as a
reliable method for determining important dietary
information over an extended time span (Graeve
et al. 1994, Lee 1995, Shi et al. 2001, Dalsgaard
et al. 2003, Richoux and Froneman 2008), unlike
stomach-content analyses that are extremely time
consuming and can only provide an indication
of the most recent ingestion of food (Sano et al.
2003). Previous studies used FAs as biomarkers
for bacteria (Rajendran et al. 1993), diatoms
(Parrish et al. 2000), dinoflagellates (Parrish et al.
2000), zooplankton (Falk-Petersen et al. 2002),
macroalgae (Johns et al. 1979, Khotimchenko and
*To whom correspondence and reprint requests should be addressed. E-mail:[email protected]
38
Prato et al. – Feeding Habits of Idotea baltica and Sphaeroma serratum
Vaskovsky 1990), and vascular plants (Wannigama
et al. 1981). FAs form structural and functional
components of membranes, and because of
biochemical restrictions on the synthesis of
FAs in many marine organisms, it is possible to
recognize FAs derived from their diet (Arts et al.
2001). These lipid components are not selectively
processed during food intake and incorporation,
and hence in many circumstance, are integrally
and markedly transferred through aquatic food
webs (Dalsgaard et al. 2003). In this way, a
predator’s FA composition can reveal dietary
sources of the lipids. Thus, FAs can be used to
follow the transfer of energy through a marine food
web and qualitatively assess the relative trophic
position of an organism (Sargent and Whittle
1981, Dalsgaard et al. 2003, Falk-Petersen et al.
2004). Because recent works reported that rotifers
and daphinids synthesize long-chain FAs such as
20:5n-3 even if these are not provided by their diet
(Weithoff and Wacker 2007, Wacker and Weithoff
2009, Martin-Creuzburg et al. 2010), this can be a
restriction to the use of trophic biomarkers.
Isopods are dominant components of
estuarine macrobenthos worldwide (Bruce 1992,
Dias and Sprung 2003, Gonçalves et al. 2005), and
in most regions of the world, they were recorded
in close association with submerged macrophyte
beds (Henninger et al. 2008, Wang et al. 2010).
However, they are recognized omnivores, capable
of utilizing a wide range of diets ranging from
carnivory (including cannibalism), to herbivory
and detritivory (Briones-Fourzán and LozanoAlvarez 1991, Newman et al. 2007). In particular,
they consume benthic microalgae, filamentous
algae, macroalgae, detritus, bacteria present on
sediment surfaces, small invertebrates, and even
conspecifics (Nicotri 1980, Franke and Janke
1998). Among isopods, Idotea baltica (Pallas)
and Sphaeroma serratum are the most common
isopods in the littoral zone of the Mar Piccolo
basin (Ionian Sea, southern Italy), where they are
numerically dominant and are important food for
many predators in this ecosystem (crustaceans,
fish, and birds) (De Nicola et al. 1989).
Previous studies were undertaken to
characterize the ecology and geographical
distribution of these isopods (Naylor 1955, Sywula
1964), feeding habits, and habitat selection (OravKotta and Kotta 2004), as well as their potential
use as a tool in ecotoxicological studies (De Nicola
et al. 1989, Prato et al. 2006, Annicchiarico et al.
2007). In Mar Piccolo, they are often associated
with macroalgae and particularly with drifting mats
39
of the green algae Enteromorpha intestinalis,
Chaetomorpha linum, and Ulva laetevirens and
red algae such as Gracilaria gracilis, G. dura, and
Gracilariopsis longissima. However, it is unclear
whether the association is favorable for the isopods
because they use algae as food, protection from
predators, or both. In addition S. serratum was
found under stones (pers. observ.), where it may
find food deposited on bottom sediments. To
date, the degree of omnivory in the diets of these
isopod species and their trophic positions in the
Mar Piccolo basin have not been assessed, and
although they are numerically the most abundant
macrobenthic component in this basin, little is
known about their diets.
The FA approach can be useful for exploring
habitat preferences, feeding strategies, and
food sources of these isopod species from Mar
Piccolo basin. The roles that FAs play as trophic
markers of lipid flow through food webs were
examined in marine ecosystems in a number of
studies (Sargent and Whittle 1981, Graeve et al.
1994 2001, Phleger et al. 1998, Dalsgaard et al.
2003). Since marked changes in environmental
conditions affect metabolic rates and can alter the
production, storage, and conversion of FAs, and
mask trophic links, FA compositions should mainly
be considered qualitative indicators of trophic links
(Dalsgaard et al. 2003).
The aim of this study was to analyze, for the
1st time, total lipid contents and FA compositions
of I. baltica and S. serratum from the Mar Piccolo
estuary, in order to elucidate the spectrum
and variety of their food sources and trophic
relationships. Since specific FAs are used as
trophic biomarkers, they can highlight the ability
of I. baltica and S. serratum to use food sources
available in their environment, which can be a key
factor in ecosystem functioning. In addition, the
FA compositions of these isopods were compared
between winter and summer collections to
determine how seasonal environmental conditions
influence their FA compositions.
Study area
Mar Piccolo is located in the northern part
of the town of Taranto (Fig. 1) with a total surface
area of 20.72 km2 structured in 2 parts, a 1st inlet
and a 2nd inlet, which have maximum depths of
13 and 10 m, respectively. There is restricted
40
circulation: water exchanges occur with the Gulf of
Taranto (on the Ionian Sea), through 2 channels,
and this water flow is subject to tidal effects with
the average variation between high and low tides
not exceeding 30-40 cm. In terms of hydrographic
characteristics, Mar Piccolo can be compared to
an estuarine ecosystem. Salinity is influenced by
the input of fresh water derived from small tributary
rivers and by freshwater springs called citri. The
low hydrodynamism and reduced water exchange
with the nearby Mar Grande create high water
stratification mainly in summer. In addition, urban
expansion and intensive agriculture have caused
increased nutrient and organic-matter levels
(Cardellicchio et al. 1991) particularly at the 2nd
inlet where the mild hydrodynamism allows organic
matter to be deposited on the bottom.
Sampling strategy
Idotea baltica and S. serratum were sampled
during winter and summer months (of 2008) in
the 2nd inlet of the Mar Piccolo estuary (Ionian
Sea, Italy; 40°29'17"E; 17°14'23"N). Sampling
was carried out once a month, and materials from
3 mo were pooled as replicates. The euryhaline
I. baltica is a littoral and sublittoral crustacean of
tidal shores, and was recorded in close association
with macrophyte beds of Chaetomorpha linum,
Enteromorpha intestinalis, Ulva laetevirens,
Gracilaria gracilis, G. dura, and Gracilariopsis
longissima. Sphaeroma serratum usually lives
in coastal marine or brackish waters, which are
Italy
Mediterranean Sea
IONIAN
SEA
1st Inlet
Sampling site
MAR PICCOLO 2nd Inlet
MAR GRANDE
often subject to variations in salinity. It lives under
stones close to the shore or among seaweed, in
5-80-cm-deep water, and it usually moves within a
range of only a few meters.
Isopods were collected under stones and by
hand from macroalgae, and small quantities of
sediment were sieved through a 1-mm stainlesssteel sieve. Individuals were placed in a clean
plastic container with water and sediment collected
in situ. This container was immediately carried
to the laboratory where the animals were placed
in aquaria. In order to remove gut contents, the
animals, prior to processing, were starved for 1 d,
and after about 24 h, samples of each species
were prepared for biochemical analysis. During
the sampling period, water temperatures were
12.2 ± 3.4 and 26.7 ± 1.5°C; salinities were 35.5
± 0.67 and 34.9 ± 0.6 psu; dissolved O2 values
were 8.1 ± 1.8 and 9.5 ± 0.4 mg/L, and pH values
were 8.3 ± 0.2 and 8.4 ± 0.2 in winter and summer,
respectively.
Sample preparation for lipid determination
All isopods analyzed were adults with total
lengths of 10.7 ± 0.8 mm for S. serratum and 14.2
± 1 mm for I. baltica. Samples of each species
were dried to a constant weight at 50°C and
ground to powder in a mortar. About 500 mg of
dry weight pooled from 35-50 samples was used
for each extraction, including 3 replicates for each
species.
Lipids were extracted using a solvent mixture
of chloroform: methanol (2: 1, v/v) following Folch
et al.’s (1957) method. The chloroform layer,
containing dissolved lipids, was collected, washed
with 0.88% potassium chloride, and completely
removed using a rotary evaporator. The total lipid
content was determined gravimetrically. Lipid
analyses were carried out in triplicate, and the
results were expressed as mg/g of dry weight (DW).
Triacylglycerols (TAGs) and total cholesterol (CHL)
were measured by the colorimetric enzymatic
Trinder method (1969), using a commercial kit
(SGM, Rome, Italy). Phospholipids (PLs) were
quantified by a colorimetric enzymatic method
(Takayama et al. 1977) with a commercial kit
(SGM). TAG, PL, and CHL levels were expressed
as a percentage of total lipids.
FA analysis
Fig. 1. Location of isopod sampling sites in Mar Piccolo basin
(southern Italy).
FAs of total lipids were transesterified to
methyl esters (FAMEs) in a boron trifluoride-
catalyzed methanol: benzene solution (1: 2, v/v).
The mixture was shaken, and then heated in
boiling water for 45 min (Allinger 1986). Samples
were allowed to cool, then 1 ml of distilled water
was added followed by vigorous shaking. FAMEs
were recovered in the upper benzene phase.
Benzene phases were concentrated under nitrogen
and kept at -20°C until further analysis.
Analysis of FAMEs was performed by gas
chromatography (GC) using an HP 6890 series
GC (Hewlett Packard, Wilmington, DE, USA)
equipped with flame ionization detector. FAMEs
were separated with a Omegawax 250 capillary
column (Supelco, Bellafonte, PA, USA) (30 m
long, 0.25-mm internal diameter, and 0.25-mm film
thickness). Helium was used as the carrier gas at
a flow rate of 1 ml/min. The column temperature
program was as follows: 150 to 250°C at 4°C/min
and then held at 250°C. FAMEs were identified by
comparing retention times with a standard (Supelco
37 Component FAME Mix). FAs were quantified
by integrating areas under peaks in the GC traces,
with calibration derived from an external standard
containing different methyl esters. FA biomarkers
Table 1. Trophic and dietary fatty acid markers
used in this paper
Source
Trophic markers
Diatomsa
20:5n-3
16:1/16:0
20:5n-3/22:6n-3 >1
Dinoflagellatesb
22:6n-3
20:5n-3/22:6n-3 <1
Bacteriac
Σ 15 + 17
Terrestrial detritus or green algaed
18:2n-6 + 18:3n-3
Carnivorye
18:1n-9/18:1n-7
20:1 + 22:1
Macroalgaef
20:4n-6
Detritusg
PUFA/SAFA
Dunstan et al. (1994) and Parrish et al. (2000). Graeve et al.
(1994), Parrish et al. (2000), and Nelson et al. (2001). cKaneda
(1991) and Rajendran et al. (1993). dBudge and Parrish (1998)
and Dalsgaard et al. (2003). ePhleger et al. (1998) and FalkPetersen et al. (2000). fKhotimchenko and Vaskovsky (1990)
and Graeve et al. (2001). gFahl and Kattner (1993). PUFA,
polyunsaturated fatty acid; SAFA, saturated fatty acid.
a
b
41
(specific FAs and ratios of FAs) of the major
potential food sources found at Mar Piccolo were
identified by comparisons with published literature
(Table 1).
Statistical analysis
For each species, the data obtained (total
lipid content, lipid classes, and trophic markers,
expressed as percent of total FAMEs) were
arcsine-transformed and analyzed using a twoway analysis of variance (ANOVA). Means were
separated at or below the 5% probability level,
using Tukey’s honest significant difference (HSD)
post-hoc test. Data were tested for normality
prior to being analyzed using the KolmogorovSmirnov test. Bartlett’s test was used to test for
homogeneity of variances (Zar 1996). Statistical
analyses were performed using SPSS software
(SPSS, Chicago, IL, USA), and 95% confidence
intervals (CI) are given.
To aid in visualizing the results obtained,
a principle component analysis (PCA) and
hierarchical cluster analysis were performed to
investigate variations in FA signatures between
species and seasons and identify the FAs most
responsible for those variations. These analyses
were carried out using STATISTICA 8 (StatSoft,
Tulsa, OK, USA).
RESULTS
Total lipid contents
Total lipid contents of I. baltica, expressed
on a DW basis, were 30.13 ± 2.77 mg/g DW in
winter and 33.45 ± 1.50 mg/g DW in summer.
Sphaeroma serratum showed similar values of
33.71 ± 1.56 mg/g DW in winter and 32.89 ±
2.46 mg/g DW in summer. Results of the statistical
analysis indicated that lipid contents of each
species did not significantly vary between winter
and summer, or between species in the same
season (ANOVA, p > 0.05) (Table 2).
Lipid classes
PLs were the major lipid class especially in I.
baltica (74.70% in winter and 65.84% in summer)
followed by TAGs that in S. serratum accounted
for 31.20% of total lipids in winter and 39.75%,
in summer (Fig. 2). Statistical analysis (two-way
ANOVA) revealed significant differences in PL
42
FAs
and TAG contents between species and seasons
(p < 0.05); however the interaction between
species and seasons was not significant (p > 0.05)
(Table 2). Sphaeroma serratum showed the
significantly highest CHL level in winter (ANOVA;
Tukey’s test p < 0.05). Post-hoc (Tukey’s) test
results are reported in figure 2.
Mean values ± standard deviation (SD) of
the FA composition are given in table 3. At least
27 FAs with numbers of carbon atoms of 1424 were identified in the species studied. On
average, unsaturated FAs (UFAs) represented the
predominant portion in I. baltica and S. serratum
90
a
80
I. baltica Winter
I. baltica Summer
S. serratum Winter
S. serratum Summer
b
70
Lipid classes %
60
c
50
c
d
40
c
30
20
b
b
a
a
a
a
10
0
TAG %
PL %
CHL %
Fig. 2. Lipid class composition of Idotea baltica and Sphaeroma serratum collected in winter and summer. Different letters (a, b, and c)
indicate a statistically significant difference between intra- and interspecific groups (Tukey’s test; p < 0.05).
Table 2. Results of a two-way ANOVA performed on lipids, triacylglycerols (TAGs), phospholipids (PLs), and
cholesterol (CHL) contents
Source
d.f.
Mean square
F Ratio
p
Total Lipid
Source
d.f.
Mean square
F Ratio
p
TAG
Species
1
0.00
1.15
ns
Species
1
1.27
111.48
***
Seasons
1
0.00
1.50
ns
Seasons
1
0.22
19.76
**
Interaction
1
0.01
3.04
ns
Interaction
1
0.03
0.27
ns
Error
8
Error
8
0.60
20.77
**
PL
Species
CHL
1
0.47
131.23
***
Seasons
1
0.03
7.99
*
Interaction
1
0.02
0.67
ns
Error
8
Species
1
Seasons
1
0.00
0.00
ns
Interaction
1
0.28
9.80
*
Error
8
Level of significance: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.
43
Table 3. Fatty acid (FA) composition (% of total FAs) of 2 isopod species (Idotea baltica and Sphaeroma
serratum) from Mar Piccolo of Taranto, Italy
Lipid content (mg/g dry weight)
14:0
15:0
16:0
17:0
18:0
20:0
22:0
24:0
SAFA
14:1
15:1
16:1
17:1
18:1n-9
18:1n-7
20:1n-9
22:1n-9
24:1n-9
MUFA
18:2n-6
18:3n-6
18:3n-3
20:2
20:3n-6
20:3n-3
20:4n-6
20:5n-3
22:2
22:6n-3
PUFA
I. baltica Winter (n = 35)
± S.D.
I. baltica Summer (n = 50)
± S.D.
30.13
2.77
33.45
1.50
4.83
1.91
20.02
1.72
9.93
2.75
2.12
2.46
45.74
2.47
0.5
1.72
0.69
11.42
9.93
0.21
1.32
1.88
30.14
6.41
0.45
2.89
1.17
0.81
1.63
4.76
1.34
0.72
3.94
24.12
0.54
0.13
0.98
0.02
0.65
0.62
1.11
0.21
4.3
0.28
0.01
0.02
0.03
1.13
0.65
0.01
0.3
0.42
2.85
0.84
0.01
0.39
0.13
0.02
0.06
0.13
0.23
0.01
0.28
2.2
5.15
0.60
32.01
1.24
8.39
0.23
0.37
0.49
48.49
2.32
0.78
9.51
0.68
10.39
11.26
0.65
0.17
2.71
38.49
3.43
1.99
1.89
0.22
0.29
0.84
1.52
1.40
0.21
1.22
13.02
0.08
0.02
1.39
0.07
0.21
0.00
0.06
0.03
2.60
0.39
0.05
0.20
0.10
1.36
1.82
0.18
0.00
0.04
1.68
0.00
0.01
0.01
0.00
0.01
0.01
0.01
0.01
0.00
0.01
0.95
S. serratum Winter (n = 50)
Lipid content (mg/g dry weight)
14:0
15:0
16:0
17:0
18:0
20:0
22:0
24:0
SAFA
14:1
15:1
16:1
17:1
18:1n-9
18:1n-7
20:1n-9
22:1n-9
24:1n-9
MUFA
18:2n-6
18:3n-6
18:3n-3
20:2
20:3n-6
20:3n-3
20:4n-6
20:5n-3
22:2
22:6n-3
PUFA
± S.D.
S. serratum Summer (n = 50)
± S.D.
33.71
1.56
32.89
2.46
3.35
1.08
19.87
1.95
8.73
1.74
2.01
1.87
40.60
0.47
0.51
9.18
2.04
10.97
6.41
1.35
1.37
3.43
35.73
5.11
1.00
5.18
1.40
0.98
0.87
3.85
1.22
0.84
3.22
23.67
0.78
0.08
1.15
0.34
0.75
0.08
0.07
0.02
3.3
0.02
0.03
0.32
0.11
0.95
0.38
0.1
0.25
0.31
2.47
0.83
0.06
0.62
0.06
0.05
0.07
0.59
0.41
0.04
0.18
2.9
3.75
0.83
24.45
1.32
7.21
0.12
0.26
0.51
38.45
0.19
0.64
11.96
0.95
9.04
10.89
1.28
1.96
3.29
40.20
4.47
1.29
4.16
0.21
0.23
0.32
4.18
2.57
0.34
3.58
21.35
0.26
0.04
1.14
0.12
0.37
0.02
0.03
0.1
1.83
0.00
0.01
0.23
0.08
0.87
1.05
0.16
0.41
0.92
1.87
0.51
0.09
0.15
0.04
0.00
0.05
0.84
0.71
0.02
0.23
1.64
SAFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.
44
in both seasons studied, and among UFAs, these
species showed a higher level of mono-UFAs
(MUFAs) than poly-UFAs (PUFAs).
The mean percentage of saturated FAs
(SFAs) significantly differed between the 2
species collected in summer (two-way ANOVA;
p < 0.05), with higher values exhibited by I. baltica.
Regarding MUFAs, I. baltica and S. serratum
demonstrated significantly higher levels in summer
than winter (two-way ANOVA; p < 0.05), and in
winter S. serratum had significantly higher levels of
MUFAs than I. baltica (two-way ANOVA; p < 0.05).
The PUFA content in I. baltica was significantly
higher in winter than summer (two-way ANOVA;
p < 0.05), while there was no consistent difference
between summer and winter for S. serratum.
Regarding comparisons between species, S.
serratum displayed a significantly higher PUFA
level than that found in I. baltica during the summer
period only (two-way ANOVA; p < 0.05) (Table 3).
Palmitic (16:0) and stearic (18:0) acids
were the most abundant SFAs recorded in the 2
isopods in both seasons, although lower palmitic
acid levels were found in winter in both species
than in summer. High amounts of MUFAs were
indicated by considerable levels of oleic (18:1n-9)
and vaccenic acids (18:1n-7) in both isopods; in
addition, considerable levels of palmitoleic acid
(16:1n-7) were detected in both species, except
for I. baltica collected in winter which showed
a lower level (1.72% of total FAs). Among the
PUFAs observed in this study, linoleic (18:2n-6),
arachidonic (AA, 20:4n-6), and docosaesaenoic
acids (DHA, 22:6n-3) were the most abundant in
I. baltica, especially in winter samples, while in S.
serratum, linoleic acid (18:2n-6), α linolenic acid
(18:3n-3), AA, and DHA were the main PUFAs in
both seasons (Table 3).
FA trophic markers
In general, the pattern of change in trophic
markers varied between seasons and between
species (two-way ANOVA), which indicates
that season and species had effects on the
concentrations of FAs in the organisms’ tissues
(Table 4).
In both isopods, the diatom biomarkers
20:5n-3, 16:1n-7+18:1n-7, 20:5n-3/22:6n-3, and
16:1n-7/16:0 were well represented, especially
in S. serratum collected in summer (Fig. 3). The
20:5n-3/22:6n-3 ratio was > 1 in I. baltica collected
in summer, while it was < 1 in S. serratum in both
seasons.
Idotea baltica showed significant differences
between seasons for almost all trophic markers
(p < 0.05), while S. serratum showed a lower
seasonal variation and only for some trophic
markers, such as 20:5n-3, 16:1n-7+18:1n-7,
20:5n-3/22:6n-3, branched and odd-numbered
FAs, and 18:1n-9/18:1n-7 (Fig. 3). Differences
in biomarker levels between I. baltica and S.
serratum were significantly higher in summer than
in winter (Tukey’s test; p < 0.05) (Fig. 3).
The 18:1n-9/18:1n-7 ratio, proposed as
an indicator of carnivorous behavior in different
aquatic organisms, presented values of 1.15
in winter and 0.92 in summer in I. baltica, and
values of 1.71 in winter and 0.83 in summer in S.
serratum, thus characterizing these species as
more carnivorous in the winter period. A two-way
ANOVA showed significant seasonal differences
(p < 0.05) (Table 4). The other 2 FA biomarkers
for a carnivorous diet, 20:1n-9 and 22:1, were
significantly higher in S. serratum than I. baltica,
in both seasons examined (p < 0.05). The FA
signature of dinoflagellates (22:6-n3) showed
moderate amounts in S. serratum in both seasons
(3.22% and 3.58% of total FAs), similar to that
observed in I. baltica collected in winter, while
in summer, its 22.6n-3 content was significantly
lower (p < 0.05). AA (20:4n-6), used as a general
marker of macroalgae, showed similar amounts in
S. serratum collected in winter and summer and in
I. baltica in winter (p > 0.05), with percentages of
3.85% and 4.18% of total FAs (4.76% of total FAs)
respectively (Fig. 3).
Specific bacterial marker FAs (the sum of oddnumbered and branched FAs) were significantly
higher in winter than summer in both isopods
(p < 0.05), but no differences were detected
between species in winter samples (p > 0.05) (Fig.
3).
In addition, 18:2n6+18:3n3, used as a
marker of terrestrial input, showed high values
in both isopods studied. The statistical analysis
revealed significant differences between seasons
in I. baltica (p < 0.05), while on the other hand, I.
baltica showed similar values of this trophic marker
to that S. serratum in winter (p > 0.05) (Fig. 3).
Figures 4 and 5 present the distributions of
the most significant trophic markers along the 2
first principal components and groupings and/or
differences among samples. Three principal
components were extracted which accounted
for 87% of the variance, and the scatterplot of
scores on the 1st 2 principal components (PC1
and PC2) showed a separation between species
45
DISCUSSION
(Fig. 4). Loading of variables on the 1st 2 principal
components showed that PUFA/SFA, 22:6n-3,
20:4n-6, 18:2n-6+18:3n-3, and branched and oddnumbered FAs, were the dominant variables for
PC1, and the 2 species showed similar values
in winter. FA trophic markers (FATMs) that
contributed most to the separation of groups along
PC2 were 20:1n-9+22:1, 20:5n-3, 16:1n-7/16:0,
and 16:1n-7+18:1n corresponding to the highest
values in S. serratum collected in summer (Fig. 4).
In isopod tissues examined, low levels of total
lipids were detected with strong similarities among
them, in both seasons. Low levels of storage lipids
(TAG) were also observed especially in winter,
and comparisons of the 2 species showed that
S. serratum had higher TAG levels than I. baltica.
Since TAGs are short-term energy reserves,
differences in TAG contents are likely indicative of
Table 4. Results of a two-way ANOVA performed on fatty acid trophic markers
Source
d.f.
Mean square
F Ratio
p
Species
1
0.14
3.55
n.s.
Species
1
Seasons
1
0.34
8.89
*
Seasons
1
Interaction
1
0.28
7.19
*
Interaction
1
Error
8
Error
8
20:5n-3
Source
d.f.
Mean square
F Ratio
p
0.11
41.69
***
0.69
256.89
***
0.03
10.80
*
16:1+18:1n-7
22:6n-3
20:4n-6
Species
1
0.44
103.94
***
Species
1
0.37
20.71
**
Seasons
1
0.68
160.82
***
Seasons
1
0.73
40.62
***
Interaction
1
0,99
236.87
***
Interaction
1
0.95
53.37
***
Error
8
Error
8
18:2n-6+18:3n-3
18:1n-9
Species
1
0.25
41.04
***
Species
1
0.02
7.53
*
Seasons
1
0.40
64.28
***
Seasons
1
0.06
19.02
**
Interaction
1
0.11
17.34
**
Interaction
1
0.01
2.18
n.s.
Error
8
Error
8
20:1n-9+22:1
15:0+17:0
Species
1
1.40
292.34
***
Species
1
0.00
0.54
n.s.
Seasons
1
0.07
9.62
*
Seasons
1
0.68
410.49
***
Interaction
1
0.30
42.28
***
Interaction
1
0.07
44.45
***
Error
8
Error
8
PUFA/SAFA
20:5n-3/22:6n-3
Species
1
0.07
18.58
**
Species
1
0.05
2.14
n.s.
Seasons
1
0.05
14.79
**
Seasons
1
0.68
27.52
**
Interaction
1
0.04
9.95
*
Interaction
1
0.10
4.02
*
Error
8
Error
8
n.s.
16:1n-7/16:0
18:1n-9/18:1n-7
Species
1
0.22
124.92
***
Species
1
0.04
1.51
Seasons
1
0.04
20.57
***
Seasons
1
0.37
12.30
**
Interaction
1
0.03
14.40
**
Interaction
1
0.12
4.03
n.s.
Error
8
Error
8
n.s., not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.
46
different feeding strategies (Lee and Patton 1989).
Lipids of both species were dominated by
PLs, which are important membrane components,
and indicates a low dependence on lipid reserves,
in agreement with data on benthic amphipods from
cold waters (Graeve et al. 1997, Kawashima et al.
1999).
25
d
I. baltica Winter
I. baltica Summer
S. serratum Winter
S. serratum Summer
20
c
15
a
a
a
10
d
:1
b
n-
+
20
:1
n-
9
18
18
:1
n-
9/
br
dod
a
c
:1
ch
an
:3
18
6+
n18
:2
PU
3/
n:5
a
7
a a
b
22
c
ed
3
n-
A
AF
n:4
/S
22
n-
3
n:6
16
7/
n:1
16
a
a b a a
6
b
ac
a
b
20
b
a
b
a
20
16
:1
n-
7+
18
20
:1
:5
n-
n-
7
3
0
a
3
ab c c
a
b
:6
a a a
c a,c
a
FA
a
b
22
5
:0
FATMs (% of total FAs, sum and ratios)
b
Like most animals, marine invertebrates have
certain lipid requirements that must be fulfilled
through their diet. EFAs cannot be synthesized
by organisms at rates sufficient to meet their
basic biochemical requirements and thus must
largely be obtained through the diet (Arts et al.
2001). FATMs are useful tools to study trophic
Fig. 3. Fatty acid trophic markers (FATMs) of the isopods Idotea baltica and Sphaeroma serratum collected in winter and summer.
Different letters (a, b, and c) indicate a statistically significant difference between intra- and interspecific groups (Tukey’s test; p < 0.05).
1.0
(A)
20:1n-9+22:1
16:1n-7/16:0
1.4
16:1n-7+18:1n-7
1.2
0.6
1.0
0.4 PUFA/SAFA
22:6n-3
0.6
0.2
-0.4
S. serr. Summer
0.8
20:4n-6
0.4
20:5n-3/22:6n-3
18:2n-6+18:3n-3
0.2 S. serr. Winter
0.0
0.0
-0.2
(B)
PC2
Factor 2
0.8
1.6
20:5n- 3
-0.2
18:1n-9/18:1n-7
-0.4
Branched and odd
-0.6
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2
Factor 1
I. bal. Summer
-0.6 I. bal. Winter
-0.8
0.4
0.6
0.8
1.0
1.2
-1.0
-1.0 -0.8 -0.6 -0.4 -0.2 -0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
PC 1
Fig. 4. Plots of loadings and scores for the principal component analysis of fatty acid trophic markers of the 2 isopod species in winter
and summer.
ecology and determine food web connections.
Contrary to more-traditional gut content analyses,
which provide information only on recent feeding,
FATMs provide information on dietary intake and
food constituents leading to the sequestration
of lipid reserves over a longer period of time
(St John and Lund 1996, Kirsch et al. 1998, Auel
et al. 2002). A previous study conducted on
Gammarus aequicauda from the same area
as this study showed the effectiveness of this
approach (Biandolino and Prato 2006). However,
because consumers selectively metabolize FAs
and can convert some forms to others, FAs can
only be used as semiquantitative food web tracers
(Dalsgaard et al. 2003). In this study, the FATM
approach showed differences in diet between I.
baltica and S. serratum collected in Mar Piccolo of
Taranto.
Sphaeroma serratum showed higher variability of food source use in both seasons, and
it did not seem to be limited by food availability
(i.e., by reduced algal biomass in winter). Indeed,
in contrast to I. baltica, S. serratum was able to
build up energy storages (in the form of TAGs),
indicating a steady food supply. The relative
contributions of carnivory, detritivory, and herbivory
to the diet of these isopod species were shown
to vary with season. The PCA grouped I. baltica
and S. serratum, collected in winter, in a cluster
separate from the remaining isopods collected
in summer, yet also showed greater intraspecific
differences between seasons. The FAs mainly
responsible for similarities in the diets of the
2 isopods in winter were greater amounts of
22:6n-3, 20:4n-6, 18:2n-6+18:3n-3, and FAs
which indicated greater dinoflagellate, macroalgal,
detrital, and bacterial inputs to the diets of both
species. This similarity between I. baltica and S.
serratum collected in winter, may have been due
to a decrease in the phytoplankton community
recorded in the Mar Piccolo basin in this season
(Marino 1988, Carrada et al. 1992, Caroppo and
Cardellicchio 1995). In summer, S. serratum
substantially shifted this feeding strategy when
diatoms and material derived from animals formed
greater components of its diet. Although the sum
of 16:1n-7+18:1n-7 significantly differed between
the 2 species and between the 2 seasons, higher
levels found in summer in both species indicated
that diatoms formed a greater component of the
diet in this season. This is in agreement with the
typical seasonal development of phytoplankton
populations in Mar Piccolo and other coastal areas
of the Mediterranean Sea, that are characterized
by a predominance of diatoms during summer
mo n t h s, f o l l o w e d b y a d e c re a se i n w i n te r
(Marino 1988, Carrada et al. 1992, Caroppo and
Cardellicchio 1995). Thus the low level of this
trophic marker in winter, potentially reflects the
reduced availability of diatoms in this season.
Again, the ratio 16:1n-7/16:0 was significantly
higher in S. serratum than in I. baltica in both
seasons. This could have been due to the
presence of diatoms in detritus samples or
benthic diatoms which are often found in greater
quantities on sediment surfaces (Alfaro et al.
2006). These results could reflect differences in
feeding strategies, that are mostly determined
by physiological requirements, which in turn are
influenced by the feeding pattern and the ability
to make use of food potentially available to them.
This may explain the high level of diatom markers
observed in S. serratum in winter, when diatoms
are quite scarce (Caroppo and Cardellicchio 1995).
Since diatoms contain a high level of 20:5n-3
(and also high levels of 16:1n-7 and 18:1n-7),
18
15
12
Distance
47
9
6
3
0
I. baltica Winter S. serratum Winter I. baltica Summer S. serratum Summer
Fig. 5. Cluster analysis of fatty acid trophic markers of I. baltica and S. serratum in winter and summer.
48
whereas dinoflagellates are usually rich in 22:6n-3,
the 20:5n-3/22:6n-3 ratio allows differentiation
between a diatom- and a flagellate-based diet
(Graeve et al. 1994, Nelson et al. 2001). Although
in this study, tissues of I. baltica and S. serratum
showed low amounts of these FAs, I. baltica
showed a ratio of > 1 for 20:5n-3/22:6n in summer,
suggesting a low consumption of dinoflagellates in
this season.
Low values of the PUFA/SAFA ratio determined in the 2 isopods were linked to high levels
of palmitic acid (16:0), suggesting a contribution
of vegetal detritus in the diet (Reemtsma et al.
1990). It is important to highlight that 16:0 is
widespread in all organisms, and most of them
(such as crustaceans) can synthesize this FA
on their own; hence it cannot be considered
a useful FA biomarker, and certain caution is
necessary in interpreting its amount. Although
the environmental and biological context in which
the isopods live allowed us to imagine the use of
detritus-based food, another explanation could
be that when the isopods have access to more
resources, they synthesize their own lipids, one of
which is 16:0.
In contrast, a recent study reported that polymethylene-interrupted (PMI)-FAs possess an
unusual methylene substitution pattern and, as
such, occur much less frequently in nature. In
marine environments, it is thought that PMI-FAs
are primarily de novo synthesized in bivalves and
carnivorous gastropods and further accumulate
and are transferred, to varying extents (depending
on diet), through the food web (Albu et al.
2011). Therefore they were identified as a useful
biomarker. Indeed, in a previous study, Prato et al.
(2010) reported that PMI-FA 22:2 was a dominant
FA among PUFAs for M. galloprovincialis from
Mar Grande of Taranto. In the present study, we
only found traces of this PMI-FA (22:2), suggesting
that the isopods are unable to synthesize these
acids, and the low amounts detected suggest the
ingestion of animal detritus (such as bivalves and
gastropods).
Sphaeroma serratum showed considerably
higher values of trophic markers typically derived
from animals (20:1n-9+22:1 and 18:1n-9/18:1n-7),
which supports evidence of a more-carnivorous
diet.
TPUFAs in green algae predominantly consist
of 18:2n-6 and 18:3n-3, and these FA compositions
are similar to those of terrestrial (vascular) plants
since they have common ancestors (Harwood
and Russel 1984, Raven et al. 1992). In this
study, the sum of 18:2n-6 and 18:3n-3, as green
algae or terrestrial markers, was high, indicating
that these isopods receive considerable inputs of
green algae present in the basin and of terrestrial
material from neighboring vegetation. In particular
in winter, S. serratum and I. baltica reflected a
greater consumption of algal material in their
diets as evidenced by the elevated presence of
algal FAs. Between the 2 species, S. serratum
showed major utilization of this food, probably
also derived from phytodetritus which is found
in the shoreline zone. Terrestrial organic matter
can also be associated with bacteria or fungi, and
constitutes an attractive and energetically utilizable
food source for peracarida and other invertebrates
(Hieber and Gessner 2002, Barlocher and Corkum
2003). In this study, the odd-branched FAs, as
an indicator of a bacterial contribution, suggested
a food supply for I. baltica and S. serratum from
decaying organic matter especially in winter. AA,
which is related to a dietary origin (macrophytes
and leaves) or to synthesis from the respective
precursor, linoloeic acid, showed high proportions
in both species, except in I. baltica collected in
summer, which showed a preference for a morereliable diet based on phytoplankton or detritus.
Although I. baltica was found exclusively on
submerged macroalgae in Mar Piccolo basin in
both summer and winter, the results suggested
that in summer, the macroalgae serve primarily as
shelter.
This study represents a 1st attempt to shed
some light on food sources commonly utilized by
I. baltica and S. serratum in the Mar Piccolo basin
(Ionian Sea, southern Italy). The FA biomarkers
proved useful in revealing differences between
isopod species in the 2 seasons investigated and
in feeding strategies adopted, such as detritivory,
herbivory, and carnivory. In conclusion, we
found that I. baltica was highly selective in its
food choice, mainly depending on algal biomass,
whereas S. serratum was a generalist which also
utilizes detritus, benthic biofilms, bacteria, and
decomposing organic material to large extent,
with diet shifts when food availability changes,
suggesting that this species could occupy a wider
niche. In addition, significant differences recorded
between the 2 species, especially in summer,
could be explained by high competition for food
resources, as the 2 species coexist in the same
intertidal area; this leads to different distributions of
resources at different times. Moreover, the results
confirm that these species play essential roles in
energy recycling in this ecosystem.
In the future, additional feeding experiments
could help clarify the ecofunctional and trophodynamic roles of I. baltica and S. serratum in this
ecosystem.
Acknowledgments: The authors wish to thank 3
anonymous reviewers for their helpful comments
on an earlier version of this manuscript.
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Feeding Habits and Trophic Niche Overlap of Aquatic Orthoptera
Associated with Macrophytes
Soledad Capello1,*, Mercedes Marchese1,2, and María L. de Wysiecki3
Instituto Nacional de Limnología (INALI-CONICET-UNL), Ciudad Univ., Santa Fe 3000, Argentina
Facultad de Humanidades y Ciencias-UNL. Ciudad Univ., Paraje El Pozo, Santa Fe 3000, Argentina
3
Centro de Estudios Parasitológicos y de Vectores (CEPAVE) (CCT-La Plata- CONICET- UNLP), Calle 2 nº 584, La Plata 1900,
Argentina
1
2
Soledad Capello, Mercedes Marchese, and María L. de Wysiecki (2012) Feeding habits and trophic niche
overlap of aquatic Orthoptera associated with macrophytes. Zoological Studies 51(1): 51-58. A dietary analysis
is a frequent 1st step in studying an animal’s ecology, because its diet directly reflects resource use and can
provide insights into habitat utilization and competitive interactions. Little is known concerning orthopteran
species that inhabit moist or wet environments, because such species do not usually become pests. We
hypothesized that aquatic orthopterans feed on only a few macrophytes, and they show trophic niche overlap.
Feeding habits of 7 orthopteran species associated with macrophytes, the botanical composition of the diets
of these insects, and their trophic niche breadth and overlap were analyzed from the Middle Paraná River,
Argentina. The diet composition by a microanalysis of feces under an optical microscope and the frequency of
occurrence of each plant, food niche breadth, niche overlap, and food specialization level of every species were
determined. Only Paulinia acuminata, Marellia remipes, and Cornops aquaticum exclusively consumed aquatic
plants. The water hyacinth (Eichhornia crassipes) was the unique macrophyte consumed by all orthopteran
species studied, although in different proportions. The greatest trophic niche breadth was shown by Coryacris
angustipennis, and the highest Berger-Parker index value was found for C. aquaticum, which also showed
high specificity. The species C. aquaticum, C. angustipennis, Conocephalus sp., and Scudderia sp. showed
niche overlap; however, they can live in the same habitats because resources are very abundant. This is the
1st analysis of the diet compositions of these species (except C. aquaticum), and it is important information to
explain orthopteran assemblages associated with macrophytes in this Argentine river.
Key words: Diet, Herbivores, Orthoptera, Water hyacinth, Paraná River.
D ietary analysis is a frequent 1st step in
exclusion by employing isolation mechanisms
(Gause 1934, Diamond 1978). Therefore, the
degree of species overlap in the utilization of
resources such as food, space, and shelter has
become a valuable approach in studying both
community structure and species coexistence.
Traditionally, overlap in resource use is quantified
as the degree of niche overlap between species,
where niche overlap is simply the joint use of a
resource (or resources) by 2 or more species
studying an animal’s ecology because it reflects
resource use and can provide insights into habitat
utilization and competitive interactions (Litvaitis
2000). Thus, partitioning of resources results in a
maximization of habitat availability and facilitation
of species coexistence which contribute to a
determination of community structure (Pianka
1974). Niche overlap may cause the competitive
exclusion of a species, or species may avoid
51
52
Capello et al. – Feeding Habits and Niche Overlapping
(Hutchinson 1957, Colwell and Futuyma 1971).
Niche overlap between species may be
viewed as the volume in multidimensional
hyperspace in which 2 or more species maintain
viable populations in the presence of one another
(Mouillot et al. 2005).
It is important to know an animal’s diet in its
habitat in order to be aware of its nutritional needs
and its interactions with other organisms. For
this reason, studies evaluating gut contents try
to identify and quantify resources that a species
uses, thereby providing information on those
resources selected from the choices available in
the environment (Tararam et al. 1993). In studies
of species interactions and community structure, it
is useful to quantify the degree to which 2 species
overlap in their utilization of space, food, and other
resources, and several measures of niche overlap
were proposed (Hurlbert 1978).
Research on both intraspecific and interspecific competition among grasshoppers is very
important for understanding the structure and
function of the community at high densities (Liu et
al. 2007). Belovsky (1985 1997) demonstrated that
interspecific competition was strong and was an
important factor determining population dynamics
and distributions of grasshopper species.
Little is known concerning orthopteran species
that inhabit moist or wet environments because
such species do not usually become pests.
Among them, the most studied species are the
acrids Cornops aquaticum and Paulinia acuminata.
Cornops aquaticum is studied due to its possible
liberation in nonnative areas as a biological control
agent of its host plant, Eichhornia crassipes
(Bennett 1970, Silveira Guido and Perkins 1975,
Ferreira and Vasconcellos-Neto 2001, Oberholzer
and Hill 2001, Adis and Junk 2003, Adis et al.
2008), which is one of most important invasive
weeds globally. Moreover, Paulinia acuminata
was studied as a potential biological control agent
for Salvinia molesta (Carbonell 1964 1980, Forno
1981, Sands and Kassulke 1986, Thomas and
Room 1986, Vieira and Adis 2000). Salvinia
molesta, one of the world’s most noxious aquatic
weeds, is notorious for dominating slow-moving
and quiescent fresh waters (Mitchell et al. 1980).
Its rapid growth, vegetative reproduction, and
tolerance to environmental stress make it an
aggressive, competitive species known to impact
aquatic environments, water use, and local
economies.
To our knowledge, there are no previous studies concerning the trophic niche chara-
c t e r i z a t i o n o f t h e s e a q u a t i c i n s e c t s . We
hypothesized that orthopterans which live on
aquatic plants feed on only a few macrophytes,
and they show trophic niche overlap. Thus,
the objectives of this study were to analyze the
botanical composition of the diets of different
orthopteran species associated with macrophytes,
and measure their trophic niche breadth and
overlap.
Study area
Samples were collected from Apr. 2006
to May 2007 in 2 floodplain lakes of the Middle
Paraná River, Argentina. The selected sites
differed in their degree of connectivity with the main
channel, being either permanently (31°38'43.77"S,
60°34'35.07"W) or temporarily connected to the
Paraná River (31°40'14.40"S, 60°34'44.43"W).
The vegetation of these floodplain lakes is
directly associated with the hydrologic regime of
the Paraná River, because the species richness
varies according to the water level (Sabattini
and Lallana 2007). In spite of differences in the
connectivity of the lakes, the most important
macrophytes at both sites were the same: E.
crassipes, Paspalum repens, S. herzogii, Pistia
stratiotes, Ludwigia peploides, Echinochloa sp.,
and Polygonum sp.
Data collection
Orthopterans are not usually considered
to be aquatic insects. However, some of their
members are linked to freshwater habitats
mainly by a relationship to an aquatic host plant.
Species that cannot develop without fresh water,
especially for egg laying and nymph development,
are considered to be primary members of the
freshwater biota (Amédégnato and Devriese
2008).
Seven orthopteran species associated with
macrophytes were selected: 5 belonged to the
suborder Caelifera (Tucayaca gracilis (GiglioTos 1897), C. aquaticum (Bruner 1906), Marellia
remipes Uvarov 1929, P. acuminata (De Geer
1773), and Coryacris angustipennis (Bruner
1900)), and 2 belonged to the suborder Ensifera
(Conocephalus sp. Brongniart 1897 and Scudderia
sp. Stål 1873).
Cornops aquaticum, P. acuminata, and M.
remipes have physiologically, ethologically, and
morphologically adaptive strategies that allow
them to permanently live in the aquatic habitats on
floating or rooted plants (Bentos-Pereira and Lorier
1991).
Diet composition
Diet compositions of these orthopterans were
determined by a microanalysis of their feces under
an optical microscope (400x) according to Arriaga
(1981a b 1986). Individuals (568 adults) were
sampled every 2 wk with an entomological net in
2006-2007. Each insect collected was immediately
placed in a paper tube for a period of 24 h, and the
feces was collected, clarified with 10% potassium
hydroxide (KOH), and mounted on a slide (Capello
et al. in press). Twenty microscope fields were
randomly selected for each sample (comprising the
feces of 1 individual) in which at least 1 piece of
epidermal tissue was present (Sheldon and Roger
1978).
The anatomy of leaves of all macrophytes
recorded in these floodplain lakes was previously
analyzed. Epidermal tissues were identified
based on cellular characteristics (epidermal cells,
stomata, trichomes, hairs, etc.), and photographs
were taken under an optical microscope. Vegetal
tissues observed in the feces of the orthopterans
were compared to reference collections to identify
the plants species consumed.
Statistical analysis
The frequency of occurrence was calculated
for each food item based on the number of fields
containing a particular food item.
Food niche breadth was calculated as the
Shannon-Wiener index (H):
FNB = - Σpi log2 pi ;
where FNB is the food niche breadth of
species i, and pi is the frequency of occurrence of
species i in the diet of the orthopteran. The larger
the numerical value of FNB is, the wider the food
niche breadth is.
The food niche overlap index was calculated
using the Pianka index in the statistical program
EcoSim 7.72 (Gotelli and Entsminger 2009). This
index is symmetrical, and it assumes values of 0
to 1, with 0 indicating that a resource is used by
a single species, and 1 indicating complete diet
overlap or certain resource consumption. Values
53
of > 0.60 indicate overlap between species.
To determine the level of food specialization
of each orthopteran species, the index of
dominance of Berger-Parker (d) was calculated by
the following formula (Magurran 1988):
d = Nmax / N;
where N is the number of all recorded food
components (taxa), and N max is the number of
specimens from taxon i (the most numerous taxon
in the diet). The Berger-Parker index (d) varies
between 1/N and 1. A value closer to 1 indicates
higher specialization in the choice of food, while
a value closer to 1/N is typical of species that are
general feeders (polyphagous).
A factorial correspondence analysis (FCA)
was applied to determine relationships between
orthopterans species and the plants consumed.
Significant differences in diet compositions
(aquatic and/or terrestrial) between orthopterans
were analyzed by the Kruskal-Wallis test at a 5%
significance level. The software XLSTAT (Win)
(free version: http://xlstat.softonic.com) was used
for these statistical analyses.
RESULTS
We e x a m i n e d 5 6 8 f e c a l s a m p l e s o f 5
g r a s s h o p p e r ’s s p e c i e s ( C . a q u a t i c u m , P.
acuminata, M. remipes, T. gracilis, and C.
angustipennis) and 2 tettigonids (Scudderia
sp. and Conocephalus sp.) to determine the
composition of their diets. Only 3 species of
grasshoppers (P. acuminata, M. remipes, and
C. aquaticum), exclusively consumed aquatic
plants, while Conocephalus sp. (38.30%) and
C. angustipennis (27.84%) consumed higher
percentages of terrestrial plants (Fig. 1).
When comparing aquatic and terrestrial
plants by the Kruskal-Wallis test in relation to
the microanalysis of feces of each species, no
significant differences were observed (p > 0.05).
Eichhornia crassipes was a unique aquatic
plant consumed by all orthopteran species
examined, although in different proportions. The
highest consumption of this macrophyte was
by C. aquaticum (91.21%), while the lowest
value was recorded by the acrid P. acuminata
(4.31%). Conversely, Azolla sp. was the aquatic
plant selected least often by orthopterans, being
consumed only by P. acuminata (Table 1).
The highest trophic niche breadth was shown
54
by C. angustipennis (1.19), and the lowest was
by C. aquaticum (0.38), with only a few vegetal
species consumed. In addition, the highest
Berger-Parker index value was reached by C.
aquaticum (0.91), thus showing high specificity
(Table 1).
The highest trophic niche overlaps (Pianka
index) obtained were between C. aquaticum and C.
angustipennis (0.842) and between Conocephalus
sp. and Scudderia sp. (0.841). On the other hand,
species showing the lowest niche overlap were P.
acuminata and T. gracilis (0.015) (Table 2).
Four groups were established according to
the results obtained by the FCA, with each species
Conocephalus sp.
Scudderia sp.
Tucayaca gracilis
Coryacris angustipennis
Cornops aquaticum
Marellia remipes
Paulinia acuminata
0%
20 %
40 %
60 %
80 % 100 %
Aquatic Plants
Terrestrial Plants
Fig. 1. Percentages of consumption of different plants (aquatics
and terrestrial) for each species of Orthoptera.
Table 1. Percentage consumed (% C) and frequency of occurrence (FO) of each plant in the diets of
Orthoptera
Caelifera
Paulinia
acuminata
n = 40
%C
FO
Marellia
remipes
n = 30
%C
Cornops
aquaticum
n = 209
FO
%C
FO
Ensifera
Coryacris
angustipennis
n = 64
%C
FO
Tucayaca
gracilis
n = 100
%C
FO
Alternanthera sp.
0
0
0
0
0
0
0.59 1.56 0
0
Azolla sp.
51.13 77.50 0
0
0
0
0
0
0
0
Eichhornia crassipes
4.31 17.50 18.65 36.60 91.21 96.66 46.54 65.62 15.90 55.33
Hydrocleis nymphoides 0
0
77.18 86.66 0
0
0
0
0
0
Hidrocotyle sp.
0
0
0
0
0
0
1.98 1.56 0
0
Ludwigia peploides
1.38 5.00 0
0
4.08 9.56 3.53 3.12 0
0
Nymphoides sp.
2.38 10.00 0
0
0
0
0
0
0
0
Oxycaryum cubense
0
0
0
0
0
0
1.02 3.12 0
0
Paspalum repens
0
0
0.17 3.33 4.23 9.09 12.85 25.00 65.30 85.33
Panicum prionites
0
0
0
0
0.33 1.91 3.13 4.68 0
0
Pistia stratiotes
3.75 10.00 1.33 10.00 0
0
0
0
0
0
Polygonum sp.
0
0
0
0
0.15 2.87 2.54 3.12 0
0
Salvinia sp.
37.06 80.00 2.97 3.33 0
0
0
0
0
0
Terrestrials plants
0
0
0
0
0
0
27.84 42.12 18.80 35.33
Shannon-Wiener index
Berger-Parker index
1.12
0.51
0.68
0.77
0.38
0.91
1.19
0.64
0.49
0.80
Scudderia
sp.
n = 65
%C
23
0
12.20
0
0
54.00
0
0
0
0
0
1.30
0
9.50
Conocephalus
sp.
n = 60
FO
%C
FO
56.92 12.00 38.33
0
0
0
36.92 7.90 33.33
0
0
0
0
0
0
87.69 41.80 66.66
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10.77 0
0
0
0
0
33.85 38.30 70.00
0.99
0.60
0.85
0.68
n, number of individuals analyzed of each species. The highest values of percentage of consumed (%C) and frequency of occurrence
(FO) of each plant in the composition of the diet of each orthoperan species can be observed in italics and underlined.
Table 2. Niche overlap among different orthopteran species (the highest values are underlined)
Cornops aquaticum
Paulinia acuminata
Marellia remipes
Coryacris angustipennis
Tucayaca gracilis
Scudderia sp.
Paulinia
acuminata
Marellia
remipes
Coryacris
angustipennis
Tucayaca
gracilis
Scudderia sp.
Conocephalus
sp.
0.069
0.234
0.036
0.842
0.058
0.195
0.270
0.015
0.055
0.537
0.240
0.033
0.047
0.305
0.087
0.151
0.023
0.028
0.472
0.204
0.841
The values that are in italics and underlined indicate a food niche overlap among the species. Only values of > 0.60 indicate overlap
between species.
55
some species, the presence of dense hairs on
the margins of the tibiae also helps reinforce their
efficiency in aquatic habitats. This modification
of the hind tibiae is more or less generalized
within all the groups linked to water, but varies
from weakly to strongly develop in most aquatic
species (Carbonell 1957a, Amédégnato 1977,
Roberts 1978). These grasshoppers also show
modifications in their ovipositor allowing them
to utilize macrophytes as substrata on which to
deposit eggs (endophitic or ephyphitic oviposition)
and produce lower egg numbers compared to
terrestrial species (Braker 1989).
C o r y a c r i s a n g u s t i p e n n i s , T. g r a c i l i s ,
Scudderia sp., and Conocephalus sp. consumed
high proportions of terrestrial plants. Thus,
by consuming aquatic and terrestrial plants,
these species act as an important energy link
between aquatic and terrestrial systems. These
interrelationships are very important in river basins
(Jackson and Fisher 1986, Gray 1989 1993),
because rivers may provide resources to terrestrial
herbivores. For example, Paratettix aztecus and P.
mexicanus (Orthoptera: Tetrigidae), small species
that generally live near river banks, obtain 88%-
exhibiting adaptive strategies to living in an aquatic
habitat. Cornops aquaticum, P. acuminata, and
M. remipes were separated in individual groups,
while the remaining species were included in a
single group. Thus, the diet of the aquatic acrids
had a specific composition, while T. gracilis, C.
angustipennis, Conocephalus sp., and Scudderia
sp. shared major proportions of common vegetal
species (Fig. 2).
DISCUSSION
Although all orthopteran species studied
consumed aquatic plants, C. aquaticum,
P. acuminata, and M. remipes fed only on
macrophytes, showing higher dependence on
aquatic habitats. These species present ethologic,
physiological, and ecological adaptations to living
in aquatic environments. These adaptations
include a general morphology with a fusiform
habitus for species living only in water and that are
used to swimming under water. These species
also have a strong hind femur and expanded hind
tibiae, including spines and modified spurs. In
2.5
H. nymp.
Marellia
2
1.5
1
F2 (27.72 %)
0.5
E.
Cornops
0
P. st.
Coryacris
O.
Tucuyaca
P.H.
Pol.Pa.
Conocephalus T.P.
Lud.
Scudderia
Alth.
-0.5
Sal.
Paulinia
Nymh. Az.
-1
-1.5
-2
-2.5
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
2.5
F1 (32.22 %)
Fig. 2. Factorial correspondence analysis (FCA) between orthopteran species ( ; and underline) and the macrophytes ( ) consumed.
E., Eichhornia crassipes; H. nymp., Hidrocleis nymphoides; P. st., Pistia stratiotes; Sal., Salvinia sp.; Az., Azolla sp.; Nymh.,
Nymphoides sp.; O., Oxycaryum cubense; H., Hidrocotyle sp.; P., Paspalum repens; Pa., Panicum prionites; Pol., Polygonum sp.; T.P.,
terrestrial plants; Lud., Ludwigia peploides; Alth., Alternanthera sp.
56
100% of their carbon from the alga Cladophora
glomerata (Bastow et al. 2002).
Picaud et al. (2003) considered that 60%
of acrid species are polyphagous, but they can
include a degree of specialization according to
the plants consumed (Crawley 1983, Chapman
1990). Thus, the high preference of M. remipes
for H. nymphoides (a scarce plant species) can be
explained by structural modifications for life on the
horizontal surface of floating leaves and for feeding
on their upper surfaces. Although this species
is phytophilous, it has characteristic geophilous
features, such as the generally depressed shape
of its body, the position of its eyes on the upper
part of the heard, and the reduced pretarsal arolia
(Carbonell 1957a b).
All of the orthopterans studied are polyphagous, because plants of different genera were
consumed. According to Bernays and Chapman
(1994) and Bernays and Minkenberg (1997), a
mix of plants in the diet can improve the nutrient
balance and increase survival and fecundity
(Unsicker et al. 2008). Although herbivorous
insects’ preferences are a balance between plant
inhibitor substances and phagostimulants, a higher
orthopteran preference was determined by inhibitor
substances (Bernays and Chapman 1994).
Consumption of E. crassipes by all species
studied compared to all other plants consumed
can be explained by the leaf roughness, pathway
of photosynthesis (C 3 plant), and higher cover
in the Paraná River system. Thus, it is a very
valuable and palatable resource for invertebrate
herbivores. However, the highest consumption
of the water hyacinth occurred by C. aquaticum,
which corresponds to a report by Ferreira and
Vasconcellos-Neto (2001) in Brazil where it was
represented by the Pontederiaceae, especially
E. crassipes. In addition, a high specificity of
C. aquaticum for different genera of the family
Pontederiaceae was reported (Silveira Guido and
Perkins 1975, Medeiros 1984, Viera and Santos
2003, Lhano et al. 2005).
Food niche breadth and overlap analysis
The highest niche breadth was exhibited
by C. angustipennis (1.19) which consumed
different proportions of 8 types of plant (aquatic
and terrestrial) and shared the principal food (E.
crassipes) with C. aquaticum. However, there was
no competition between the 2 species due to this
food niche overlap because the water hyacinth is
not a limited resource; it is the most abundant plant
in the Paraná River system.
Paulinia acuminata, commonly considered a
possible agent of biological control for S. molesta
(Carbonell 1964 1980, Forno 1981, Sands and
Kassulke 1986, Thomas and Room 1986, Vieira
and Adis 2000) also presented a high niche
breadth (1.12) by selecting 6 aquatic plant species
for its diet, including E. crassipes, L. peploides,
and Nymphoides sp. as a new record. Carbonell
et al. (2006) reported S. auriculata, Pistia
stratiotes, Lemna sp., Limnobium leavigatum,
Spidorella intermedia, and Azolla filiculoides in the
diet spectrum of P. acuminata.
Conocephalus sp. and Scudderia sp. showed
similar food niche breadths and niche overlap.
This similarity in diet compositions might be a
key reason for the high competition between
these species (Liu et al. 2007), but they are
able to partition terrestrially available resources
and coexist. In spite of limited information on
orthopterans that live on aquatic plants, food niche
overlap between many orthopteran species was
reported in terrestrial environments (Ueckert and
Hansen 1971, Sheldon and Rogers 1978).
The coexistence in the same habitats of the
7 orthopteran species studied can be explained by
positive associations that favor mutual occurrence
and by the high availability of abundant resources.
Acknowledgments: The authors dedicate this
work to the memory of Dr. J. Adis. This study was
financed in part by CONICET.
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Species Composition and Seasonal Occurrence of Recruiting Glass Eels
(Anguilla spp.) in the Hsiukuluan River, Eastern Taiwan
Nico Jose Leander1, Kang-Ning Shen1, Rung-Tsung Chen3, and Wann-Nian Tzeng1,2,4,*
Institute of Fisheries Science, National Taiwan Univ., Taipei 106, Taiwan
Department of Life Science, College of Life Science, National Taiwan Univ., Taipei 106, Taiwan
3
Endemic Species Research Institute, Chichi, Nantou 552, Taiwan
4
Department of Environmental Biology and Fisheries Science, National Taiwan Ocean Univ., Keelung 202, Taiwan
1
2
Nico Jose Leander, Kang-Ning Shen, Rung-Tsung Chen, and Wann-Nian Tzeng (2012) Species
composition and seasonal occurrence of recruiting glass eels (Anguilla spp.) in the Hsiukuluan River, eastern
Taiwan. Zoological Studies 51(1): 59-71. There are 16 species and 3 subspecies of freshwater eel (Anguilla
spp.) found in the world. Among them, 4 species, A. japonica, A. marmorata, A. bicolor pacifica, and A.
celebesensis were reported from Taiwan. Anguilla japonica is an important aquaculture species and is
abundant on the west coast of Taiwan, while the rest are tropical species and are more abundant on the east
coast. In addition, A. celebesensis, which was identified in Taiwan in the past, appears to be the new species
A. luzonensis (A. huangi ) described from northern Luzon, the Philippines. To clarify these issues, the species
composition, relative abundances, and seasonal occurrences of anguillid eels on the east coast of Taiwan
were investigated based on 1004 glass eel specimens collected from the estuary of the Hsiukuluan River,
eastern Taiwan in 2005-2009. Eel species were identified using morphological characters such as caudal fin
pigmentation patterns, the position of the origin of the dorsal fin, and body proportions. The reliability of the
morphological method for species identification was checked by a DNA analysis. Anguilla marmorata was the
most abundant eel species in the Hsiukuluan River, making up 98.4% of the total catch, while there were very
few A. bicolor pacifica (1.6%) and A. japonica (< 1%). Anguilla marmorata recruited mainly to the estuary during
spring to summer but was found year-round, while A. bicolor pacifica recruited mainly during autumn. Results of
the DNA analysis did not support the occurrence of A. luzonensis and/or A. celebesensis based on differences
in the distance between the origin of the dorsal and anal fins as a percent of total length (ADL/%TL). Anguilla
celebesensis, which was identified in the past, was not found in this study and might just be the newly described
eel species, A. luzonensis, or just a phenotypic variation of A. marmorata. Differences in abundances and
geographic distributions of these eel species were explained by their temperature preferences, species origins,
and current systems in the coastal waters of Taiwan. http://zoolstud.sinica.edu.tw/Journals/51.1/59.pdf
Key words: Japanese eel, Giant mottled eel, Indonesian shortfin eel, Glass eels, Temporal and spatial
distribution
F
reshwater eels (of the genus Anguilla)
have been a constant source of fascination
to humans, and despite decades of intensive
research, many aspects of their biology still
remain a mystery. There are 16 species and 3
subspecies of freshwater eels in the world (Ege
1939, Castle and Williamson 1974, Watanabe
et al. 2009), and among them, 4 species are
reported from Taiwan: Japanese eel A. japonica,
giant mottled eel A. marmorata, Indonesian short
fin eel A. bicolor pacifica, and Indonesian mottled
eel A. celebesensis (Tzeng 1982 1983a, Tzeng
and Tabeta 1983, Han et al. 2001). Anguilla
japonica is an important aquaculture species and
*To whom correspondence and reprint requests should be addressed. Tel: 886-2-33662887. Fax: 886-2-23639570.
59
60
Leander et al. – Anguilla spp. in Eastern Taiwan
is more abundant on the southern, western, and
northern coasts of Taiwan. Anguilla marmorata
in Taiwan is rarely studied because it was on the
endangered species list until 2009. On the other
hand, the 2 other species are very rare (Tzeng
et al. 1995, Tzeng and Chang 2001). Anguilla
japonica is a temperate species, while the
other 3 species are tropical ones (Tesch 1977).
Recently, a new eel species, A. luzonensis, was
found in the Pinacanauan River, a tributary of the
Cagayan River in northern Luzon, the Philippines
(Watanabe et al. 2009). This new species has
variegated skin pigmentation and broad maxillary
bands of teeth similar to those of A. celebesensis;
thus, it is difficult to distinguish these 2 species
morphologically, although they show statistically
significant differences in 6 proportional characters
and 2 meristic characters (Watanabe et al. 2009).
Anguilla luzonensis inhabits the Philippines and
can possibly extend to adjacent areas such as
Taiwan. It was hypothesized that the spawning
area of this species is in the North Equatorial
Current (NEC) region of the western North
Pacific (Aoyama 2009). Shortly afterwards, Teng
et al. (2009) also described a new species of
anguillid eel from an aquaculture farm in Taiwan
which was named A. huangi. The eels at this
aquaculture farm were reportedly imported from
Luzon, the Philippines. These 2 new species
have overlapping morphological characters and
geographical distributions with A. celebesensis.
Both new species are genetically distinct from all
known anguillid eel species (Minegishi et al. 2009,
Teng et al. 2009, Watanabe et al. 2009).
In Taiwan, fishermen collect glass eels (elver)
of Japanese eel in estuaries for aquaculture. They
classify glass eels into white and black types
according to the pigmentation pattern on the tail
bud and caudal fin (Tzeng 1983a 1985, Tzeng
and Tabeta 1983). White-type eels are comprised
solely of A. japonica, while black-type eels are
supposedly comprised of A. marmorata, A. bicolor
pacifica, A. celebesensis, and possibly the new
species as well. White-type elvers are usually
collected for aquaculture, and several studies were
conducted on this species including ones which
examined its metamorphosis, estuarine arrival
(Tzeng 1990, Cheng and Tzeng 1996), fluctuations
in estuarine recruitment in relation to environmental
conditions (Tzeng 1984a 1985, Chen et al. 1994,
Tzeng 2006, Han et al. 2009), exploitation rates
(Tzeng 1984b), population genetic structure (Tseng
et al. 2006, Chang et al. 2007, Han et al. 2010),
population dynamics, and fishery management (Lin
and Tzeng 2008, Lin et al. 2009). However, little
information is available on the tropical black-type
eel population, which might be due to the fact that
fishing for and aquaculture of A. marmorata were
illegal on the island before Apr. 2009 because it
was listed as an endangered species according to
the Wildlife Conservation Act of Taiwan. Studies
of species identification, catch composition, and
recruitment dynamics are essential for fisheries
management of tropical eels.
This study aimed to clarify the species
composition and seasonality of tropical eels
recruiting in eastern Taiwan. Because A.
celebesensis and A. luzonensis are more or less
morphologically similar, it was hypothesized that
they can be distinguished from A. marmorata
using morphological measurements such as
the ano-dorsal fin length (ADL) in relation to the
total length (ADL/%TL), with the latter having
a larger ADL/%TL than the former. In addition,
to understand if the A. celebesensis specimen
described by Tzeng (1982) was misidentified with
the new recently described species (Teng et al.
2009, Watanabe et al. 2009), their morphometric
characters and mitochondrial DNA cytochrome
(Cyt) b sequences were investigated, and Anguilla
eel species in Taiwan were revised.
Sample collection
The anguillid glass eel specimens used in
this study were collected by the Taiwan Endemic
Species Research Institute (TESRI) of Nantou
County, Taiwan. Samples were collected monthly
in 2005 and 2007-2009 at 2 stations in the lower
reach of the Hsiukuluan River, eastern Taiwan
(Fig. 1, Table 1). The Hsiukuluan River is the
largest river in eastern Taiwan with a length of
81 km and a drainage area of 1790 km2 (Shiao et
al. 2003). The 1st station was located in the river
mouth, and a traditional triangle net was used for
glass eel collection; the 2nd station was located
2 km upstream where a fish way trap was set up
in an artificially dug water channel that was about
20 m long, 1.5 m wide, and 25 cm deep. Glass
eels were collected together with amphidromous
shrimp (Chen et al. 2009). After collection,
specimens were immediately preserved in 75%
ethanol. Environmental parameters such as
dissolved oxygen (DO), salinity (conductivity), pH,
water temperate, water velocity, and turbidity were
61
also recorded at every sample collection.
N
Morphometric measurements and species
identification
Hsiukuluan River
120° 121°122°E
2
25°N
1
24°
23°
1000 m
Fig. 1. Map showing glass eel sampling stations (1 and 2) in
the lower reach of the Hsiukuluan River in eastern Taiwan. The
bars indicate the Juisiu and Long Rainbow Bridges.
Glass eel species were identified using
morphological characters and pigmentation
patterns summarized in figure 2 which was
modified from Tzeng (1982 1983a) and Tzeng
and Tabeta (1983). Morphological characters,
including total length, pre-dorsal fin length (PDL),
pre-anal fin length (PAL), and ADL, were measured
to the nearest 0.1 mm (Fig. 3). Total length
(TL) was determined by measuring the distance
between the tip of the snout and the end of the
tail, while the PDL was determined by measuring
the distance from the tip of the snout to the origin
Table 1. Species composition of Anguilla glass eels collected in the lower reach of Hsiukuluan River in
eastern Taiwan in 2005-2009. (A) and (B) indicate possible proportions of A. marmorata and A. luzonensis
and/or A. celebesensis (uncertain). Values in parentheses indicate the number of individuals used for the
measurement of ADL/%TL to discriminate A. marmorata and A. luzonensis and/or A. celebesensis (uncertain)
and number of individuals used for molecular identification in A and B
Sampling period
2005
2007
2008
2009
June
July
Sept.
Apr.
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
Jan.
Feb.
Mar.
Apr.
May
June
July
Aug.
Sept.
Total
Stn., station.
Sample size
Species composition
Stn. 1
Stn. 2
Total
0
7
0
0
0
31
0
56
4
36
280
62
1
1
42
64
17
61
69
4
1
151
19
8
1
48
23
13
0
1
0
0
0
0
0
2
0
0
0
0
0
2
151
26
8
1
48
54
13
56
5
36
280
62
1
1
44
64
17
61
69
4
3
0
0
0
0
0
0
0
0
0
0
2
1
1
0
0
1
0
0
0
0
0
736
268
1004
5
A. marmorata (A)
+ uncertain (B)
A
B
Unidentified
0
0
0
0
0
0
0
0
0
0
10
2
0
0
0
0
0
1
0
0
0
151
26 (26)
8 (8)
1 (1)
48 (48)
54 (14)
13 (13)
56
5 (5)
36 (22)
268 (67)
59 (54)
0
1
44 (24)
63 (60)
17 (9)
60 (48)
69 (18)
4 (4)
3 (3)
24
6
1
46
12
11
4
17
56
38
0
19
53
9
41
17
2
2
1
1
0
1
0
1
1
2
5
9
0
0
5
0
1
0
0
0
1
1
0
1
2
1
0
3
6
7
0
5
2
0
6
1
2
1
13
985 (424)
A. japonica A. bicolor pacifica
358 (2) 27 (4)
39
62
of the dorsal fin. The PAL was determined by
measuring the distance from the tip of the snout
to the origin of the anal fin. The ADL, on the other
hand, was the difference in distance between
the origin of the dorsal and anal fins in percent of
the total length (ADL/%TL). In addition, tail bud
and caudal fin cutaneous pigmentation patterns,
which appear during the glass eel pigmentation
process, were also used for species identification.
Developmental stages from glass eel to elver
were also determined according to the extent (or
absence) of skin pigmentation over the head,
tail, and other body regions following methods
described by Strubberg (1913), Bertin (1956), and
Tesch (1977 2003).
Glass eels were classified into long- and
short-finned types according to the value of
ADL/%TL. Individuals with ADL/%TL values of
< 5% were classified as short-finned eels (the predorsal fin origin is closer to the anus than jaw),
while individuals with AD/%TL values of > 5%
were classified as long-finned eels (the pre-dorsal
fin origin is closer to the jaw than anus) (Ege
1939, Tesch 2003). Anguilla bicolor pacifica is a
short-finned eel with pigmentation in the tail bud
that extends to the caudal fin, while the rest are
long-finned eels. The 3 long-finned eel species
were then separated according to the cutaneous
pigmentation patterns on the posterior part of
the body. Anguilla japonica has no pigmentation
at this stage, while both A. marmorata and A.
luzonensis and/or A. celebesensis have more or
less the same pigmentation patterns in the tail
bud. Anguilla marmorata and A. luzonensis and/or
A. celebesensis were then separated according
to ADL/%TL values. The ADL/%TL was reported
to differ between these species but there is some
Fin length
Short
Long
A. bicolor pacifica
Caudal cutaneous pigmentation
Present
Absent
ADL/%TL
A. japonica
< 13/uncertain
> 13
mtDNA identification
A. marmorata
degree of overlap (Tzeng 1982, Teng et al. 2009,
Watanabe et al. 2009). Individuals with ADL/%TL
values of > 13 were classified as A. marmorata,
while those with values of < 13 were classified as A.
luzonensis and/or A. celebesensis. Furthermore,
the reliability of using ADL/%TL to discriminate A.
marmorata, A. luzonensis, and/or A. celebesensis
was tested by molecular identification. In total, 6
individuals with minimum, medium, and maximum
values of ADL/%TL were chosen for the DNA
analysis.
DNA extraction, polymerase chain reaction
(PCR) amplification, and phylogenetic analysis
Total genomic DNA was extracted from
muscle tissues of individuals with minimum,
medium, and maximum values of ADL/%TL
using a DNA purification and extraction kit.
A pair of oligonucleotide primers, H15341
( 5 ' - T G C TA A C G AT G C C C TA G T G G - 3 ' ) a n d
L151341 (5'-CTAGTCAACCTACTAATGGG-3')
was used to amplify a fragment of Cyt b using
PCR amplification (Han et al. 2002). PCR amplification was carried out in a 25-µl reaction mixture
containing 0.5 µl template DNA, 2.5 µl 10x reaction
buffer, 0.5 µl dNTP, 1 µl of each forward and
reverse oligonucleotide primers, 0.25 µl DNA Taq
polymerase, and 19.25 µl double-distilled water.
The thermal profile consisted of initial denaturation
at 94°C for 3 min followed by 35 cycles of
denaturation at 94°C for 30 s, annealing at 50-55°C
for 1 min, and extension at 72°C for 30 s, with a
final extension at 72°C for 10 min. PCR products
were electrophoresed on a 1% agarose gel and
stained with ethidium bromide (EtBr) for band
characterization via ultraviolet trans-illumination.
Sequencing reactions were performed using an
ABI PRISM 377 Auto DNA Sequencer (Applied
Biosystems, Foster City, CA, USA).
The generated sequences were compared
to the mitochondrial (mt)DNA Cyt b sequences
of all known species and subspecies of Anguilla
PD
AD
PA
SL
A. celebesensis
A. luzonensis
A. marmorata
Fig. 2. Schematic diagram of the methods used for anguillid
glass eel species identification.
TL
Fig. 3. Diagram showing morphometric measurements of the
glass eel. PD, pre-dorsal length; AD, ano-dorsal length, PA,
pre-anal length, SL, standard length, and TL, total length.
retrieved from GenBank (accession nos.:
AB038556, AB469437, and AP007233-49) to
determine their phylogenetic relationships using
the Neighbor-joining (NJ) method with the Kimura
two-parameter model as implemented in MEGA 4.1
(Tamura et al. 2007). The resultant topology was
assessed by bootstrapping with 1000 replications.
Data analysis
The morphometric datasets were subjected
to normality and equal variance tests because of
the unequal sample sizes. If the dataset passed
the test, significant differences were examined
using a one-way analysis of variance (ANOVA)
followed by pairwise multiple comparisons using
the Holm-Sidak method. On the other hand, if the
dataset failed the test, significant differences were
examined using a Kruskal-Wallis ANOVA on ranks
followed by multiple comparisons using Dunn’s
method. Holm-Sidak and Dunn’s post-hoc tests
were conducted to detect pairwise differences
between species with an overall alpha level of
0.05. All statistical analyses were carried out using
SigmaStat software vers. 3.5 (Systat Software,
San Jose, CA, USA). In addition, morphological
data of A. celebesensis measured by Tzeng (1982)
were compared to the new species, A. luzonensis,
to check if they were synonymous. Also, vertebral
counts determined for A. celebesensis (in Tzeng
1982), A. luzonensis (in Watanabe et al. 2009),
and A. huangi (in Teng et al. 2009) were compared.
63
(424 individuals) was chosen to discriminate
the species. Individuals with ADL/%TL values
of > 13 were classified as A. marmorata (A)
while those with ADL/%TL values of < 13 were
classified as A. luzonensis and/or A. celebesensis
(uncertain or B) (Table 1). On the other hand,
unidentified individuals were labeled as uncertain.
Individuals with ADL/%TL values of > 13 totaled
358 individuals, while those with values of < 13
totaled only 27 individuals. Based on pigmentation
patterns and morphometric analyses, very few A.
japonica (5 individuals) or A. bicolor pacifica (13
individuals) specimens were identified. A subset
(A)
(B)
RESULTS
Species composition
In total, 1004 anguillid eels were collected
and examined in this study (Table 1). Species
were preliminarily identified using the caudal
pigmentation pattern and ADL/%TL values (Table
1). Cutaneous pigmentation patterns on the
caudal part of glass eels differ among species
and can be classified into 3 types (Fig. 4): type 1
lacks pigmentation on both the tail bud and caudal
fin, i.e., A. japonica (Fig. 4A); type 2 has large
patches of small (stellate) melanophores on the
caudal fin, i.e., A. bicolor pacifica (Fig. 4B); and
type 3 has a large patch of diffused melanophores
on the tail bud, i.e., A. marmorata, A. luzonensis,
and/or A. celebesensis (Fig. 4C). Pigmentation
patterns alone cannot be used to distinguish these
3 pigmented tropical eel species so a subset
(C)
Fig. 4. Caudal fin and tail bud pigmentation patterns of anguillid
glass eels. (A) Anguilla japonica, (B) A. bicolor pacifica, and (C)
A. marmorata, A. luzonensis, and/or A. celebesensis. Scale
bar = 1.5 mm.
64
of samples with ADL/%TL values of < 13 and of
> 13 were chosen for DNA analyses to check the
reliability of using ADL/%TL to discriminate A.
marmorata, A. luzonensis, and/or A. celebesensis.
Molecular identification
The phylogenetic tree of all currently
recognized species and subspecies of Anguilla
constructed using the entire mitochondrial DNA
genome is shown in figure 5A. The resultant
topology clearly indicates clustering of A.
luzonensis and A. huangi, and this was strongly
supported by 100% bootstrap probability. Also,
the phylogenetic tree supported the occurrence
of the new eel species that is genetically distinct
from all known species and subspecies of
Anguilla. In addition to this, the complete mtDNA
genome sequence homology test using the
BLAST algorithm (Zhang et al. 2000) showed 99%
similarity between A. luzonensis and A. huangi,
indicating a very high degree of genetic similarity
between them.
On the other hand, the phylogenetic analysis
did not support the occurrence the new species
and/or A. celebesensis in Taiwan based on
species-specific differences in ADL/%TL values.
(A)
Sequences of eel species with ADL/%TL values
of < 12%, of 12%-13%, and of > 13% all clustered
with A. marmorata in the phylogenetic tree with
100% bootstrap probability (Fig. 5B), indicating
that the proposed species-specific differences in
ADL/%TL may just be a phenotypic variation of A.
marmorata, and the use of ADL/%TL to distinguish
A. luzonensis and/or A. celebesensis might not be
reliable.
Comparison of morphometric characters
among eel species
Based on groupings according to caudal
cutaneous pigmentation patterns, morphometric
measurements of different species were determined. Because the molecular analysis did not
support the occurrence of A. luzonensis and/or A.
celebesensis, individuals with ADL/%TL values
of < 13 were now classified as A. marmorata
and were not included in the morphometric
comparisons. Ranges and mean TLs differed
among eel species (Table 2). TLs of long-finned
eels ranged 46.47-58.04 (mean ± standard
deviation: 49.43 ± 2.32) mm in A. marmorata,
45.45-57.79 (53.01 ± 4.54) mm in A. japonica, and
40.40-47.00 (44.50 ± 3.04) mm in A. celebesensis
(B)
A. huangi
100
100 A. australis australis
A. australis schmidtii
A. luzonensis
98
A. dieffenbachii
A. interioris
A. bengalensis labiata
93
100
100
96
A. bengalensis bengalensis
77
A. marmorata
A. obscura
A. bicolor bicolor
100
100
100
A. mossambica
A. celebesensis
92
A. megastoma
A. japonica
Indo-Pacific Group
A. reinhardtii
A. bicolor pacifica
A. obscura
A. japonica
A. bicolor bicolor
62
100
A. megastoma
A. interioris
A. borneensis
91
100
A. mossambica
A. anguilla
100
84
A. rostrata
100
A. australis australis
100
0.01
A. huangi
A. luzonensis
A. marmorata
Atlantic Group
HKLRM042809 (Fin ratio: 11.4)
100
A. dieffenbachii
98
A. bicolor pacifica
A. bengalensis labiata
100 A. bengalensis bengalensis
A. celebesensis
100
A. rostrata
A. borneensis
A. reihardtii
82
A. anguilla
100
99
Oceania Group
100
A. australis schmidtii
HKLLR072507a (Fin ratio: 23.6)
0.01
Fig. 5. Phylogenetic tree of the genus Anguilla inferred from the entire mitochondrial genome (A) and cytochrome b (B) sequences.
Individuals with maximum (25.8% and 23.6%), medium (14.1% and 12.4%), and minimum (11.3% and 11.4%) values of the ratio of the
ano-dorsal length to the percent total length (ADL/%TL) are also shown (B). Bootstrap probabilities are indicated near the nodes.
(mean, 5.33 ± 1.43; range, 3.81-7.02 mm), A.
marmorata (7.70 ± 0.95; 6.20-9.91 mm), and
A. celebesensis (4.63 ± 1.16; 3.30-5.70 mm;
Tzeng 1982) (Table 2). Anguilla marmorata
(A)
3
n=5
2
1
0
(B)
Frequency
(Tzeng 1982). On the other hand, the TL of A.
bicolor pacifica ranged 45.86-49.22 (47.29 ±
1.06) mm. TLs of all 4 species examined significantly differed (ANOVA, p < 0.05). Lengthfrequency distributions of all species are presented
in figure 6. Anguilla japonica had the largest mean
TL, followed by A. marmorata, A. bicolor pacifica,
and A. celebesensis.
The PDL of the short-finned eel A. bicolor
pacifica ranged 16.52-18.55 (17.82 ± 0.63) mm,
the largest among all eel species examined (Table
3). In long-finned eel species, the PDL ranged
12.04-15.05 (13.21 ± 1.59) mm in A. japonica,
9.00-14.40 (11.43 ± 0.94) mm in A. marmorata, and
12.50-13.70 (13.03 ± 0.50) mm in A. celebesensis.
Significant differentiation in PDL was observed
among species (Kruskal-Wallis: H = 36.51, d.f. = 3,
p < 0.001; Table 3). No significant differentiation
was observed between A. bicolor pacifica and
A. japonica or between A. celebesensis and A.
marmorata (p > 0.05; Table 2). Anguilla marmorata
was observed to have the smallest PDL, while A.
bicolor pacifica had the largest. PAL, on the other
hand, did not significantly differ between short- and
long-finned eel species (p > 0.05; Table 2).
The PAL of A. bicolor pacifica ranged
16.77-18.74 (18.04 ± 0.60) mm, while those of
A. japonica, A. marmorata, and A. celebesensis
ranged 15.85-19.46 (18.54 ± 1.56), 17.0223.92 (19.13 ± 1.32), and 15.80-19.40 (17.65 ±
1.58) mm, respectively. No significant differentiation in PAL was observed among species
(ANOVA, p > 0.05; Table 3).
According to the ADL, anguillid eels collected
were classified into a short-finned eel, i.e., A.
bicolor pacifica, with a mean (± SD) ADL of 0.22
± 0.11 (0.08-0.37) mm, which was significantly
smaller than those of long-finned eels, A. japonica
65
6
5
4
3
2
1
0
n = 13
(C)
35
30
25
20
15
10
5
0
n = 184
(D)
2
n=4
1
0
41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
Total length (mm)
Fig. 6. Length frequency distribution of glass eels of Anguilla
japonica (A), A. bicolor pacifica (B), and A. marmorata (C)
caught in the lower reach of the Hsiukuluan River of eastern
Taiwan and A. celebesensis (D) (Tzeng 1982). n = sample
size.
Table 2. Comparison of morphometric characters among the 5 Anguilla species. All values are in
millimeters except for ADL/%TL. Values inside parentheses beside the species names indicate the sample
sizes used for morphometric comparisons. Different superscript letters indicate a significant difference at
p < 0.05
Mean ± S.D. (range) mm
Total length
Pre-dorsal fin length
Pre-anal fin length
Ano-dorsal fin length
ADL/%TL
1
A. japonica (5)
A. bicolor pacifica (13)
A. marmorata (30)
A. celebesensis1 (4)
53.01 ± 4.54 (45.45-57.79)d
13.21 ± 1.59 (12.04-15.05)b
18.54 ± 1.56 (15.85-19.46)a
5.33 ± 1.43 (3.81-7.02)a
10.03 ± 2.43 (8.19-12.97)a
47.29 ± 1.06 (45.86-49.22)b
17.82 ± 0.63 (16.52-18.55)b
18.04 ± 0.60 (16.77-18.74)a
0.22 ± 0.11 (0.08-0.37)a
0.43 ± 0.22 (0.17-0.79)a
49.43 ± 2.32 (46.47-58.04)c
11.43 ± 0.94 (9.00-14.40)a
19.13 ± 1.32 (17.02-23.92)a
7.70 ± 0.95 (6.20-9.91)b
15.57 ± 1.77 (13.27-20.35)b
44.50 ± 3.04 (40.40-47.00)a
13.03 ± 0.50 (12.50-13.70)a
17.65 ± 1.58 (15.80-19.40)a
4.63 ± 1.16 (3.30-5.70)b
10.30 ± 1.97 (8.17-12.13)b
Measurements from Tzeng (1982).
66
had the largest ADL followed by A. japonica, A.
celebesensis, and A. bicolor pacifica. Significant
differentiation in ADL was observed among species
(Kruskal-Wallis, H = 40.18, d.f. = 3, p < 0.001;
Table 2). No significant difference was observed
between A. celebesensis and A. marmorata
or between A. bicolor pacifica and A. japonica
(p > 0.05; Table 2).
The ADL/%TL value of A. marmorata ranged
13.27%-20.35% (15.57% ± 1.77%) while values of
the other eel species ranged 0.17%-0.79% (0.43%
± 0.22%) in A. bicolor pacifica, 8.19%-12.97%
(10.03% ± 2.43%) in A. japonica, and 12.13%18.17% (10.30% ± 1.97%) in A. celebesensis.
Significant differentiation in ADL/%TL values was
observed (Kruskal-Wallis, H = 40.16, d.f. = 3,
p < 0.001; Table 2). No significant difference was
(A)
Pre-dorsal fin length
21
19
17
15
13
11
9
7
5
(B)
Ano-dorsal fin length
14
12
10
8
6
4
2
0
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
Total length
Fig. 7. Relationships among the pre-dorsal fin length, anodorsal fin length, and total length in glass eels of Anguilla
japonica ( ), A. bicolor pacifica ( ), A. marmorata ( ), and A.
celebesensis ( from Tzeng 1982).
found between A. celebesensis and A. marmorata
or between A. japonica and A. bicolor pacifica
(p > 0.05; Table 2).
Figure 7 indicates that the short-finned eel A.
bicolor pacifica can easily be distinguished from
A. marmorata, A. japonica, and A. celebesensis
using body proportions, especially the PDL and
ADL in relation to TL. The PDL/TL (%) (Fig. 7A)
was 36.02%-37.69% (mean, 37.68%) in the shortfinned eel A. bicolor pacifica, while in the longfinned eel species it ranged 19.37%-24.81%
(23.12%) in A. marmorata, 26.49%-26.04%
(24.92%) in A. japonica, and 27.68%-30.94%
(29.28%) in A. celebesensis. On the other hand,
ADL/TL (%) more-significantly differed between
short- and long-finned eel species (p < 0.05, Table
2; Fig. 7). The ADL/TL (%) (Fig. 7B) was 0.17%0.75% (0.46%) in the short-finned eel, A. bicolor
pacifica, while in the long-finned eel species it
ranged 13.34%-17.07% (15.58%) in A. marmorata,
8.38%-12.15% (10.05%) in A. japonica, and
8.17%-12.13% (10.40%) in A. celebesensis.
Comparisons of vertebral counts between
the new species A. luzonensis (Watanabe et
al. 2009) and A. huangi (Teng et al. 2009) and
the supposedly A. celebesensis specimens
described by Tzeng (1982) are shown in table
3. Numbers of vertebrae showed overlapping
counts. Total vertebral counts ranged 101-110
(105.3) in A. celebesensis, 103-107 (104.8) in
A. luzonensis, and 103-106 (104.9) in A. huangi.
Abdominal vertebral counts ranged 39-42 (34.3)
in A. celebesensis, 40-42 (41.1) in A. luzonensis,
and 40-41 (40.6) in A. huangi. Caudal vertebral
counts, on the other hand, ranged 62-68 (65) in A.
celebesensis and 61-66 (63.8) in A. luzonensis.
Caudal vertebral counts of A. huangi were not
available.
Developmental stages
Anguillid eels immigrating to the Hsiukuluan
River were at the transition stage from glass eel
Table 3. Comparison of vertebral counts of Anguilla celebesensis, A. luzonensis, and A. huangi
Species
A. celebesensis a
A. luzonensis b
A. huangi c
a
Vertebral counts
Total
Abdominal
Caudal
101-110 (105.3)
103-107 (104.8)
103-106 (104.9)
39-42 (34.3)
40-42 (41.1)
40-41 (40.6)
62-68 (65)
61-66 (63.8)
N/A
Tzeng (1982), bWatanabe et al. (2009), cTeng et al. (2009). N/A, not available.
to elver with various pigmentation stages (Table
4). At station 1 (river mouth), the majority of
individuals caught were at stage VA (62%) followed
by stages VB (34.1%), VIA1 (2.2%), and VIA2 (1.7%).
These stages were characterized by the respective
absence (stage VA) or presence (stage V B) of a
cerebral nerve-cord spot and pigmentation on the
tail and caudal fin (Bertin 1956). The pigmentation
stage suggested that the glass eels had recently
arrived at the river mouth. On the other hand, at
station 2 (approximately 2 km upstream from the
river mouth), the majority of individuals caught
were at stage VIA1 (35.2%) followed by stages VB
(27.8%), VI A2 (13%), VI A4 (11.1%), VI A3 (7.4%),
VA (3.7%), and VIB (1.9%). Stages VB-VIA4 (elver
stage) were characterized by a progressive
pigmentation on the dorsolateral part of the elver.
Seasonal occurrence
Relative abundances of glass eels varied with
sampling stations, months, and years (Table 1).
Four anguillid eel species were identified based on
caudal pigmentation patterns and morphometric
measurements. Anguilla japonica is a temperate
species, and relatively few (< 1%) were found
in eastern Taiwan, although it is abundant on
the west coast of Taiwan. Anguilla marmorata
was most abundant among the 3 tropical eel
species, making up 98.4% of the total catch,
followed by a very few (1.3%) A. bicolor pacifica.
But because the phylogenetic analysis did not
support the occurrence of the new species and/or
A. celebesensis, the relative abundance of A.
67
marmorata should be more than 98.4%.
It was found that different eel species recruit
to the Hsiukuluan River at different times of the
year: A. marmorata recruited mainly during spring
and summer but was found year-round, while
A. bicolor pacifica recruited during autumn. The
temperate species, A. japonica, recruited mainly
during winter.
DISCUSSION
Pigmentation stage in relation to upstream
migration
The pigmentation stage of glass eels differed
among sampling stations. It was observed that the
stages of glass eels became more advanced as
they moved upstream. Station 1 is brackish water
to seawater, while station 2 is fresh water. It is still
not known whether these changes in pigmentation
stages are due to the effect of salinity change,
a time-delay effect of recruitment, or both. But
the effect of a time delay can be checked by the
difference in the number of otolith daily increments
of elvers between stations.
Why is A. japonica abundant on the west coast
while A. marmorata is abundant on the east
coast of Taiwan?
The species composition of glass eels
recruiting to the Hsiukuluan River, eastern Taiwan,
was dominated by A. marmorata which greatly
Table 4. Pigmentation stages of different Anguilla eel species collected from 2 stations in the lower reach of
the Hsiukuluan River, Taiwan
Station
Species
Pigmentation stage
n
VA
VB
VIA1
VIA2
VIA3
VIA4
VIB
1
A. japonica
A. bicolor pacifica
A. marmorata
Uncertain
Total
% composition
5
12
144
18
179
5
11
85
10
111
62.0
0
1
53
7
61
34.1
0
0
4
0
4
2.2
0
0
2
1
3
1.7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
A. japonica
A. bicolor pacifica
A. marmorata
Uncertain
Total
% composition
0
1
44
9
54
0
0
2
0
2
3.7
0
0
11
3
14
27.8
0
0
17
2
19
35.2
0
0
7
0
7
13.0
0
0
1
3
4
7.4
0
0
5
1
6
11.1
0
0
1
0
1
1.9
68
differs from that reported from rivers in southern,
western, and northern Taiwan where A. japonica
dominates (Tzeng 1982 1983b, Tzeng and Chang
2001). The low relative abundance of A. japonica
in this study was doubtful at first, because it might
have been due to sampling bias due to fisherman
sorting out A. japonica for aquaculture before
handing the glass eel samples to the researchers.
But a recent study by Han et al. (pers. comm.)
in the same river system also revealed that the
relative abundance of A. japonica was very low
(< 1%). But why does the dominant species of
recruiting glass eels differ between the east and
west coasts of Taiwan? This scenario can be
further explained by the different geographical
distributions, temperature preferences, and
habits of the different eel species. During the
leptocephalus stage, A. marmorata and A. japonica
might not have different distributions because they
use the same spawning ground to the west of
Mariana Island, and their larvae are transported by
the NEC and Kuroshio Current to their destinations
(Kuroki et al. 2009, Miller et al. 2009). But after
metamorphosing from leptocephalus to glass
eels, the temperate A. japonica migrates with the
cold China Coastal Current to the west coast of
Taiwan (Cheng and Tzeng 1996), while the tropical
species, A. marmorata, A. bicolor pacifica, and the
newly described A. luzonensis prefer the east coast
which is influenced by the warm Kuroshio Current.
Previous studies also revealed that A. japonica
elvers were more abundant on the northern,
western, and southern coasts of Taiwan than the
east coast (Tzeng 1996, Tzeng and Chang 2001).
Those reports indicated that species-specific
differences in geographical distribution of glass
eels were closely correlated to the coastal current
systems which differ between the east and west
coasts of Taiwan (Tzeng 1996). Also, the speciesspecific recruitment, abundance, and distribution
of A. marmorata and A. japonica in Taiwan can
also be explained by differences in the duration of
their leptocephalus stage, age at metamorphosis,
and growth rate. The somatic growth rate is
faster and the age at metamorphosis is younger
in A. marmorata than A. japonica (Leander et al.
unpubl. data). Thus, the former can recruit earlier
at a younger age. This must be the reason why
A. marmorata can recruit abundantly in northern
Luzon, the Philippines and along the east coast
of Taiwan, while very few A. japonica are known
to recruit there, because the latter is still in the
leptocephalus stage and drifting with the Kuroshio
Current. The drifting A. japonica leptocephali
metamorphose into glass eels beyond Taiwan, and
some of them then enter the westward branch of
the Kuroshio Current that takes them to continental
waters of East Asia where they migrate with the
cold, southerly flowing China Coastal Current to
the northern, western, and southern coasts of
Taiwan. This scenario was validated by the peak
catch of elvers that coincided with the period of the
lowest winter temperatures when the northeastern
monsoon-driven China Coastal Current was
strongest (Tzeng 1985) and from the daily ages of
elvers arriving at estuaries along the west coast of
Taiwan being older in the south than in the north
(Cheng and Tzeng 1996). In addition to this,
Tzeng and Chang (2001) also suggested that the
comparatively abundant freshwater discharges and
wider shelf area along the west coast of Taiwan
can potentially attract elvers to migrate upstream,
unlike on the east coast where conditions might
be less attractive for inshore migration and
recruitment of elvers because the warm Kuroshio
is very close to the shore, the salinity is higher, and
the continental shelf is narrower. Compared to A.
marmorata, the abundance of A. bicolor pacifica
was very low, such that they can be considered
an occasional species. This is because the origin
or the spawning ground of these eel species is far
from Taiwan.
Differences in habitat use and seasonal occurrence
Previous studies indicated that the peak
catch of A. japonica elvers in Taiwan occurred
during winter from Nov. to Feb. (Tzeng 1983b
1996, Tzeng et al. 1995), while the peak catch of
A. marmorata elvers in the Hsiukuluan River in
eastern Taiwan occurred mainly during spring and
summer (Lin 2001, Han et al. unpubl. data). This
difference in the recruitment season also supports
different temperature preferences of these 2
species. In addition, the habitat use of the adult A.
japonica and A. marmorata living sympatrically in a
river also differs (Shiao et al. 2003) with the former
usually occupying lower reaches of the river while
the latter occupies upper reaches.
Differences in recruitment patterns of the
different species of glass eel might be due
to differences in their spawning season and
migration. In temperate eel species, spawning
occurs over a limited period, i.e., Feb. to Apr. in
A. rostrata (McCleave et al. 1987), Mar. to June
in A. anguilla (McCleave and Kleckner 1987), Apr.
to Nov. in A. japonica (Tsukamoto 1990), Aug. to
Dec. in A. dieffenbachi (Jellyman 1987), and Sept.
to Feb. in A. australis (Jellyman 1987). With these
limited spawning periods, recruitment of their glass
eels is therefore limited to certain seasons. On the
other hand, tropical eel species have a spawning
season that persists almost throughout the year,
and this year-round spawning behavior may extend
the period of recruitment of their glass eels to
estuarine habitats to year round, as was described
in previous studies (Tabeta et al. 1976, Arai et
al. 1999a b 2001, Shen and Tzeng 2007). In
addition, fluctuations in daily catches of glass eels
in estuaries are greatly influenced by the spawning
duration, oceanic currents, and differences in early
life history traits such as the age at metamorphosis
and age at recruitment, as well as environmental
cues such as the moon phase, tidal currents, and
water temperature (Tzeng 1985, Cheng and Tzeng
1996, Wang and Tzeng 1998 2000).
Misidentification of A. celebesensis in Taiwan
Previous studies (Tzeng 1982, Tzeng and
Tabeta 1983) reported the occurrence of A.
celebesensis in natural waters of Taiwan, but it
was never found again in any recent studies. It is
possible that A. celebesensis glass eel specimens
described in previous studies from the northern
Philippines, Taiwan, and southern China (Tabeta et
al. 1976, Tzeng 1982, Ozawa et al. 1989, Arai et
al. 1999b 2003, Lue et al. 1999) were misidentified
and were probably A. luzonensis. The leptocephali
of A. celebesensis have never been identified in
the NEC region, and its spawning area was found
to be located in the Celebes Sea and Tomini Bay
of Indonesia which is far from the NEC region
(Kuroki et al. 2006, Watanabe et al. 2009). Also,
recent studies indicated that A. celebesensis
appears to have short spawning migrations and
larval durations (Miller et al. 2009).
The newly described eel species, A.
luzonensis and A. huangi, were synonymous
because they showed a very high degree of
similarity in morphometric characters, which was
also strongly supported by the phylogeny of all
currently recognized species and subspecies
of Anguilla constructed using the entire mtDNA
genome sequence for which sequences of A.
luzonensis and A. huangi exhibited 99%-100%
similarity. Because it is agreed that both names
refer to the same species, therefore these names
should be formally synonymized. According to
Article 23 of the International Code of Zoological
Nomenclature, the precedence between 2 or
69
more names is determined by the dates on which
the works containing the names or acts were
published, unless that name has been invalidated
or another name is given precedence by any
provision of the code or by any ruling of the
commission. In other words, the 1st nomenclatural
act or the 1st published name is given precedence,
and in the case of the new eel species, the name
luzonensis was published in Mar. 2009 while
huangi was published in Nov. 2009, so the name
luzonensis has priority and precedence over the
2nd name. Also, luzonensis is a better name for an
eel species discovered in Luzon, the Philippines.
It seems that the new species was found in
Taiwan before but was probably misidentified as
A. celebesensis in 1982 because of the similarity
in pigmentation patterns and almost identical
morphometric characters (Table 3). Numbers of
vertebrae (total, abdominal, and caudal vertebrae)
measured in A. celebesensis by Tzeng (1982),
A. luzonensis by Watanabe et al. (2009), and
A. huangi by Teng et al. (2009) also showed
overlapping counts (Table 4). Unfortunately,
further comparisons using genetic approaches are
not possible because A. celebesensis specimens
described by Tzeng (1982) are no longer available.
But with the obvious similarities in pigmentation
patterns, morphometric measurements (i.e., PDL,
PAL, and ADL/%TL), and vertebral counts, there
is no doubt that A. celebesensis found by Tzeng
(1982) in natural waters of Taiwan might be the
new eel species, A. luzonensis.
In summary, there are 3 species of anguillid
eels found on the east coast of Taiwan based
on species-specific pigmentation patters and
morphometric measurements. But the results
of the genetic analysis did not support the
occurrence of a 4th species (A. luzonensis and/or
A. celebesensis) indicating that the use of speciesspecific differences in ADL/%TL to distinguish the
new species is not reliable. Further investigations
on the species composition of recruiting anguillid
eels on a wider scale are warranted to validate
the occurrence of A. luzonensis and/or A.
celebesensis in natural waters of Taiwan. The
newly described A. luzonensis might have been
misidentified as A. celebesensis in previous
studies because these species are almost identical
in terms of morphological features. Geographical
distributions differ among glass eels species. In
the Hsiukuluan River, eastern Taiwan, the species
composition of the recruiting anguillid eels was
dominated by A. marmorata with A. japonica and
A. bicolor pacifica as minor species, which greatly
70
differs from what was previously reported in rivers
of northern, western, and southern Taiwan where
A. japonica dominates. The recruitment season
of A. marmorata is mainly from early summer to
autumn but can occur almost year round, while
that of A. japonica is during winter. Anguilla
bicolor pacifica mainly recruits during autumn.
These results suggest that tropical eels have a
unique geographical distribution and recruitment
season which greatly differs from those of the
temperate eel, A. japonica. This information is
essential for fishery regulation and management
implementation.
Acknowledgments: The authors are grateful
to the staff of the Division of Habitats and
Ecosystems, Taiwan Endemic Species Research
Institute (TESRI), (Chichi, Taiwan) for sample
collection, to students and staff of the Fisheries
Biology Laboratory, Institute of Fisheries Science,
National Taiwan Univ. (Taipei, Taiwan) for their
assistance, and to the anonymous reviewers for
providing valuable suggestions on this manuscript.
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Offspring of Older Males are Superior in Drosophila bipectinata
Mysore S. Krishna*, Hassan T. Santhosh, and Shridhar N. Hegde
Drosophila Stock Center, Department of Studies in Zoology, Univ. of Mysore, Manasagangotri, Mysore 560 006, Karnataka, India
Mysore S. Krishna, Hassan T. Santhosh, and Shridhar N. Hegde (2012) Offspring of older males are superior
in Drosophila bipectinata. Zoological Studies 51(1): 72-84. Drosophila bipectinata offspring of old- and youngaged males reared in low (LLD) and high larval densities (HLD) were employed to test a good gene model of
female preference for male age classes. It was noted that with both LLD and HLD, offspring of old-aged male
had significantly greater pre-adult fitness (egg-larval hatchability and larval-adult viability) and adult fitness
(son’s mating success, mating latency, mating ability, progeny production, and longevity, and daughter’s mating
success, fecundity, and longevity) over sons of young-aged males, which suggested that larval density did not
have an effect on the inheritance of characters from parent to offspring. Among larval densities, LLD offspring
performed significantly better in all traits studied than did HLD offspring. Thus, D. bipectinata females prefer oldaged males to obtain greater fitness benefits for their offspring, thereby confirming the good-gene model.
Key words: Female preference, Male age, Pre-adult fitness, Adult fitness.
I
t was widely proposed that in species with
no parental care, females may choose to mate
with older males to obtain good genes for their
offspring (Price and Hansen 1998). In species
of the genus Drosophila, males do not generally
provide parental care or any resources to mating
females except sperm and accessory gland
proteins. Therefore species of Drosophila can be
used as model organisms to test the ‘good-gene’
hypothesis. Food shortages due to larval densities
in Drosophila are known to influence almost all
fitness traits (Ribo et al. 1989); however, it is
unknown whether or not food shortages have any
influence on offspring fitness of female preferences
for male traits. Male traits in Drosophila, such as
size, age, color, pigmentation, courtship song, and
parental care, are known to be used by females in
mating to derive both direct and indirect benefits
(Hegde and Krishna 1997). The expression of
these traits not only depends on the genotype but
also on other factors such as the developmental
environment (i.e., larval density and temperature)
and parental age (Mousseau and Dingle 1991).
The most compelling studies of Drosophila
found that larger males have a higher mating
success than their smaller counterparts, both in
laboratory and field conditions (Partridge et al.
1987, Santos et al. 1992, Hegde and Krishna
1997, Krishna and Hegde 2003). Male age is
another trait known to influence female mating
preferences: some studies found that females of D.
melanogaster and D. pseudobscura prefer to mate
with older-aged males (Moulin et al. 2001, Avent et
al. 2008). One potential explanation is that females
are likely to gain indirect benefits because older
males are highly viable and have demonstrated
their survival ability, and there is a general positive
correlation between male longevity and genetic
viability (Hansen and Price 1995, Kokko 1997,
Beck et al. 2002). Little attention has been paid
to the parental age effect in studies of Drosophila
except for the work of Parsons (1964). Further,
72
Krishna et al. – Male Age Influence on Offspring Fitness in Drosophila
few Drosophila studies on age effects directly
examined the effect of parental age on progeny
fitness; instead they considered physiological
changes associated with changes in parental
age, molecular aspects, selection experiments,
and comparisons of populations generated from
individuals of different ages (Comfort 1953,
Parsons 1962, Wattiaux 1968, Lints and Hoste
1974, Ganetzky and Flanagan 1978, Rose and
Charlesworth 1981, Lubkinbill et al. 1984, Partridge
and Fowler 1992, Roper et al. 1993, Chippindale et
al. 1994, Orr and Sohal 1994). Very few attempts
have been made to study the influence of male
age on offspring fitness in Drosophila (Price and
Hansen 1998, Avent et al. 2008). Even in those
studies, a single fitness trait was studied, and
therefore it is difficult to test the good-gene model
of female preference for male age classes.
Herein, we approached this issue using D.
bipectinata. In this species, females prefer to mate
with old-aged males more frequently over youngaged males (Somashekar and Krishna 2011).
Therefore in the present study, offspring (sons and
daughters) of young- and old-aged males mated
separately with 5-6-d-old virgin females were used
to test the good-gene model (Arnold 1983) in low
(LLD) and high larval density (HLD) situations.
Experimental stock
An experimental stock of D. bipectinata
established from progeny obtained from 3
isofemale lines collected at Mysore, Dharwad, and
73
Bellur was used. This stock was cultured using
40 flies (20 males and 20 females) in half-pint
milk bottles (250 ml) containing wheat cream agar
medium in 21 ± 1°C and a relative humidity of 70%
using a 12: 12-h light: dark cycle for 3 generations.
Assignment of age classes
Before assigning age classes to males, the
longevity of unmated males of the experimental
stock was studied. Unmated males were transferred separately and individually into a vial
containing wheat cream agar medium and
maintained there in the same laboratory conditions
as described above. These flies were transferred
to a new vial containing wheat cream agar medium
once a week, and this process was continued until
the death of the fly. Fifty replicates were run, and
the mean longevity of unmated males (number of
days a male lived from the time of its eclosion) of
D. bipectinata was calculated. It was found that
the mean longevity of D. bipectinata was 58 ±
5 d. Furthermore, males of D. bipectinata showed
courtship activities within 11-12 h of their eclosion,
and they remained sexually active for 50-55 d
(Table 1). However, in nature, it is very difficult for
the flies to live for even 50 d. Hence, we assigned
male age classes as follows: young-aged males
were 2-3 d old, and old-aged males were 46-47 d
old. In our study, 90% of unmated males survived
for more than 50 d.
The first emerging flies were aged for 47-48 d.
When these flies reached 44 d old, the next set of
flies was isolated and was aged for 2-3 d. These
young- and old-aged males were separately mated
with 5-6-d-old virgin females to obtain offspring
Table 1. Courtship activities (no.) of 11-12-h- and 50-55-d-old males of D. bipectinata
Parameter
Male courtship activities
Tapping
Scissoring
Vibrating
Licking
Circling
Female courtship activities
Ignoring
Extruding
Decamping
Male age
11-12 h
50-55 d
t value
9.32 ± 0.31
9.68 ± 0.45
9.10 ± 0.26
3.76 ± 0.17
3.34 ± 0.26
14.28 ± 0.27
13.98 ± 0.61
14.40 ± 0.35
7.46 ± 0.18
5.88 ± 0.17
11.966**
5.594*
11.968**
14.976**
7.944*
7.34 ± 0.23
5.68 ± 0.27
3.82 ± 0.16
3.60 ± 0.24
2.42 ± 0.16
1.86 ± 0.11
11.241**
10.184**
9.731*
* Significant at p < 0.01 and ** p < 0.001.
74
which were cultured in the same environment.
This experimental design eliminated the problem
of testing the flies at different times. However with
this experimental design (horizontal variation), it is
impossible to exclude differences in the histories
of young- and old-aged males. Furthermore, 90%
of published works studying horizontal variations
used the same experimental design.
Male age influence on offspring pre-adult fitness
Eggs were collected from separately crossing
young- and old-aged males with 5-6-d-old virgin
females using Delcour’s procedure (1969). One
hundred eggs were seeded in a vial (7.6 × 2.5 cm)
containing wheat cream agar medium to create an
LLD. Similarly, 400 eggs were seeded in a vial (7.6
× 2.5 cm) containing wheat cream agar medium
to create an HLD. These vials were cultured and
maintained in laboratory conditions as described
above. The number of eggs hatching into 1st
instar larvae was counted. Then 100 1st instar
larvae were transferred to a new vial containing
wheat cream agar medium, and the number of
progeny that emerged from these larvae was also
counted. This was considered the larva-adult
viability. In total, 50 replicates were created for
offspring of young- and old-aged males. A twoway analysis of variance (ANOVA) of a general
linear model was used on offspring egg-larval
hatchability and larval-adult viability from the SPSS
10.0 program (SPSS, Chicago, IL, USA).
Experiment 1: Male age influence on the son’s
mating success using a female choice experiment with LLD and HLD
Effect of paint on the son’s mating success
The effect of paint on the son’s mating
success was tested before commencing the
experiment by painting one of 2 sons of youngand old-aged males, and allowing them to mate
for 1 h. Fifty replicates were used, and the results
indicated that painting one of the competing sons
in a female-choice experiment had no effect on the
performance of the flies (Table 2).
To study the influence of the male age on the
son’s mating success, in each trial, a 5-6-d-old
virgin female (obtained from the main experimental
stock) along with 2 unmated 5-6-d-old sons of
young- and old-aged males were individually
aspirated into an Elens-Wattiaux mating chamber
(1964). The thorax of sons of the young-aged
male was painted with Indian ink in 1 trial, while in
the other trial, the thorax of sons of the old-aged
male was painted with Indian ink following the
procedure of Hegde and Krishna (1997), and then
the males were observed for 1 h. When mating
occurred, copulating pairs were aspirated out from
the mating chamber. Sons rejected by females
in the female mate-choice experiment were also
individually transferred to new vials containing
wheat cream agar medium. Both selected and
rejected sons by females in the female matechoice experiment were later used to measure
wing length following the procedure of Hegde and
Krishna (1997). Fifty replicates were used for each
combination in the female mate-choice experiment,
and a Chi-squared analysis was applied, a paired
t-test was also run on the mean wing length of
selected and rejected sons. Separate experiments
were carried out for both LLD and HLD.
Experiment 2: Male age influence on son’s
mating latency and copulation duration
A son at 5-6 d old and a 5-6-d-old virgin
female (obtained from the main experiment stock)
were individually aspirated into an Elens-Wattiaux
chamber and observed for 1 h. Any pair that had
not mated within 1 h was discarded. If mating
occurred, we recorded the mating latency (time
between the introduction of the male and female
together into the mating chamber until initiation of
copulation of the pair) and copulation duration (time
Table 2. Effects of paint on the mating success of sons of young- and old-aged males of D. bipectinata
Female
5-6 d
5-6 d
ns
, non-significant.
Sons of different male age classes
Young, Young
Old, Old
Mating success (%)
Painted male
Non-painted male
χ2 value
23 (46%)
28 (56%)
27 (54%)
22 (44%)
0.32ns
0.72ns
75
between initiation of copulation to termination of
copulation by the pair).
Experiment 3: Daughter fitness mating success
and fecundity
Male age influence on progeny production
Effect of painting on the daughter’s mating
success
Soon after copulation, the mated female as in
experiment 2 was individually transferred to a new
vial containing wheat cream agar medium that was
refreshed every 5 d, and the number of progeny
obtained was recorded.
Male age influence on the son’s mating ability
Soon after mating as in experiment 2,
a mated son was allowed to mate with a 2nd
female (a 5-6-d-old virgin female obtained from
the experimental stock). If mating occurred with
the 2nd female, the pair was allowed to complete
copulation, and then the female was checked for
insemination as described above. This process
was continued, and the number of females
inseminated by each son in 1 h was recorded
as the son’s mating ability (number of females
inseminated by each son in 1 h).
Male age influence on son’s longevity
Soon after recording the sons’ mating ability,
these sons were individually transferred to new
vials containing food medium which was refreshed
once a week until their death to record the
longevity.
Fifty replicates were separately conducted
for each of the sons of young- and old-aged males
to examine such parameters as the son’s mating
latency, copulation duration, progeny production,
mating ability, and longevity. Experiments were
also carried out separately for LLD and HLD. A
two-way ANOVA was used on these data using the
SPSS 10.0 program.
The effect of paint on a daughter’s mating
success was tested before commencing the
experiment by painting one of 2 daughters of
young- and old-ages males and allowing them
to mate for 1 h. Fifty replicates were used, and
the results indicated that painting of one of the 2
daughters in the male-choice experiment did not
have an effect on the performance of the flies (Table
3).
In each trial, a 5-6-d-old unmated male
(obtained from the main experimental stock) and
2 virgin 5-6-d-old daughters of young- and oldaged males were individually aspirated into an
Elens-Wattiaux mating chamber (1964). The
thorax of a daughter of a young-aged male was
painted with Indian ink in 1 trial, while in the other
trial, that of a daughter of an old-aged male was
painted with Indian ink following the procedure
of Hegde and Krishna (1997), and then the flies
were observed for 1 h. When mating occurred,
copulating pairs were aspirated out of the mating
chamber. Fifty separate replicates were each used
for sons of young- and old-aged males. Separate
experiments were carried out for LLD and HLD. A
Chi-square analysis was applied to the daughter’s
mating success data.
Experiment 4: Male age influence on the daughter’s fecundity
Daughters at 5-6 d old of young- and old-aged
males and 5-6-d-old unmated males (obtained
from the main experiment stock) were individually
aspirated into an Elens-Wattiaux chamber and
observed for 1 h. Any pair which did not mate
within 1 h was discarded. If mating occurred,
then soon after copulation, the mated female was
Table 3. Effects of paint on the mating success of daughters of young- and old-aged males of D. bipectinata
Male
5-6 d
5-6 d
, non-significant.
ns
Daughters of different male age classes
Young, Young
Old, Old
Mating success (%)
Painted male
Non-painted male
χ2 value
24 (48%)
27 (54%)
26 (52%)
23 (46%)
0.08ns
0.32ns
76
individually transferred to a new vial containing
wheat cream agar medium that was refreshed
once every 24 h until her death. The number of
eggs obtained was counted, and the daughter’s
longevity was recorded. Fifty separate replicates
were used for daughters of young- and old-aged
male classes. Experiments were carried out
separately for LLD and HLD. A two-way ANOVA
in the SPSS 10.0 program was used on the above
data.
Male age influence on the son’s mating success
RESULTS
Male age influence on offspring pre-adult fitness
Egg-larval hatchability / larva-adult viability (in numbers)
Figure 1 shows pre-adult fitness in terms of
the mean egg to larval hatchability and larval to
adult viability in offspring of young- and old-aged
males. It was noted that the offspring of old-aged
males had greater pre-adult fitness in both LLD
F value for Egg-larval hatchability
F1 = 109.467, d.f. = 1,196; P < 0.001
F2 = 196.648, d.f. = 1,196; P < 0.001
F3 = 0.644, d.f. = 1,196; P > 0.05
F vaalue for Larval-adult viability
F1 = 54.334, d.f. = 1,196; P < 0.001
F2 = 56.646, d.f. = 1,196; P < 0.001
F3 = 5.662, d.f. = 1,196; P > 0.05
80
LLD
HLD
70
60
50
40
30
20
10
0
offspring of young offspring of old age offspring of young offspring of old age
age male
male
age male
male
Hatchability (in %)
and HLD than did offspring of young-aged males.
Pre-adult fitness with the LLD was greater than
that with the HLD. Significant variations in egg
to larval hatchability and larval to adult viability
were found between offspring of young- and oldaged males and also between the LLD and HLD.
But an insignificant variation was found for the
interaction of male age and larval density by a twoway ANOVA of the general linear model using the
SPSS 10.0 program.
Viability (in %)
Fig. 1. Male age influence on offspring pre-adult fitness (in %)
in low (LLD) and high larval densities (HLD) of D. bipectinata.
F1, F value between age classes; F2, F value between larval
densities; F3, F value for the interaction between age classes
and larval densities.
Females of D. bipectinata chose to mate
with sons of old-aged males more frequently than
sons of young-aged males (Table 4). The mating
success of sons of old-aged males was 80%
(n = 50) in an LLD and 76% (n = 50) in an HLD,
while mating success of sons of young-aged males
was 20% (n = 50) in an LLD and 24% in an HLD
when sons of young- and old-aged males were
involved in the crosses. A Chi-squared analysis
applied to the mating success data showed
significant variations between sons of young- and
old-aged males and also between the LLD and
HLD.
Mean wing lengths of selected and rejected
sons by females in LLD and HLD of D. bipectinata
are given in table 5. In both LLD and HLD for all
combinations, the mean wing length of rejected
sons was slightly longer than that of selected sons,
but the difference was insignificant between the
mean wing lengths of selected and rejected sons.
Male age influence on the son’s mating
activities and longevity
Mean values of the mating latency and
copulation duration of sons of old- and young-aged
males are given in figure 2. Sons of old-aged
males showed less time for mating latency and
copulated longer compared to sons of young-aged
males. Among larval densities, flies from the LLD
Table 4. Mating success (%) of sons of young- and old-aged males in low (LLD) and high larval
densities (HLD) of D. bipectinata
Larval density
LLD
HLD
** Significant at p < 0.001.
Sons of young-aged males
Sons of old-aged males
χ2 value
20%
24%
80%
76%
18.0**
12.5**
took a shorter time to begin mating and copulated
longer than flies from the HLD. A two-way ANOVA
using the general linear model was carried out
on the above parameters and showed significant
variations between sons of young- and old-aged
males, between LLD and HLD, and also for the
interaction between male age classes and larval
densities.
Figure 3 shows the mean sons’ mating
abilities of young- and old-aged males of D.
bipectinata. It was noted that sons of old-aged
males inseminated a greater number of females
in a given unit of time than sons of young-aged
males. The sons’ mating ability data subjected to
a two-way ANOVA using the general linear model
showed significant variations between sons of
young- and old-aged males and between larval
densities, but an insignificant variation was noted
for the interaction between male age classes and
larval densities.
Mean longevity data of sons of young- and
LLD
HLD
10
8
6
4
2
Sons of young age
male
Sons of old age
male
Mating latency (in min)
Male age influence on fecundity and fertility of
females mated with sons of young- and oldaged males
Mean values of fecundity and fertility of
females mated with sons of young- and old-aged
males in the LLD and HLD of D. bipectinata are
given in figure 5. The fecundity and fertility of
females mated with sons of old-aged males were
found to be greater than those of females mated
with sons of young-aged males. The fecundity
F value for Copulation duration
F1 = 34.44, d.f. = 1,196; P < 0.001
F2 = 40.31, d.f. = 1,196; P < 0.001
F3 = 7.83, d.f. = 1,196; P < 0.05
12
0
old-aged males are given in figure 4. Sons of
old-aged males lived longer compared to sons of
young-aged males. A two-way ANOVA using the
general linear model on the sons’ longevity data
showed significant variations between sons of
young- and old-aged males and between larval
densities, but an insignificant variation was found
for the interaction between male age classes and
larval densities.
Sons of young age
male
F1 = 23.70, d.f. = 1,196; P < 0.001
F2 = 14.91, d.f. = 1,196; P < 0.01
F3 = 0.00, d.f. = 1,196; P > 0.5
Mean ± SE of Son mating abiliity (in numbers)
Mating latency / copulation duration (in minutes)
F value for Mating latency
F1 = 106.5, d.f. = 1,196; P < 0.001
F2 = 2.12, d.f. = 1,196; P > 0.05
F3 = 18.55, d.f. = 1,196; P < 0.01
Sons of old age
male
3.5
LLD
HLD
3
2.5
2
1.5
1
0.5
0
Sons of young age male
Sons of old age male
Son mating ability (in no.)
Copulation duration (in min)
Fig. 2. Male age influence on the son’s mating latency (in min)
and copulation duration (in min) in low (LLD) and high larval
densities (HLD) of D. bipectinata. F1, F value between age
classes; F2, F value between larval densities; F3, F value for
the interaction between age classes and larval densities.
77
Fig. 3. Male age influence on the son’s mating ability (in no.)
in low (LLD) and high larval densities (HLD) of D. bipectinata.
Table 5. Mean wing length (mm) of selected and rejected sons of young- and old-aged males in low (LLD)
and high larval densities (HLD) of D. bipectinata
Larval density value
Females
Males
Wing length of selected sons (mm)
Wing length of rejected sons (mm)
t
LLD
5-6 d old
Young, Old
1.596 ± 0.013
1.597 ± 0.015
0.103NS
HLD
5-6 d old
Young, Old
1.46 ± 0.009
1.47 ± 0.009
1.42NS
, non-significant.
NS
78
and fertility of flies from the LLD were found to be
greater than those of flies from the HLD. A twoway ANOVA using the general linear model applied
to the fecundity and fertility data showed significant
variations between young- and old-aged males
and between the LLD and HLD. However, an
insignificant variation was found for the interaction
between sons of male age classes and larval
densities.
Male age influence on the daughter’s mating
success
Table 6 gives the mating success data
of daughters of young- and old-aged males.
Daughters of old-aged males had greater mating
success than daughters of young-aged males. The
mating success rates daughters of old-aged males
were 74% in the LLD and 70% in the HLD (n = 50),
while those of young-aged males were 26% in the
LLD and 30% in the HLD. Mating success data of
daughters of young- and old-aged males showed
significant variations between daughters of youngand old-aged males and also between the LLD
and HLD.
Male age influence on the daughter’s fecundity
Table 7 and figure 6 shows the mean fecundity of daughters of young- and old-aged males
in the LLD and HLD. It was noted that daughters
of old-aged males had greater fecundity compared
to daughters of young-aged males. Among larval
densities, flies from the LLD had greater fecundity
compared to those from the HLD. Significant
variations were found in the fecundity of daughters
of young- and old-aged males and between the
LLD and HLD. Insignificant variations were found
in fecundity from the interaction between sons of
male age classes and larval densities.
Similar results was also found in mean
longevity of daughters of different male age
F1 = 21.88, d.f. = 1,196; P < 0.001
F2 = 43.94, d.f. = 1,196; P < 0.001
F3 = 0.819, d.f. = 1,196; P > 0.05
LLD
HLD
60
50
40
30
20
10
0
F value of Fertility
F1 = 143.10, d.f. =1,196; P < 0.001
F2 = 58.26, d.f. =1,196; P < 0.001
F3 = 3.71, d.f. =1,196; P > 0.05
250
70
Fecundity / Fertility (in numbers)
Mean ± SE of son's longevity (in days)
80
F value of Fecundity
F1 = 74.57, d.f. = 1,196; P < 0.001
F2 = 23.77, d.f. = 1,196; P < 0.001
F3 = 0.12, d.f. = 1,196; P > 0.05
Sons of old age male
Son’s longevity (in days)
150
100
50
Sons of young age
male
Sons of old age Sons of young age
male
male
Fecundity (in no.)
Fig. 4. Male age influence on the son’s longevity (in d) in low
(LLD) and high larval densities (HLD) of D. bipectinata. F1,
F value between age classes; F2, F value between larval
HLD
200
0
Sons of young age male
LLD
Sons of old age
male
Fertility (in no.)
Fig. 5. Fecundity (in no.) and fertility (in no.) of females mated
with sons of young- and old-aged males in low (LLD) and high
larval densities (HLD) of D. bipectinata. F1, F value between
age classes; F2, F value between larval densities; F3, F value
for the interaction between age classes and larval densities.
Table 6. Mating success (%) of daughters of young- and old-aged males in low (LLD) and high larval
densities (HLD) of D. bipectinata
Larval density
Daughters of young-aged males
Daughters ofold-aged males
χ2 value
LLD
26%
74%
11.52**
HLD
30%
70%
8.0**
classes in the LLD and HLD (Table 7 and Fig. 7).
DISCUSSION
In D. bipectinata, offspring of females mated
with young- and old-aged males reared in LLD
and HLD were analyzed to test the good-gene
model associated with female preference for male
age on the one hand and whether or not a father
produces successful offspring on the other. Lifehistory traits such as pre-adult traits (egg to larval
hatchability and larva to adult viability) and adult
fitness traits (son’s mating success, mating ability,
progeny production, and longevity and daughter’s
mating success, fecundity, and longevity) are
important components of fitness traits that a father
can give to his offspring. Figure 1 reveals that in
both LLD and HLD, offspring of old-aged males
F1 = 188.993, d.f. = 1,196; P < 0.001
F2 = 223.09, d.f. = 1,196; P < 0.0001
F3 = 0.347, d.f. = 1,196; P > 0.05
F1 = 129.62, d.f. = 1,196; P < 0.001
F2 = 295.45, d.f. = 1,196; P < 0.0001
F3 = 0.445, d.f. = 1,196; P > 0.05
LLD
HLD
200
150
100
50
0
had significantly greater egg to larval hatchability
and larval to adult viability compared to offspring
of young-aged males. This suggests that offspring
pre-adult fitness increases with increasing male
age. Our results also support the work of Pervez
et al. (2004), who while studying a predatory
ladybug, demonstrated a positive effect of male
age on egg viability, and they found that eggs
sired by 20-30-d-old males had significantly higher
viability than those sired by younger males. In
contrast to this, in D. melanogaster, Price and
Hansen (1998) found decreased larval viability in
eggs sired by older males compared to younger
males. Similarly, the negative effects of male
age on offspring fitness were also shown in other
species. Serre and Robaire (1998) reported a
significantly higher neonatal death rate in progeny
of older male Norway rats. Jones et al. (2000)
observed a higher egg hatching success in lekking
Daughters of young age male
Daughters of old age male
Mean ± SE of daughter's longevity (in days)
Mean ± SE of daughter's fecundity (in numbers)
250
79
70
LLD
HLD
60
50
40
30
20
10
0
Daughter’s fecundity (in no.)
Daughters of young age male
Daughters of old age male
Daughter’s longevity (in days)
Fig. 6. Male age influence on daughter’s fecundity (in no.) in
low (LLD) and high larval densities (HLD) of D. bipectinata.
Fig. 7. Male age influence on daughter’s longevity (in d) in
low (LLD) and high larval densities (HLD) of D. bipectinata.
Table 7. Fecundity (no.) and longevity (d) of daughters of young- and old-aged males in low (LLD) and high
larval densities (HLD) of D. bipectinata
Parameter value
Fecundity (no.)
Longevity (d)
LLD
HLD
t value
LLD
HLD
t value
Daughters of young-aged males
Daughters of old-aged males
t
171.52 ± 1.88
143.28 ± 1.61
11.32**
55.34 ± 0.69
40.88 ± 0.75
13.76**
199.74 ± 2.03
169.18 ± 2.27
10.10**
64.02 ± 0.71
50.64 ± 1.03
9.98**
9.80**
10.45**
8.49**
7.86**
80
sandfly females mated to young- and middleaged than to older males. In the present study,
greater egg to larval hatching ability and larval to
adult viability of eggs sired by female mated with
old male may reflect the higher quality of zygotes
sired by older fathers. However, like in Jones et al.
(2000), better egg hatchability might be a result of
fertilization success.
The reproductive success of offspring of
young- or old-aged males depends on their preadult fitness and also on adult traits, i.e., son
and daughter success in mating, mating ability,
progeny production, and longevity (Krishna and
Hegde 2003). Table 4 shows that females of
D. bipectinata were able to discriminate sons of
young- and old-age classes, and they preferred
to mate more frequently with sons of old-aged
males over sons of young-aged males. This
result was found to be similar in both LLD and
HLD suggesting that the inheritance of successful
mating traits from father to son was not influenced
by larval density. The observed greater mating
success of sons of old-aged males could be due
to differences in the attractiveness of sons of
young- and old-aged males. Studies showed that
females that mated with attractive males provided
better fitness benefits to their offspring such as
greater longevity (Norris 1993, Petrie 1994), faster
growth rate (Reynolds and Gross 1992), increased
fecundity of daughters (Reynolds and Gross 1992,
Moore 1994), and increased attractiveness of sons
(Weldell and Tregenza 1999). In species in which
male attractiveness is not associated with many
morphological features, male attractiveness can be
measured using components involved in courtship
behavior, i.e., mating latency, copulation duration,
and levels of activities during courtship, which
can be used to measure the attractiveness of the
male (Hegde and Krishna 1997). Males which
show greater activities during courtship are more
attractive (Hegde and Krishna 1997). Species
of the genus Drosophila do not show body color,
pigmentation, or morphological feature variations
with male age or attractiveness. Hence the activity
level of courting males may be used to indicate the
attractiveness of males. In D. bipectinata, sons
of old-aged males showed greater activity; they
mated faster and copulated longer than sons of
young-aged males during courtship, suggesting
that sons of old-aged males were more attractive
than sons of young-aged males. Even in the
parental generation of D. bipectinata, old-aged
males had greater courtship activities compared
to young-aged males suggesting that old-aged
males were more attractive than younger-aged
males (Somashekar and Krishna 2011). This
attractiveness was also passed on to their sons.
Our results support the argument of Fisher
(1950) who while working on D. melanogaster,
pointed out that successful fathers could produce
successful sons and pass a legacy of greater
mating success on to their offspring. Our results
in D. bipectinata also support studies of females
which mated with attractive males: they provided
fitness benefits to their offspring in the form of
greater longevity (Norris 1993, Petrie 1994), faster
growth rates (Reynolds and Gross 1992, Moore
1994, Welch et al. 1998), increased fecundity of
daughters (Reynolds and Gross 1992, Moore
1994), and increased attractiveness of sons
(Wedell and Tregenza 1999).
Since 5-6-d-old sons of young- and oldaged males were cultured and maintained in the
same temperature and environmental conditions,
the observed greater mating success cannot
be attributed to difference in mating history,
experience, or maintenance of sons of different
male age classes; instead, it could be due to
the difference in the male age classes. Another
study of Drosophila also showed that male flies
which inseminate more females in a given time
also produce more progeny than male flies which
inseminate a smaller number of females (Krishna
and Hegde 2003). In D. bipectinata, we noted that
sons of old-aged males inseminated more females
in a given time and lived longer than sons of
young-aged males (Figs. 3, 4). This suggests that
sons of old-aged males had greater reproductive
success than sons of young-aged males.
In D. bipectinata, the observed indirect
genetic benefits might have been achieved either
by passing good genes on to their offspring, thus
ensuring more-viable sons and daughters or
the heritability of male attractiveness, i.e., moreattractive fathers sired more-attractive sons. In
contrast to this in D. pseudobscura, Avent et al.
(2008) found that females of D. pseudobscura
could not discriminate sons of young- or old-aged
males. Even in D. melanogaster, the mating ability
of old-aged males was found to be less than that
of sons of young- and intermediate-aged males
(Hansen and Price 1995). This suggests that
species-specific differences may exist with regard
to the influence of male age on offspring fitness.
We also noted that females that mated with sons of
old-aged males had significantly greater fecundity
and fertility than those mated with sons of youngaged males (Fig. 5). Thus, these studies on D.
bipectinata confirm that successful fathers produce
successful sons.
Daughters of old-aged males had greater
mating success, fecundity, and longevity in both
LLD and HLD than daughters of young-aged males
(Tables 6, 7, Figs. 6, 7). This confirms that in D.
bipectinata, females select old-aged males in their
parental generation to obtain better offspring. The
notion that mating with aged males may impose
costs to the female somehow contributes to the
view that male age is an honest signal of male
genetic quality, because older males have proven
their superior ability to survive (Manning 1985).
This was not true in our study, as D. bipectinata
females mating with old-aged males might not
incur costs to the female; instead, females
preferred to mate with old-aged male than youngaged males. Therefore in D. bipectinata, male
age is an honest signal of male genetic quality. A
female preference for old males was demonstrated
in several species.
Among larval densities, offspring of old- and
young-aged males in an LLD had significantly
greater egg to larval hatchability and larval to adult
viability compared to offspring in an HLD (Fig.
1). This suggests that rearing larval density has
a significant influence on egg to larval hatchability
and larval to adult viability. This supports earlier
studies on the influence of larval density on eggadult viability in Drosophila (Barker 1973, Barker
and Podger 1970). Thus in D. bipectinata,
although food shortages due to larval rearing
densities influence offspring pre-adult fitness, it did
not influence the inheritance of characters from
father to offspring.
Thus pre-adult and adult fitness traits of D.
bipectinata in LLD and HLD suggest that offspring
fitness increases with increased male age. In
contrast to results of Hansen and Price (1995), our
results in D. bipectinata support the good-gene
model because preferred old age males provide
indirect benefits through the production of higherquality offspring. Hence in D. bipectinata, females
can use age as a reliable signal of heritable
variations in male quality.
The experimental design eliminated potential
maternal effects by mating 5-6-d-old females
to young- and old-aged males. The results of
our experiments in D. bipectinata are not in
accordance with mutation accumulations in the
germ line as the sole cause of genetic differences
between ages, as there was no evidence for
reduced pre-adult or adult fitness traits. Instead,
we found increased offspring fitness. Furthermore,
81
this increased offspring fitness may also be
attributed to the removal of deleterious mutation as
male age increases. On the other hand beneficial
mutations might have increased with increasing
male age.
According to Hansen and Price (1999), age
and sex variations in the mutation load are very
dependent on the average effect of new mutations,
and they argued that the mutation load most likely
increases with age. Our results in D. bipectinata
do not agree with this; instead deleterious mutation
appeared to have been weeded out as males
aged. Therefore offspring of old age males had
greater fitness.
Jones et al. (2007), while studying hide
beetles, found that sperm viability and sperm
transfer vary with male age but were smaller
than those of an intermediate age. It is not
known whether the observed greater egg to
larval hatchability and larval to adult viability in D.
bipectinata can account for the total sperm viability
or quantity of sperm transferred with male age.
Studies of D. melanogaster found that the sex
ratio of offspring produced by females was biased
with respect to the age of males to which they
were mated (Long and Pischedda 2005). They
showed that females mated to old males produced
a greater proportion of daughters than did females
mated to young males. In the present study in
D. bipectinata, we did not count the numbers of
daughters and sons produced by females mated
with sons of either young- or old-aged males. It
is not known whether the sex ratio of offspring
produced by females of D. bipectinata was biased
with respect to the age of males.
In hide beetles, Jone and Elgar (2004) found
that intermediate-aged males had greater mating
success, and females mated to intermediateaged males had greater fecundity and fertilization
success, but they did not study offspring fitness.
In contrast in D. bipectinata, females mated to oldaged males had greater fitness traits (i.e., fecundity
and fertility) than females mated to young-aged
males. In the present study, even the offspring of
old-aged males had greater fitness. This suggests
that the female preference for male age varies
among different species and genera. Furthermore,
Liu et al. (2011) also showed that female cabbage
beetles did not discriminate their partners on the
basis of age classes.
In the Mexican fruit fly, Perez-Staples et
al. (2010) found that females did not receive
direct benefits by mating with old and sexually
experienced males but may have obtained indirect
82
benefits. In contrast in D. bipectinata, females
mating with old-aged males received direct
benefits in term of greater fecundity and fertility
and also indirect benefits in terms of greater
offspring fitness.
It was suggested that if deleterious mutations
accumulate in the germline with age, they will
decrease the genetic quality of sperm and impose
a cost on female fitness. If these mutations also
affect sperm’s competitive ability or production,
then females will benefit from polyandry instead
of a preference for male age (Radwan 2003,
Gasparini et al. 2010). Female multiple mating
is also common in Drosophila; however, it is not
known whether females of D. bipectinata which
first mate with old-aged males will undergo
multiple mating more frequently or not. In contrast
to our study, Prokop et al. (2007) found decreased
offspring fitness of female bulb mites mated with
old-aged males compared to females mated with
sons of young-aged males.
In sexual selection, it was noted that many
sexual displays, i.e., song traits, had a great
influence on female mate preferences and were
found to vary with male age (Verburgt et al. 2011).
But it is not known whether or not offspring of
different male age classes show variations in
secondary sexual characters that are paternally
inherited.
In the present study, both pre-adult and adult
fitness components were found to be significantly
greater in offspring of old-aged males compared
to offspring of young-aged males. This suggests
that there was an increased in breeding values
of all the life-history traits of old-aged males
studied. Our result do not support the work of
Price and Hansen (1998) who suggested that
the breeding value of D. melanogaster was
reduced with an increasing male age. However,
Price and Hansen (1998) studied only 3 different
characters of egg-adult viability, son mating ability,
and daughter fecundity. Two of the 3 characters
showed decreased values in offspring of old-aged
males compared to offspring and intermediateaged males. The Hansen and Price (1995) model
was based on quantitative genetics and did not
consider genetic details. However in 1999 while
studying age and sex distributions of the mutation
load, Hansen and Price assumed that mutations
have an overall deleterious effect on the total
fitness components.
In Drosophila, mutations with a large effect
were excluded as only 2%-5% of all Drosophila
zygotes carry a new lethal mutation (Crow
and Simmons 1983). Kondrashov and Houle
(1994) showed that the deleterious effects of
mutations can be elevated in harsh environments.
Furthermore, the majority of mutations in
Drosophila may be caused by transposable
elements (Green 1988), and these mutations may
typically have weak deleterious effects (Keightley
1996).
Fitness distributions with male age in D.
bipectinata also showed that fitness parameters
increased with increasing age.
Life-history theories or tradeoff models which
predict negative genetic correlations between
fitness components may be common (Charlesworth
1990, Houle 1991). In the present study, we tested
11 different fitness components (four of sons,
three of daughters, and two of pre-adult fitness)
in offspring of young- and old-aged males. In D.
bipectinata, we noted that there was no tradeoff in
8 different fitness components between offspring
of young- and old-aged males. Therefore, our
results do not agree with the tradeoff model, and
instead support the view of Manning (1985) and
other hypotheses of increasing fitness of offspring
with increased male age. The present study also
supports an earlier study of Partridge (1980) who
found increased larval viability among offspring of
females that were allowed to choose their mates.
Thus in D. bipectinata, females select old-aged
males to obtain indirect genetic benefits, which
supports the good-gene model.
Acknowledgments: The authors are grateful to
the Chairman, Department of Studies in Zoology,
Univ. of Mysore, for providing facilities. Dr. M.S.
Krishna is also grateful to the University Grants
Commission, New Delhi, India, for financial
assistance (through a major research project) to
carry out this work. We are also highly grateful
to the editor and 2 anonymous referees for their
valuable suggestions to our manuscript.
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Reproductive Isolation among Acropora Species (Scleractinia:
Acroporidae) in a Marginal Coral Assemblage
Nuwei Vivian Wei1,2, Hernyi Justin Hsieh3, Chang-Feng Dai2, Carden C. Wallace4, Andrew H. Baird5,
and Chaolun Allen Chen1,2,6,*
Biodversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan
Institute of Oceanography, National Taiwan Univ., Taipei 106, Taiwan
3
Marine Biology Research Center, Taiwan Fishery Research Institute, Penghu 880, Taiwan
4
Museum of Tropical Queensland, Townsville Q4810, Australia
5
ARC Centre of Excellence for Coral Reef Studies, James Cook Univ., Townsville Q4811, Australia
6
Life Science Institute, National Taitung Univ., Taitung 904, Taiwan
1
2
Nuwei Vivian Wei, Hernyi Justin Hsieh, Chang-Feng Dai, Carden C. Wallace, Andrew H. Baird, and
Chaolun Allen Chen (2012) Reproductive isolation among Acropora species (Scleractinia: Acroporidae)
in a marginal coral assemblage. Zoological Studies 51(1): 85-92. Hybridization was proposed as being an
important source of evolutionary novelty in broadcast-spawning reef-building corals. In addition, hybridization
was hypothesized to be more frequent at the periphery of species’ ranges and in marginal habitats. We tested
the potential for hybridization in 2 ways: observations of the time of spawning and non-choice interspecific
fertilization experiments of 4 sympatric Acropora species in a non-reefal coral assemblage at Chinwan Inner
Bay (CIB), Penghu Is., Taiwan. We found that colonies of more than 1 species rarely released gametes at the
same time, thus limiting the opportunities for cross-fertilization in the wild. On the few occasions when different
species released gametes in synchrony, interspecific fertilization in experimental crosses was uniformly low (the
proportion of eggs fertilized ranged 0%-4.58% with a mode of 0%), and interspecific-crossed embryos ceased
development and died within 12 h after initially being fertilized. Ecological and experimental analyses indicated
that reproductive isolation exists in these 4 Acropora species even though they have the opportunities to spawn
synchronously, suggesting that hybridization is not very frequent in this marginal coral habitat at CIB.
Key words: Acropora, Hybridization, Synchronous spawning, Marginal coral community.
T
he Indo-Pacific scleractinian genus
Acropora is one of the most comprehensively
studied coral groups in terms of evaluation of
hybridization as a mechanism contributing to
species richness. Acropora is a diverse genus
of more than 100 species, up to 76 of which can
occur in sympatry (Wallace 1999). Furthermore,
35 sympatric Acropora species were reported to
release gamete bundles within a 2-h period during
multi-species spawning events on the Great Barrier
Reef (GBR) (Willis et al. 1985, Babcock et al.
1986), and similar multi-species spawning events
occur in most specious coral assemblages (Baird
et al. 2009), thus providing an opportunity for
interspecific breeding (Willis et al. 2006, Fukami et
al. 2008). Willis et al. (2006) in a review of artificial
cross-fertilizations of Indo-Pacific Acropora,
estimated that 45% of the species examined could
form hybrids, although in most crosses, only a
few eggs were fertilized. They concluded that the
capacity to hybridize is common in Indo-Pacific
Acropora (Willis et al. 2006). Similarly, while there
85
86
Wei et al. – Hybridization of Reef-Building Corals
are only 3 endemic Acropora in the Caribbean,
morphological and genetic data suggest that
A. prolifera is a hybrid of A. cervicornis and A.
palmata (van Oppen et al. 2000, Vollmer and
Palumbi 2002), and all 3 species are believed to
be capable of interbreeding (reviewed in Willis et
al. 2006).
Although hybridization has received a great
deal of attention as a potential mechanism for
speciation in Acropora, there is evidence to
suggest that hybridization in nature is less common
than in vitro. For example, fine-scale temporal
differences in gamete release and differences in
the time taken for egg/sperm bundles to break up
are sufficient to prevent cross-fertilization among
species that spawn on the same night (Fukami
et al. 2003, Wolstenholme 2004). Indeed, some
species that spawn as little as 1.5 h apart are
genetically distinct (van Oppen et al. 2002, Fukami
et al. 2004). Furthermore, when eggs are offered
a choice of both intra- and interspecific sperm,
hybrid embryos are very rarely produced (Willis et
al. 2006). Similarly, sperm display less activity in
response to heterospecific eggs than to conspecific
ones. These observations suggest that corals
possess many prezygotic barriers that reduce the
chance of hybridization in the field. In addition,
population genetic analyses of A. hyacinthus and A.
cytherea from allopatric populations indicated that
these 2 species constitute distinct entities, despite
producing a high proportion of hybrid embryos in
artificial crosses and extensively sharing alleles
according to phylogenetic analyses (Marquez et
al. 2002a b). Such cases may represent incipient
species (i.e., of very recent origin) in which
reproductive barrier are incomplete. Alternatively,
they may represent cases of incomplete lineage
sorting in reproductively isolated species that still
retain different degrees of ancestral polymorphism.
The extent of hybridization or genetic isolation
may vary among different biogeographical regions
and also as a function of the number of congeneric
species participating in multi-species spawning
events. Willis et al. (2006) speculated that when
many species spawn simultaneously, there will be
strong selection for efficient gamete recognition;
whereas at sites where gametes of fewer species
interact, selection may be less stringent. In other
words, they predicted that rates of hybridization
would be more frequent at the periphery of coral
species’ distribution ranges, where numbers of
species are lower than on the GBR and Okinawa,
where numbers of sympatric Acropora species are
relatively high (Willis et al. 2006).
In the present study, the reproductive properties of 4 sympatric Acropora, A. muricata,
A. hyacinthus, A. humilis, and A. valida were
investigated in Chinwan Inner Bay (CIB), Penghu I.,
Taiwan. Penghu I. is surrounded by a subtropical
non-reefal coral community (Chen 1999) with low
coral diversity compared to tropical reefs (Hsieh
2008). CIB is a semi-enclosed bay where the
coral assemblage at depths of 2-6 m is dominated
by these 4 Acropora species (Hsieh 2008).
Synchronous spawning of scleractinian corals
occurs from late Apr. to early June, depending
on the lunar phase (Chen unpubl. data). Using
observations of spawning times and artificial
crosses of these 4 species, we examined the
hypothesis that hybridization is more frequent in a
marginal habitat at the periphery of these species’
distribution range.
Study site
Field observations of coral spawning and
cross-fertilization experiments were conducted
between 2002 and 2005 at the Marine Biology
Research Center (MBRC), a facility of the Taiwan
Fisheries Research Institute, located adjacent to
the coral assemblage in CIB, Penghu I., Taiwan
(23°31'N, 119°33'E) in the Taiwan Strait. The
coral assemblage in the CIB has developed
atop volcanic rocks and is characterized by the
dominance of Acroporiidae in shallow waters,
including A. muricata, A. hyacinthus, A. valida,
A. humilis, and Montipora cactus (Hsieh 2008).
Spawning of these species has been monitored
since 1999 by tagging colonies and field
observations, revealing that most scleractinian
corals in CIB release gamete bundles around the
full moon in late Apr., early May, late May, or early
June, depending on the lunar phase (Table 1,
Chen et al. unpubl. data).
Tank observations of spawning and crossing
experiments
Five days before the predicted date of a
spawning event, 4-6 colonies of A. muricata,
A. hyacinthus, A. valida, and A. humilis were
collected, placed in individual buckets, and
incubated in tanks with through-flow seawater.
At the time of collection, separate colonies of
each species sampled were tagged in the field.
87
at the MBRC in order to mimic the conditions
of natural wave agitation and ambient sea
temperatures. Approximately 4 h after crossing,
vials were retrieved, and the ratio of fertilization
was determined. The percent fertilization was
calculated from the number of embryos among
the total number of propagules counted. Only
embryos that had reached the 4- or 8-cell stage
(Miller and Ball 2000) were scored as having
been successfully fertilized. Additional counts
were made at 8-12 h after the initial fertilization, to
confirm continuous embryo development until at
least the “prawn-chip” stage (Miller and Ball 2000).
Observations were continued at 24, 48, 72, and
96 h to record the development of embryos into
planula larvae.
When spawning in the tanks was observed, the
tagged colonies were checked the following day to
determine whether or not the corresponding field
colonies had also released gametes. On the date
of the predicted spawning night, the seawater was
switched off to avoid contamination by sperm from
the ocean. Time of sunset, colony setting, and
bundle release were recorded.
Cross-fertilization experiments were conducted following Willis et al. (1997). Gamete
bundles were collected immediately following
spawning. Eggs and sperm were separated using
plankton mesh sieves, and eggs were washed
in at least 10 changes of sperm-free seawater.
Sperm density was estimated, and suspensions of
106 sperm/ml were prepared. This concentration
produced the best results in non-choice crossing
experiments (Willis et al. 1997). Breeding trials
were followed by the instruction matrix described in
Willis et al. (1997). Intraspecific, interspecific, and
self-fertilization trials consisted of approximately
100 eggs added for 2 h of spawning to the diluted
sperm stock in 25-ml screw-cap glass vials.
Controls were conducted using washed eggs
incubated in sperm-free water in order to verify that
the seawater used was sperm-free.
Vials containing gametes were placed
in baskets with floats and attached to a jetty
RESULTS
Spawning day and time
The spawning day of Acropora spp. in CIB,
Penghu Is., ranged from 7 d before to 9 d after
the full moon in Apr. and May (Table 1). In 2002,
1 colony of A. muricata and 1 colony of A. humilis
spawned in the tank 3 d before the full moon in
Apr., followed by 1 colony of each of the 3 species
Table 1. Spawning day, relationship to the full moon (-, day before the full moon; +, day after the full moon),
sunset times of spawning days, and beginning of spawning times of 4 sympatric Acropora spp. in Chinwan
Inner Bay, Penghu, Taiwan in 2002-2005. Number of colonies observed in the laboratory are shown in
parentheses. Blanks indicate that spawning was not observed in those species in the laboratory. Sunset
times were retrieved from the database of the Central Weather Bureau, Taiwan
Species
Date
2002
24 Apr. (-3)
Sunset
18:23
A. muricata
20:00 (1)
A. humilis
20:15 (2)
25 Apr. (-2)
18:23
27 Apr. (0)
18:35
20:15 (1)
20:00 (1)
20:05 (3)
20:30 (4)
20:30 (6)
20:45 (4)
20:30 (4)
20:30 (1)
20:30 (2)
20:15 (1)
20:40 (3)
20:30 (2)
20:50 (3)
20:50 (6)
21:00 (1)
20:10 (1)
2004
20:40 (3)
20:50 (3)
21:00 (2)
20:40 (4)
20:30 (2)
20:30 (2)
20:30 (3)
May 1 (+8)
18:26
20:40 (1)
20:45 (6)
Bundle setting Interval after
2005
time
setting
19:10-19:50
19:00-19:50
19:00-20:00
19:00-19:50
30-70 min
35-60 min
35-80 min
45-100 min
May 2 (+9) May 15 (-7)* May 16 (-6) May 19 (-3) May 20 (-2)
18:26
20:40 (3)
20:50 (3)
21:00 (1)
May 23 (+8)*
18:21
20:00 (1)
18:33
20:50 (2)
21:00 (2)
Apr. 23 (+7)
18:21
A. hyacinthus
18:31
20:40 (3)
Apr. 20 (+4)
18:26
20:00 (1)
May 11 (+8) May 13 (+10)
May 2 (+5)
18:26
20:00 (1)
Sunset
A. muricata
A. humilis
A. valida
A. hyacinthus
29 Apr. (+2)
18:26
20:00 (1)
Date
28 Apr. (+1)
18:23
A. valida
Species
2003
18:33
20:30 (3)
18:33
20:40 (4)
18:35
21:00 (3)
21:30 (1)
21:30 (3)
18:35
21:00 (2)
21:15 (2)
21:30 (3)
88
releasing gamete bundles 2 nights before the
full moon, the night of full moon, and 1 night
after the full moon. More than 1 colony of each
species spawning synchronously was observed
for 3 species on the 2nd night after the full moon.
However, synchronous spawning of 4 species was
only observed on the 5th night after the full moon.
In 2003, 3 Acropora spp. were observed to release
gamete bundles in the tanks on the 4th and 7th
nights after the full moon. All 4 species spawned
on the 8th night. In 2004, only 3 species spawned
in the tanks on the 8th and 10th nights after the full
moon in May. In 2005, spawning was even less
synchronous with 1 and 2 species respectively
spawning on the 8th and 9th nights after the full
moon in Apr., and 3 species spawning 2 d before
the full moon in May. No synchronous spawning
was observed among any of the 4 species in
2005 (Table 1). All tagged colonies that released
gamete bundles in the tank also released gamete
bundles in the field.
With all 4 species, gamete bundles appeared
in the polyp mouth between 19:10 and 20:00 in
2002-2005 (Table 1). Bundle release occurred
between 20:00 and 21:30, i.e., 30 to 100 min after
sunset. A 10-30-min time delay was observed
between species that spawned on the same night.
In most cases, A. muricata was the 1st species to
spawn, and A. humilis was the last during nights of
synchronous spawning.
Crossing experiments
The results of cross-fertilization experiments
are summarized in table 2. Intraspecific crosses
(excluding selfing) consistently produced high
fertilization rates, ranging from 94.73% in A.
muricata to 99.33% in A. hyacinthus. Selfing
occurred at a very low rate (0.63%-0.89%) except
with A. humilis (5.97%). Interspecific fertilization
was consistently very low, ranging from 0% with
A. humilis sperm × A. hyacinthus eggs to 4.58% in
the cross of A. valida sperm × A. hyacinthus eggs.
All interspecific fertilizations were associated with
high standard errors, suggesting that successful
fertilization rates were highly variable among
the crosses, and the mode was 0%. While
intraspecific-crossed embryos continued to
develop into “prawn-chip” stage at 12 h after initial
fertilization, all interspecific-crossed embryos had
died by 2 d, and no further embryo development
stage was observed in the vials. All intraspecificcrossed embryos had successfully developed to
swimming planula larvae by 96 h.
DISCUSSION
In the present study, results of spawning
dates, times, and interspecific crossing
experiments indicated that reproductive isolation
Table 2. Fertilization trials within and between species of 4 sympatric Acropora spp. The mean fertilization
percentages of all individual crosses conducted between 2002 and 2005 in Chinwan Inner Bay were
averaged for each combination, and values are shown with the standard error. Values for the intraspecific
crosses are the averages of both reciprocal combination of eggs and sperm. n is the number of colonies
used in the trials
Egg
A. muricata (n = 21)
A. muricata
A. humilis
A. valida
94.73 ± 9.15
0.18 ± 1.04
0.13 ± 0.50
(112/112)
A. humilis (n = 9)
1.25 ± 2.97
(1/59)
A. valida (n = 15)
2.11 ± 7.03
(5/105)
A. hyacinthus (n = 11)
1.47 ± 3.33
(4/81)
Cross of the same colony
Controls
(no sperm)
Sperm
0.77 ± 2.65
(1/60)
(0/63)
97.28 ± 4.35
(24/24)
0.00 ± 0.00
(0/21)
1.26 ± 2.88
(2/48)
5.97 ± 8.20
(6/27)
(0/105)
1.74 ± 4.11
(1/21)
95.53 ± 5.98
(78/78)
4.58 ± 10.57
(7/46)
0.63 ± 1.28
(0/36)
A. hyacinthus
0.08 ± 0.38
(0/90)
1.88 ± 4.36
(4/48)
1.37 ± 4.80
0.00 ± 0.00
(0/63)
0.00 ± 0.00
(0/27)
0.00 ± 0.00
(2/46)
(0/36)
99.33 ± 1.55
0.00 ± 0.00
(54/54)
(0/33)
0.89 ± 1.67
(0/33)
exists among the 4 dominant Acropora species,
and hybridization is likely to be very rare among
Acropora in CIB, Penghu I., a non-reefal marginal
coral community in Taiwan (Chen 1999, Hsieh
2008).
Prezygotic isolation: species diversity and
spawning times
Acropora species diversity and spawning
patterns in CIB greatly differ from those on the
GBR, Australia and at Akajima I., Okinawa, Japan
where species diversity is relatively high and many
species spawn in synchrony (Willis et al. 1985,
Babcock et al. 1986, Hayashibara et al. 1993).
Up to 76 Acropora species occur in sympatry,
and 35 of them have been recorded to release
gamete bundles within 2 h of each other during
spawning events on the GBR (reviewed in Willis
et al. 2006). Similar patterns were observed at
Akajima I., Okinawa, where 10-12 of 35 Acropora
species were recorded to spawn on the same
nights (Hayashibara et al. 1993, Hatta et al. 1999).
In contrast, at CIB and other adjacent islands, A.
muricata, A. valida, A. humilis, and A. hyacinthus
are the dominant species in the coral assemblage
(Hsieh 2008). Spawning of these 4 species was
observed from the 7th night before to the 9th night
after a full moon depending on the month and year
(Table 1). In most cases, 2 or 3 species spawned
1.5-2 h after sunset, and synchronous spawning
of all 4 species was only observed on 1 night
each in 2002 and 2003, suggesting that Acropora
spawning at CIB is less synchronous than at the
GBR and Okinawa. Although the time of gamete
bundle release was similar among species
(0-30 min), successful interspecific fertilization
rates remained low. In contrast to the hypothesis
of Willis et al. (2006), reproductive barriers caused
by gamete recognition appear to be strong even
in regions such as Taiwan where there are few
congeneric Acropora species and where gametes
rarely interact because of year-to-year variations
in lunar nights of spawning. A similar pattern of
non-synchronous gamete release by sympatric
Acropora species was also observed at a highlatitude coral community at Otuski, Kochi, Japan
(Takemura et al. 2007). At a high-latitude coral
community in Shirahama, Wakayama, Japan,
spawning times of 2 A. solitaryensis ecomorphs
differed by 13-18 min, but there was a significantly
low in vitro fertilization rate between ecomorphs.
In contrast, 1 ecomorph, the arborescent A.
solitaryensis, can cross with A. pruinosa with a
89
significantly high fertilization rate in vitro (Suzuki
and Fukami 2011). However, A. pruinosa spawned
approximately 1 h earlier than both ecomorphs
of A. solitaryensis in vivo at Shirahama; thus, it is
unlikely that these 2 high-latitude Acropora would
hybridize in the field. Overall, when the ecological
aspects of spawning are considered, such as
fine-scale differences in the timing of gamete
release and bundle breakup (Wolstenholme et al.
2003, Wolstenholme 2004), as well as species
differences in sperm aging, dispersal, and dilution
of gametes (Levitan et al. 2004), interspecific
fertilization might occur very rarely in the field.
Postzygotic isolation: low interspecific
crossing rates, zygotic morality, and hybrid
inviability
Even though there is potential for Acropora
species to spawn synchronously and interspecifically fertilize in vitro, postzygotic isolation
mechanisms, such as gametic incompatibility,
zygotic morality, hybrid inviability, hybrid sterility,
and hybrid breakdown, still work against
hybridization frequently occurring among species.
In the case of Acropora at CIB, gametic
incompatibility, zygotic mortality, and hybrid
inviability might prevent hybridization from
occurring. First, interspecific fertilization rates,
expressed as the proportion of eggs fertilized
ranged 0%-4.58% with a mode of 0%, and were
low among the 4 Acropora species at CIB (Table 3).
These extremely low fertilization rates suggest that
gametic incompatibility plays an important role in
prezygotic isolation. This result is similar to those
in much of the previous literature, which indicates
that few species crosses have high rates of fertilization, even between species that potentially
can cross often (Table 3). This scenario is further
supported by recent experiments on sperm choice
and activity, which showed that eggs “prefer” to be
fertilized by sperm from the same species (Morita
et al. 2006, Willis et al. 2006) and that sperm are
preferentially activated by conspecific eggs (Morita
et al. 2006, Willis et al. 2006). Second, even
though there is a low frequency of interspecific
fertilization, breakdown of interspecific embryos
after 12 h suggests that either zygotic mortality,
i.e., an egg is fertilized but the zygote does not
develop, or hybrid inviability, i.e., a hybrid embryo
forms but is of reduced viability, continually prevent
the development processes from being completed.
90
Table 3. Summary of interspecific fertilization success of Acropora species in the literature
Species
A. hyacinthus × A. cytherea
Fertilization success (%)a
50
Species groups b
hyacinthus
Clades c
C
Locality
f
Magnetic I. (19°15'S, 146°50'E)
Palm I. (18°31'S, 146°19'E),
Australia
A. pulchra × A. millepora
A. selago × A. millepora
A. pulchra × A. elseyi
A. pulchra × A. cytherea
A. cytherea × A. selago
A. valida × A. elseyi
A. pulchra × A. hyacinthus
A. pulchra × A. nasuta
A. millepora × A. muricata
A. millepora × A. cytherea
A. selago × A. tenuis
A. hyacinthus × A. valida
A. millepora × A. valida
A. nasuta-A × A. nasuta-B
45
40
24
10
8
6.4
6.2
2.5
2.2
1.7
1.4
1.3
0.3
0.5 ± 1.7 d
aspera
selago/ aspera
aspera/ echinata
aspera/ hyacinthus
hyacinthus/ selago
nasuta/ echinata
aspera/ hyacinthus
aspera/ nasuta
aspera/ muricata
aspera/ hyacinthus
selago
hyacinthus/ nasuta
aspera/ nasuta
nasuta
C
C
C, D
C
C
A, D
C
C, A
C, B
C
C
C, A
C, A
A
A. nasuta-B × A. nasuta-C
0.0 ± 0.2 e
0.3 ± 0.6 d
nasuta
A
A. nasuta-A × A. nasuta-C
0.1 ± 0.3 e
96.8 ± 2.5 d
nasuta
A
A. muricata-A × A. muricata-B
1.1 ± 3.1 e
1.2 ± 1.5 d
muricata
B
A. nasuta × A. muricata
1.6 ± 1.5 e
0.5 - 75 d
nasuta/ muricata
A. digitifera × A. nasuta
0.2 - 94.5 e
0.2 - 14.3 d
humilis/ nasuta
h
Akajima I., Okinawa, Japan (30°N, 123°E)
A
humilis/ muricata
A, B
A. intermedia × A. nasuta
A. intermedia × A. muricata
A. intermedia × A. digitifera
A. florida × A. nasuta
A. florida × A. muricata
A. florida × A. digitifera
A. florida × A. intermedia
0.7 - 12.6 e
0
0
0
0 - 0.1
0
0-1
71.5 ± 28.5 d
robusta/ nasuta
robusta/ muricata
robusta/ humilis
florida/ nasuta
florida/ muricata
florida/ humilis
florida/ robusta
A, B
B
B, A
C, A
C, A
C, A
C, B
A. tenuis × A. yongei
0e
24.2 ± 48.5 d
selago
C
A. tenuis × A. donei
46.4 ± 53.5 e
22.0 ± 37.1 d
selago
C
A. yongei × A. donei
A. tenius × A. verweyi
A . tenius × A. austera
A. solitaryensis (AR) × A. solitaryensis (PL)
A. solitaryensis (AR) × A. pruinosa
12.9 ± 31.5 e
0
0
0
< 1.0
88.0 ± 22.0 d
A. solitaryensis (PL) × A. pruinosa
48.7 ± 28.9 e
0d
A. muricata × A. humilis
2.3 ± 3.2 e
0.71 ± 2.27
selago
selago/ verweyi
selago/ rudis
Akajima I., Okinawa, Japan (30°N, 123°E)
A, B
0 - 3.6 e
0d
A. digitifera × A. muricata
g
C
C, D
C, A
i
Shirahama (33°43'N, 135°19'E)
j
Chinwan, Penghu I., Taiwan
muricata/ humilis
B, A
(23°31'N;119°33'E)
A. muricata × A. hyacinthus
A. muricata × A. valida
A. humilis × A. hyacinthus
A. humilis × A. valida
A. hyacinthus × A. valida
0.74 ± 2.40
1.12 ± 5.07
1.57 ± 3.69
1.03 ± 3.26
2.98 ± 8.32
muricata/ hyacinthus
muricata/ nasuta
humilis/ hyacinthus
humilis/ nasuta
hyacinthus/ nasuta
B, C
B, A
A, C
A
C, A
Data adopted from information summarized in the literature. b Species groups defined by Wallace (1999). c Phylogenetic clade
information summarized by Wallace (1999). d Sperm from the former species crossed with eggs of the latter species. e Reciprocal
crosses. f Willis (1997). g Hatta et al. (1999). h Fukami et al. (2003). i Suzuki and Fukami (2011). j This study. -, Not available.
a
Hybridization is not promoted in Acropora in
marginal coral communities
It was hypothesized that when many
congeneric species spawn simultaneously in
sympatry, there will be strong selection for efficient
gametic recognition, compared to sites where
gametes of fewer species spawn in synchrony.
This led to the speculation that hybridization may
occur more frequently at the periphery of species’
ranges (Willis et al. 2006, Richards et al. 2008).
However, this scenario was not supported when
we compared the in vitro interspecific fertilization
success rates across geographic regions (Table 3).
First, if we take 5% as the “significant” criterion for
fertilization success, at the GBR, where Acropora
diversity is high (76 species) (Wallace 1999),
eight of 14 (57.14%) crosses exhibited significant
fertilization success (Willis 1997). At Akajima I.,
Okinawa, Japan, of 35 Acropora species recorded,
seven of 19 (36.84%) crosses exhibited significant
fertilization success (Hatta et al. 1999, Fukami et
al. 2003). In contrast, at CIB, Penghu I., Taiwan
and Shirahama, Japan, where Acropora species
diversity is relatively low (Hsieh 2008, Takuma
pers. comm.), fertilization success rates were
low (with mode of 0%) compared to those of the
GBR and Akajima I., except for the cross between
A. pruniosa and the arborescent morph of A.
solitaryensis (Suzuki and Fukami 2011). These
data reveal an opposite trend to the prediction by
Willis et al. (2006) that hybridization will be more
frequent at the periphery of a species’ range.
Second, not only is species diversity relatively
low, but most species in marginal habitats
(including CIB) are also distantly related according
to morphological phylogenies (Wallace 1999).
At the GBR, most instances of high fertilization
success are between species pairs from the same
clade (clade C) (Table 3). At Akajima I., Okinawa,
high fertilization success occurs either by a 1-way
cross within the same clade (e.g., A. nausta-A
× A. nasuta-C) or with high variation between
clades (e.g., A. nasuta × A. muricata) (Table 3).
At Penghu, the total number of coral species is
75, with only A. muricata, A. humilis, A. valida,
and A. hyacinthus dominant in shallow waters
(Hsieh 2008). These 4 species are classified in 3
clades that had a mode of fertilization successes
equal to 0%. Similar species diversities were also
reported at other high-latitude coral communities.
From the Solitary Is. (> 29°S), a high-latitude
coral community in eastern Australia, 14 Acropora
species were recorded (Wilson and Harrison
91
2003). From Otsuki, Kochi Prefecture, Japan
(> 33°N), 8 Acropora species were recorded
(Nomura and Takemura 2005, Takemura et
al. 2007). Acropora species within these
assemblages are often distantly related. This
differs from the situation in the Caribbean, where
co-occurring species are closely related, such as
with the A. cervicornis group or the Montastraea
annularis complex. Even though those species
are closely related and may occasionally hybridize,
mechanisms to prevent hybridization from
occurring have also developed in the M. annularis
complex (Levtian et al. 2004). Future studies of
reproductive compatibilities of Acropora species
are needed in order to confirm that hybridization
is less likely to occur in marginal communities of
high-latitude regions compared to the GBR and
Okinawa, such as at the Solitary Is., Australia and
Otsuki, Japan.
In summary, observations of spawning days
and times and interspecific fertilization trials of 4
sympatric Acropora species were conducted at
a non-reefal coral community in CIB, Penghu I.,
Taiwan. The results showed that Acropora spp.
in CIB spawned less synchronously compared
to previous reports of species in the GBR and
Okinawa, and interspecific fertilization was
extremely low. Ecological and reproductive
analyses indicated that the 4 sympatric Acropora
species maintain clear species boundaries, and
hybridization does not appear to be as frequent at
marginal habitats as formerly hypothesized. Thus,
the scenario that hybridization facilitates IndoPacific Acropora species expanding their ranges
and adapting to changing environments should be
considered with caution.
Acknowledgments: Many thanks are given to
the staff of the Marine Biology Research Center,
a facility of the Taiwan Fishery Research Institute,
and Penghu County Marine Life Prorogation Center
for providing hospitality during our field trips. We
thank C.W. Chen and Y.Y. Chuang for assistance
with the spawning and crossing experiments, K.
Shashank and T. Mezaki for providing references
of Acropora spawning in Otuski, Kochi, Japan, and
members of the Coral Reef Evolutionary Ecology
and Genetics (CREEG) laboratory, Biodiversity
Research Center, Academia Sinica (BRCAS),
Hybridization Discussion Group, J. Reimer, Y.
Loya, and 2 anonymous referees for constructive
comments. N.V. Wei was supported by a predoctoral fellowship from the National Science
Council (NSC), Taiwan and BRCAS. This study
92
was supported by research grants from the NSC
and Academia Sinica to CAC. This is the CREEGRCBAS contribution no. 60.
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Veron JEN. 1995. Corals in space and time: the biogeography
and evolution of the Scleractinia. Ithaca, NY and London:
Cornell Univ. Press.
Vollmer SV, SR Palumbi. 2002. Hybridization and the evolution
of reef coral diversity. Science 296: 2023-2025.
Wallace CC. 1999. Staghorn corals of the world: a revision
of the coral genus Acropora (Scleractinia; Astrocoeniina;
Acroporidae) worldwide, with emphasis on morphology,
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CSIRO Publishing.
Willis BL, RC Babcock, P Harrison, JK Oliver, CC Wallace.
1985. Patterns in the mass spawning of corals on the
Great Barrier Reef. Proc. Fifth Int. Coral Reef Symp.
Tahiti 4: 343-348.
Willis BL, R Babcock, P Harrison, C Wallace. 1997. Experimental hybridization and breeding incompatibilities within
the mating systems of mass spawning corals. Coral
Reefs 16: S53-S65.
Willis BL, MJH van Oppen, DJ Miller, SV Vollmer, DJ Ayre.
2006. The role of hybridization in the evolution of reef
corals. Annu. Rev. Ecol. Evol. Syst. 37: 489-517.
Wolstenholme JK. 2004. Temporal reproductive isolation and
gametic compatibility area evolutionary mechanisms in the
Acropora humilis species group (Cnidaria; Scleractinia).
Mar. Biol. 144: 567-582.
Wolstenholme JK, CC Wallace, CA Chen. 2003. Species
boundaries within the Acropora humilis species group
(Cnidaria; Scleractinia): a morphological and molecular
interpretation of evolution. Coral Reefs 22: 155-166.
Wilson JR, PL Harrison. 2003. Spawning patterns of
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Ser. 260: 115-123.
Pseudosiderastrea formosa sp. nov. (Cnidaria: Anthozoa: Scleractinia) a
New Coral Species Endemic to Taiwan
Michel Pichon1, Yao-Yang Chuang2,3, and Chaolun Allen Chen2,3,4,*
Museum of Tropical Queensland, 70-102 Flinders Street, Townsville 4810, Australia
Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan
3
Institute of Oceanography, National Taiwan Univ., Taipei 106, Taiwan
4
Institute of Life Science, National Taitung Univ., Taitung 904, Taiwan
1
2
Michel Pichon, Yao-Yang Chuang, and Chaolun Allen Chen (2012) Pseudosiderastrea formosa sp. nov.
(Cnidaria: Anthozoa: Scleractinia) a new coral species endemic to Taiwan. Zoological Studies 51(1): 93-98.
Pseudosiderastrea formosa sp. nov. is a new siderastreid scleractinian coral collected in several localities in
Taiwan. It lives on rocky substrates where it forms encrusting colonies. Results of morphological observations
and molecular genetic analyses are presented. The new species is described and compared to P. tayamai and
Siderastrea savignyana, and its morphological and phylogenic affinities are discussed.
Key words: Pseudosiderastrea formosa sp. nov., New species, Scleractinia, Siderastreid, Western Pacific
Ocean.
A
Pseudosiderastrea, described as P. formosa sp.
nov.
siderastreid scleractinian coral was
collected from several localities around Taiwan
and nearby islands, where it is relatively rare.
The specimens present some morphological
similarities with Pseudosiderastrea tayamai Yabe
and Sugiyama, 1935, the only species hitherto
known from that genus, and with Siderastrea
savignyana Milne Edwards and Haime, 1849,
which is the sole representative in the Indian
Ocean of the genus Siderastrea de Blainville,
1830. In order to ascertain its taxonomic position,
morphological observations were carried out on a
suite of 33 specimens at the Museum of Tropical
Queensland, Townsville, Australia and at the
Biodiversity Research Center, Academia Sinica,
Taipei, Taiwan. Molecular phylogenetic analyses
were also conducted at the Biodiversity Research
Center. The results presented below indicate
that these specimens belong to a new species of
MATERIAL AND METHODS
Specimens were collected by scuba diving at
Wanlitung (21°59'48"N, 120°42'10"E) and the outlet
of the 3rd nuclear power plant (21°55'51.38"N,
120°44'46.82"E) on the southeastern coast
of Taiwan in Kenting National Park, Chi-Fai
(23°7'0.59"N, 121°23'49.58"E) in Taitung County,
and at Yeiyu (22°3'1"E, 121°30'35") at Orchid I.
(Lanyu in Chinese). Specimens for morphological
studies were bleached to remove soft tissues
by dipping them in household bleach (sodium
hypochlorite) for 24 h. They were then rinsed with
fresh water and thoroughly dried. Morphological
observations were carried out using a Leica
*To whom correspondence and reprint requests should be addressed. E-mail:[email protected]; [email protected]
93
94
Pichon et al. – Pseudosiderastrea formosa in Taiwan
MX8 stereomicroscope, equipped with an ocular
graticule. Scanning electron microscopy (SEM)
was performed at James Cook Univ., Townsville,
Australia on a JEOL 5410LV instrument.
Samples for the molecular phylogenetic
analyses were collected from Bocas del Toro,
Panama (S. radians and S. siderea); Isla Uraba,
Panama (S. glynni); Brazil (S. stellata); Kiunga,
Kenya (S. savignyana); and Kaohsiung (P.
tayamai) and Wanlitung, Taiwan (P. formosa sp.
nov.). For each sample, a piece of about 5 × 5 cm
was stored in a modified guanidine solution or
75% ethanol. DNA extraction methods were as
specified by Fukami et al. (2004). Cytochrome
(Cyt) b sequences were amplified by a polymerase
chain reaction (PCR) with the primer set, AcCytbF
( 5 ' - G C C G T C T C C T T C A A ATATA A G - 3 ' ) a n d
AcCytbR (5'-AAAAGGCTCTTCTACAAC-3')
(Fukami et al. 2008), with the following PCR
conditions: 94°C for 2 min, followed by 35 cycles
at 94°C for 45 s, 50°C for 30 s, and 72°C for 60 s,
and ending with a final phase at 72°C for 10 min.
PCR products were directly sequenced.
Sequences were aligned using codons,
and their genetic distances were calculated
using MEGA5 (Tamura et al. 2011). Cyt b, a
highly variable mitochondrial coding region
in Siderastrea, was selected to analyze the
divergence of Pseudosiderastrea specimens
and their evolutionary status with the closely
related genus, Siderastrea (Fukami et al. 2008).
Using the final dataset, which contained 16 Cyt b
sequences of 771 base pairs (bp), the best fitting
evolutionary models were determined by the
Akaike information criterion (AIC) test in ModelTest
(Posada et al. 1998). A phylogenetic analysis
was performed using PhyML 3.0 (Guindon et al.
2010) for maximum-likelihood (ML) and MrBayes
(Ronquist et al. 2003) for Bayesian inference (BI),
under the GTR+I model of DNA evolution. The ML
was performed using Shimodaira and Hasegawa
(SH-like) branch support with 1000 bootstrap
replicates. Sequences obtained from this study
were submitted to GenBank with accession nos.
JN600483-98.
For the BI, 6 runs with 5 × 106 generations
each were calculated, while topologies were saved
every 100 generations. One-fifth of the 50,000
topologies were discarded as burn-in, and the
remaining ones were used to calculate posterior
probabilities.
RESULTS
Family Siderastreidae Vaughan and Wells, 1943
Genus Pseudosiderastrea Yabe and Sugiyama,
1935
Pseudosiderastrea formosa sp. nov.
Synonomy : Siderastrea savignyana Dai & Horng 2008, p. 165.
Material examined: Holotype: Museum of
Tropical Queensland G 64378 Taiwan, Wanlitung
21°59.85'N, 120°42.22'E. Depth 3 m, Coll.
A. Chen, 20 Nov. 2009. Paratypes: Museum
of Tropical Queensland. G 64374-7 Taiwan,
Wanlitung 21°59.85'N, 120°42.22'E. Depth 3 m,
Coll. A. Chen, 20 Nov. 2009.
Other material: Museum of Tropical Queensland. G 64352-64, Taiwan, Lanyu (Orchid I.); G
64365-73 Taiwan, Kenting. Biodiversity Research
Center Museum. ASIZC0000958-9, Taiwan, ChiFai.
Description: Colony small, thin, and tightly
encrusting substratum. Most specimens examined
being fragments of colonies up to 5 cm in
maximum dimension. Holotypic colony (fragment)
47 × 22 mm (Fig. 1). Growing margins very thin,
often showing incomplete calcification of corallite
structures. Corallites cerioid and uniform in
shape and size within each specimen examined.
Corallite shape possibly varying from subcircular to
polygonal and even squarish in some specimens.
In latter, arrangement of corallites tending to be
in linear rows. Corallite size range 1.8-4.4 mm in
calicular diameter (mean maximum diameter, 2.8
± 0.2 mm). Septa wedge-shaped (Fig. 2), and
hexamerally arranged in 3 cycles, sometimes part
of 4th (S1 and S2 > S3 and S4). Smaller corallites
with only 18 septa, whereas larger corallites with
up to 34 septa. S3 and S4 tending to curve and
flanking S1 and S2, sometimes deeply fused
in fossa. Such a fusion pattern never involving
more than 3 septa, and rarely present more than
twice in any given corallite. A number of corallites
completely lacking any septal fusion. Septa slightly
exserted, continuous, and convex over corallite
edge. Near corallite edge, septa only moderately
inclined towards calicular center, and then sloping
more steeply towards columellar pit. Septal axial
edges bearing conspicuous ornamentation (Fig. 2)
composed of 7-10 granules, sometimes flattened
transversally (Fig. 3). Septal faces entire and
ornamented with small, pointed granules (Fig.
3). Fossa up to 2 mm deep, containing a welldeveloped, convex, massive columella. Columella
circular and up to 1 mm in diameter (average
diameter, 0.8 mm) sometimes reaching up to 1/3
of corallite diameter (Fig. 4). However, some
corallites with a slightly elongate columella,
composed of 2-4 smooth elements. Columella
often visible below oral disc in live specimens.
Wall solid and similar in thickness to septal outer
edge (0.3 mm). Synapticular ring absent within
corallite wall. However, a few synapticulae
possibly present in some corallites, and in such
specimens, some synapticulae also present on thin
growing margin of corallum in a few incompletely
developed peripheral corallites. Corallum white to
light beige. Living colonies grayish-green, beige,
or pink.
Etymology: The species name formosa
(Latin formosus: beautiful, elegant) refers to the
regular and neat aspect of the corallum. It is also
reminiscent of the old name for Taiwan, where this
species is thought to be endemic.
Distribution: Known only from Taiwan and
5 mm
Fig. 1. P. formosa sp. nov., holotype MTQ G 64378. Wanlitung,
Taiwan.
95
nearby islands, incrusting bare rocky outcrops at
< 10 m deep (Fig. 5), where it may co-occur with
P. tayamai.
Remarks: Overall, skeletal characters display
100 μm
Fig. 3. P. formosa sp. nov. MTQ G 64373. Scanning electron
microscopic image. Note the laterally flattened septal
dentations and conical ornamentation on the septal sides.
5 mm
Fig. 4. P. formosa sp. nov., holotype. Note the non-exserted
septa, solid wall, and well-developed columella.
1 mm
Fig. 2. P. formosa sp. nov. MTQ G 64365. Scanning electron
microscopic image. Note the exserted septa and welldeveloped septal ornamentation.
Fig. 5. P. formosa sp. nov. from Wanlitung. A small colony
living on rocky substrate.
96
little variation among specimens, and only minor
differences were observed. They principally
concern the size of the corallites and number
of septa, development of the septal margin
ornamentation, more or less exsert character
of the septa above the common wall, and the
size of the columella. The series of specimens
examined; however, is rather homogeneous, and
no significant variations among the 3 geographic
locations where the specimens were collected
were noted.
Molecular phylogenetic analyses
Fifty-two variable sites containing 50
parsimoniously informative sites were found in 16
sequences of the Siderastrea-Pseudosiderastrea
group examined. Pairwise genetic distances
were calculated under the setting of the Kimura
2-parameter model, and averaged 0.03 between
the Pseudosiderastrea and Siderastrea groups.
The overall distance within Siderastrea was
0.012, while that within Pseudosiderastrea was
only 0.003. Most species of Siderastrea occur in
the Atlantic Ocean (Caribbean and Brazil) (Budd
et al. 1994), and their pairwise genetic distance
was smaller than that found in the single IndoPacific species S. savignyana (Atlantic group:
0.00037; S. savignyana: 0.00086). Within the
genus Pseudosiderastrea, the genetic distance
75/98
between P. tayamai and P. formosa sp. nov.
was 0.004, which is much higher than that of
species comparisons among Atlantic species of
Siderastrea.
Porites porites, Dendrophyllia sp., and
Stephanocoenia michelinii were used as outgroups
in the phylogenetic analysis. The resulting ML
and BI topologies were similar for the Siderastrea
and Pseudosiderastrea groups (Patristic distance
correlation = 0.95) (Fourment et al. 2006) (Fig.
6), and consisted of 4 clades: clade I included
Siderastrea species from the Atlantic Ocean
group (Forsman et al. 2005); clade II included all
specimens of S. savignyana collected from the
Indian Ocean; and clades III and IV contained all
specimens of Pseudosiderastrea. All clades had
strong statistical support (≥ 75%) in both the ML
(bootstrap) and BI (posterior probability) analyses.
DISCUSSION
The genus Pseudosiderastrea was establ i s h e d b y Ya b e a n d S u g i y a m a ( 1 9 3 5 ) f o r
t h e s p e c i e s P. t a y a m a i f r o m A r u I s . , b u t
was subsequently treated as a subgenus of
Anomastraea (Vaughan and Wells 1943, Wells
1956). However, more recently it was again
treated as a genus in its full right (Veron and
Pichon 1979). In the original description, Yabe
Sgl_3108 Siderastrea glynni
Sra_2832 Siderastrea radians
Sra_2834 Siderastrea radians
99/99
Ssi_2831 Siderastrea siderea
I
Ssi_2832 Siderastrea siderea
99/100
Sst_2844 Siderastrea stellata
Sst_2846 Siderastrea stellata
Ssa_3155 Siderastrea savignyana
II
Ssa_3153 Siderastrea savignyana
100/100
75/96 Ssa_3154 Siderastrea savignyana
Psp_5349
77/96
III
Psp_ 5350
Pseudosiderastrea from Wanlitung
Psp_5353
99/100
Pta_5348 Pseudosiderastrea tayamai
IV
77/ 71
AB441313 Stephanocoenia michelinii
AB441324 Dendrophyllia sp.
Outgroup
-/100
NC_008166 Porites porites
Fig. 6. Phylogenetic analyses based on Bayesian inference and maximum likelihood of the partial mitochondrial cytochrome (Cyt) b
gene. Ten Siderastrea and 6 Pseudosiderastrea specimens were included and separated into 4 clades, including clades I and II for
Siderastrea and clades III and IV for Pseudosiderastrea. Stephanocoenia, Dendrophyllia, and Porites were chosen as outgroups.
and Sugiyama (1935) remarked that P. tayamai
was close to the Atlantic S. radians and S.
siderea, which were the only Siderastrea species
available for them to compare. According to Yabe
and Sugiyama (1935), the major morphological
differences between these 2 genera were the
absence of septal perforations and the reduced
development of synapticulae in Pseudosiderastrea.
They also remarked that Pseudosiderastrea has
similar features to Anomastraea, the latter differing
by the presence of perforated septa and septal
dentation increasing in size towards the center
of the calice, with a tendency to form pali-like
structures.
The relative regularity of the corallite shape
within each colony of P. formosa sp. nov., in
the material examined, is reminiscent of S.
savignyana Milne Edwards & Haime (1849), which
is particularly widespread in the western Indian
Ocean (Fig. 7). By comparison, Pseudosiderastrea
specimens most often display a more-irregular
corallite shape, although occasionally some
regularly shaped corallites were noted (see Veron
and Pichon 1979, fig. 145). However, the solid
walls and septa, and the almost total absence of
synapticulae and synapticular rings leave no doubt
as to the generic position of our specimens, which
clearly belong to the genus Pseudosiderastrea,
for which they represent a new species.
Pseudosiderastrea formosa sp. nov. differs from P.
tayamai (Fig. 8) in having more-regularly-shaped
corallites, a smaller number of septa which are
slightly wedge-shaped and seldom fused at their
inner margin, coarser septal ornamentation, and a
very developed, highly conspicuous columella.
Fig. 7. Siderastrea savignyana. Specimen from Kuwait clearly
showing the well-developed synapticular rings. (Photo: P.
Harrison)
97
Molecular phylogenetic affinities
The Pseudosiderastrea spp. clade grouped
as a sister group to Siderastrea spp. (Fig. 6),
and as such, the monophyletic status of both
genera is confirmed. Using cytochrome oxidase
subunit 1 (COI) and Cyt b, Fukami et al. (2008)
reexamined the familial and generic relationships
of many scleractinian representatives, and
found that the Pacific “Siderastrea” (samples
collected from Wanlitung, Taiwan), which in fact
belonged to P. formosa sp. nov., and the Atlantic
Siderastrea specimens were a monophyletic
group. The monophyletic origins of Siderastrea
and Pseudosiderastrea were also confirmed by the
COI phylogeny of scleractinian corals proposed
by Kitahara et al. (2010). Those results clearly
indicated that Pseudosiderastrea and Siderastrea
have a very recent common ancestor.
Following morphological observations
provided herein, the Cyt b phylogeny indicated
that P. formosa sp. nov. and P. tayamai belong to
the same genus based on monophyletic support
of Cyt b phylogeny (clades III and IV, Fig. 6). The
genetic distance of Cyt b between P. formosa sp.
nov. and P. tayamai (p = 0.004) was relatively
larger than the interspecific distance for species
in the Atlantic clade (clade I), which contains the
most recently diverged Siderastrea species from
the Atlantic Ocean, S. glynni (p = 0-0.0006 for Cyt
b in this study) (Forsman et al. 2005). The smaller
distance we showed in Pseudosiderastrea is due
to the slower evolution of mitochondrial DNA in
anthozoans (Shearer et al. 2002). Comparing our
results with others using the same marker, the
Fig. 8. Pseudosiderastrea tayamai (MTQ G 64630) from
Kaohsiung, Taiwan, showing irregularly shaped and sized
corallites and frequent fusion of the predominantly lamellar
septa.
98
genetic distance between P. formosa sp. nov. and P.
tayamai was equivalent to the interspecific distance
of Cyt b found in Acropora (p = 0.004 between P.
formosa sp. nov. and P. tayamai, p = 0.0039 in
interspecific comparisons of Acropora; Chen et al.
2008). The genetic distance between P. formosa
sp. nov. and P. tayamai implies that the genetic
divergence of these 2 species is sufficiently large
to support P. formosa sp. nov. being a different
species from P. tayamai.
Acknowledgments: The authors acknowledge
the assistance of Dr. P. Muir (S.E.M. and pictures)
and Ms. B. Done (collection manager), both of
the Museum of Tropical Queensland, Townsville,
Australia. We are also grateful to Dr. H. Fukami
and Dr. Z. Forsman for gifts of Siderastrea DNA
samples from Panama, to Dr. D. Obura for
providing material from Kenya, his input in the
early stages of the project, and comments on the
manuscript, to Dr. P. Harrison for permission to
reproduce an illustration of S. savignyana, and
to 2 anonymous reviewers, Dr. D. Miller, and
members of the Coral Reef Evolutionary Ecology
and Genetics (CREEG) laboratory, Biodiversity
Research Center, Academia Sinica (BRCAS) for
constructive comments. A collection permit was
granted by the Kenting National Park, Ministry
of the Interior, Pingtung, Taiwan. This study was
made possible by grants from Academia Sinica
and the National Science Council, Taiwan to C.A.C.
This is the CREEG-BRCAS contribution no. 72.
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Population Structure and Historical Demography of the Whiskered
Velvet Shrimp (Metapenaeopsis barbata) off China and Taiwan Inferred
from the Mitochondrial Control Region
Ta-Jen Chu1, Daryi Wang2, Hsien-Lu Huang3, Feng-Jiau Lin4, and Tzong-Der Tzeng5,*
Department of Leisure and Recreation Management, Chung Hua Univ., 707 Wu-Fu Road, Sec. 2, Hsinchu 300, Taiwan
Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan
3
Department of Nutrition and Health Science, Fooyin Univ., 151 Chinhsueh Road, Ta-Liao, Kaohsiung 831, Taiwan
4
Department of Life Sciences, National Cheng Kung Univ., Tainan 701, Taiwan
5
Department of Leisure, Recreation and Tourism Management, Shu-Te Univ., 59 Hun-Shan Rd., Hun-Shan Village, Yen-Chau,
Kaohsiung 824, Taiwan
1
2
Ta-Jen Chu, Daryi Wang, Hsien-Lu Huang, Feng-Jiau Lin, and Tzong-Der Tzeng (2012) Population structure
and historical demography of the whiskered velvet shrimp (Metapenaeopsis barbata) off China and Taiwan
inferred from the mitochondrial control region. Zoological Studies 51(1): 99-107. Sequence analyses of a
344-base-pair segment of the mitochondrial control region were conducted to elucidate the population structure
and historical demography of the whiskered velvet shrimp (Metapenaeopsis barbata) off China and Taiwan. Six
populations including 187 individuals were separately collected from the northern East China Sea (ECS), waters
off Kagoshima (KS, Japan), Taichung (TC, west-central Taiwan), Cheding (CD, southwestern Taiwan), Xiamen
(XM, southern China) and Hong Kong (HK). The haplotype diversity (h) was high for all populations (96.95%),
with values ranging 89.1% (CD) to 98.9% (KS). Nucleotide diversity (π) of all populations was 1.524%, with
values ranging 0.714% (TC) to 1.554% (ECS). All FST values among the 6 populations were significant except
for the ones from the pairs TC-CD, XM-HK, KS-XM, and KS-HK. The haplotype network was divided into 2
clades: clade I included individuals from all populations but YZR, and clade II did not include specimens from
CD or TC. Neutrality tests and mismatch distribution analyses both suggested that this species had experienced
a population expansion. Three distinct groups were yielded by the AMOVA tests, pair-wise FST analyses, and
the UPGMA tree of the 6 sampled areas. The 1st group included the ECS, the 2nd contained TC and CD, and
the 3rd included the KS, XM, and HK populations. Based on the continuity of the geographic position and gene
flow, the 2nd and 3rd groups should be considered a single population.
Key words: Metapenaeopsis barbata, Mitochondrial DNA, Population structure, Population expansion.
U
nderstanding the population genetic
structure is an important component of successful
and sustainable long-term management of fishery
resources (Hillis et al. 1996), and recent advances
in population genetic (Excoffier et al. 1992) and
historical demographic analyses (Rogers and
Harpending 1992, Fu 1997) can be very helpful in
providing valuable and complementary information
to catch- and age-composition data (Pauly et al.
2002).
Analyses of population genetic structures
of marine biota have frequently revealed that
organisms with a high dispersal capacity have
little genetic distinction over large geographic
scales (Hellberg 1996). Such studies suggest
that there are high levels of gene flow among
*To whom correspondence and reprint requests should be addressed. Tel: 886-7-6158000 ext. 4211. Fax: 886-7-6158000 ext. 4299.
99
100
Chu et al. – Population Structure of Metapenaeopsis barbata
marine populations. However, there is growing
evidence that widespread marine organisms are
more genetically structured than expected given
their high dispersal potential and apparent lack of
barriers to dispersal in the ocean (Palumbi 1997,
Benzie 1999, Briggs 1999). Thus, there may be
limits to the actual dispersal of marine organisms
with high dispersal potential (Benzie and Williams
1997). Those limits widely vary with species,
habitats, local ocean conditions, and historical
events, and they may produce sufficient chances
for genetic distinction (Palumbi 1994).
The whiskered velvet shrimp
(Metapenaeopsis barbata) is a small penaeid
species with a hard integument. It can be found
at 20-70 m in depth on rocky, sandy, and muddy
bottoms (Holthuis 1980, Yu and Chan 1986).
This shrimp is distributed throughout the IndoPacific region: Japan, the Korean Peninsula, Hong
Kong, Taiwan, Thailand, the Malay Archipelago
(including Indonesia), the southwestern coast of
India, and the Red Sea (Holthuis 1980, Miquel
1984). The whiskered velvet shrimp also plays
an important role in capture fisheries of China,
Taiwan, and other Asian countries (Wu 1984 1985,
Holthuis 1980). The life history of the whiskered
velvet shrimp, with an offshore planktonic larval
phase, estuarine post-larval and juvenile phases,
and offshore adult and spawning phases (Dall et
al. 1990), may allow moderate gene flow among
populations.
Various approaches have been adopted
to examine population structures of marine
organisms, including studies of the distribution and
abundance of various life-history stages, marking
and tagging, morphological characters, allozymes,
and DNA markers. Because each technique has
its merits and disadvantages, integrating the results
of several methods in a multi-pronged approach
to stock identification may maximize the likelihood
of correctly defining stocks (Pawson and Jenning
1996). Several studies on the fishery biology of
the whiskered velvet shrimp in the East China Sea
(ECS) and Taiwan Strait (TS) were conducted (Wu
1984, Tzeng and Yeh 1995, Tzeng et al. 2005), but
only 1 paper analyzed morphological characters in
an attempt to determine the stock structure of this
species (Tzeng et al. 2001). Two morphologically
distinguishable populations of whiskered velvet
shrimp in the ECS and TS were discriminated.
However, variations in morphological characters
can be affected by both genetic and environmental
factors, so that discrimination of populations based
on morphological variations must be verified by
genetic evidence to confirm that the variations
reflect the true degree of reproductive isolation
rather than environmental isolation (Pepin and
Carr 1992).
Mitochondrial (mt)DNA has many attributes
that make it particularly suitable for population
genetic studies, including its rapid rate of
evolution, a lack of recombination, and its
maternal inheritance (Hoelzel et al. 1991). Since
the control region of mtDNA was shown to be
the most variable region in both vertebrates and
invertebrates, this region is an ideal marker for
characterizing geographical patterns of genetic
variations within and among prawn populations
(Simon 1991). In this paper, sequence analyses
of a segment of the mitochondrial control region
were conducted to elucidate the population
genetic structure and historical demography of
the whiskered velvet shrimp in adjacent waters of
China and Taiwan.
Sampling
Six whiskered velvet shrimp populations
including 187 specimens were collected from
commercial shrimp trawlers (Fig. 1, Table 1). They
were separately sampled from the northern ECS,
waters off Kagoshima (KS, Japan), Taichung (TC,
west-central Taiwan), Cheding (CD, southwestern
Taiwan), Xiamen (XM, southern China), and Hong
Kong (HK). Specimens were immediately iced or
frozen after capture and later kept at -75°C before
DNA extraction.
DNA extraction, amplification, and sequencing
Total DNA was extracted from frozen muscle
tissues using a standard DNA proteinase K
digestion/phenol-chloroform extraction procedure
(Kocher et al. 1989). The complete control region
was amplified using the primers P30 (5'-GATC
TTTAGGGGAATGGTGTAATTCCATTG-3') and
P24 (5'-GTGTAACAGGGTATCTAATCCTGG-3'),
which respectively bind to the tRNAMet and 12S
rRNA genes. Thermal cycling was performed in
a GeneAmp 2400 thermal cycler (Perkin-Elmer,
Norwalk, CT, USA), and polymerase chain reaction
(PCR) conditions consisted of initial denaturation
at 95°C for 5 min; then 39 cycles of denaturation
at 95°C for 50 s, annealing at 50°C for 1 min,
and extension at 72°C for 1.5 min; followed by
Sequence analyses
a final extension at 72°C for 10 min. Amplified
DNA was separated through electrophoresis on
1.5% agarose gels and purified with the Gene
Clean II kit (Bio101, La Vista, CA, USA). Doublestranded DNA was sequenced on an ABI 377 DNA
sequencer (Applied Biosystems, Foster City, CA,
USA) with the P24 primer.
N
101
DNA sequences were aligned with ClustalX,
vers. 1.83 (Thompson et al. 1997), then subsequently optimized by eye in BIOEDIT, vers. 7.0.5.3
(Hall 1999). Control region sequences were
confirmed by comparing them with the complete
Clade I
32°N
Clade II
ECS
KS
China Coastal Current
30
Kuroshio Current
East China Sea
SCS Warm Current
28
CHINA
26
TC
XM
24
TAIWAN
CD
Taiwan Strait
22
HK
South China Sea
114
116
118
120
122
124
126
128
130°E
Fig. 1. Sampling localities and haplotype frequencies of whiskered velvet shrimp off China and Taiwan. Numbers of clades I and II in
each sampling site are shown in table 1.
Table 1. Codes of sampling sites, sample size (n), number of haplotypes (nh), number of clades, gene
diversity (h), and nucleotide diversity (π) with the 95% confidence interval (CI), Tajima’s D, and Fu’s Fs
statistics in 6 whiskered velvet shrimp populations
Code
Sampling site
n
ECS
East China Sea
30
XM
Waters off Xiamen
39
TC
Waters off Taichung
34
CD
Waters off Cheding
45
HK
Waters off Hongkong 25
KS
Waters off Kagoshima 14
Clade I
115
Clade II
72
Total
187
nh
No. of clade I No. of clade II
25
24
19
23
18
13
50
52
101
* p < 0.05; ** p < 0.01, ns; not significant (p > 0.05).
0
22
34
45
10
6
30
17
0
0
15
8
h (%)
π (%)
Tajima’s D
Fu’s Fs
98.2 ± 1.600
90.4 ± 4.200
93.2 ± 2.600
89.1 ± 3.800
97.0 ± 2.500
98.9 ± 3.100
92.8 ± 1.500
98.2 ± 0.700
96.95 ± 0.65
1.554 ± 0.138
1.170 ± 0.104
0.714 ± 0.099
0.727 ± 0.109
1.484 ± 0.108
1.348 ± 0.114
0.746 ± 0.061
1.362 ± 0.090
1.524 ± 0.068
-1.14700ns
-0.17860ns
-1.69561*
-2.04709**
0.05348ns
-0.06866ns
-2.11381**
-1.49631*
-1.70788*
-18.99674**
-15.46402**
-14.24433**
-18.54332**
- 8.61357**
- 8.18931**
-26.79920**
-25.65157**
-25.14003**
102
published mtDNA sequence of Penaeus monodon
(Wilson et al. 2000). Subsequent analyses
were based on a segment of the control region
sequence obtained from 187 individuals. The
nucleotide composition and numbers of variable
sites were assessed with MEGA3 (Kumar et
al. 2004). The genetic diversity (h), nucleotide
diversity (π), number of polymorphic sites (S),
and average number of nucleotide differences (K)
(Nei 1987) in each sample were calculated using
DnaSP vers. 4.10 (Rozas et al. 2003).
To examine whether any 2 populations
genetically differed from each other, pairwise F ST statistics (Wright 1965) among the 6
populations were estimated and tested using the
program, ProSeq (Filatov 2002). The statistical
significance of the estimate was tested using 1000
permutations. A dendrogram of the 6 sampling
areas was constructed using the unweighted pairgroup method with arithmetic means (UPGMA)
based on FST values using MEGA3.
An analysis of molecular variance (AMOVA)
implemented in ARLEQUIN vers. 3.01 (Excoffier
et al. 2005) was performed to test geographic
divisions among samples. Various groupings
of samples were suggested by (1) the UPGMA
tree of sampling areas, (2) F ST values between
samples, and (3) the geographic distribution. The
significance of these Φ statistics was evaluated by
104 random permutations of sequences among
them.
A network of haplotypes was constructed
using the median-joining method (Bandelt et al.
1999) in Network vers. 4.2.0.1 available at www.
fluxus-engineering.com. To check for deviations
from neutrality, Tajima’s D (Tajima 1989) and
Fu’s Fs statistical tests (Fu 1997) were run to
assess evidence for population expansion using
ARLEQUIN. Meanwhile, concordance of the data
with the distribution underlying the expansion
model was assessed. Historical demographic
expansion was investigated by examining the
frequency distributions of pair-wise differences
between sequences (mismatched distribution)
with ARLEQUIN. Rough dates of the population
expansion were estimated using the formula
τ = 2μT (Rogers and Harpending 1992), where T is
the time since expansion, τ is the expansion time,
and 2μ is μ (the mutation rate) × generation time ×
number of bases sequenced.
RESULTS
Amplification of whiskered velvet shrimp
genomic DNA with the P30 and P24 primers
produced a PCR product of approximately 1200
nucleotides base pairs long. We were able to
obtain a 344-bp segment of the control region
for each specimen. The nucleotide composition
of the fragment was AT-rich (A, 40.57%; G,
6.91%; C, 5.21%; T, 47.31%), as is usual for this
region in many invertebrate species. In total,
69 variable sites, including 28 singletons and 41
parsimoniously informative sites, were observed.
The average number of nucleotide differences
(K) of all populations was 5.243, with values
ranging 2.455 (TC) to 5.347 (ECS). The haplotype
diversity (h) was high for all populations (96.95%),
with values ranging 89.1% (CD) to 98.2% (ECS).
Nucleotide diversity (π) for all populations was
1.524%, with values ranging 0.714% (TC) to
1.554% (ECS) (Table 1).
All F ST values among pairs of populations
were significant except the 4 pairs of TC-CD, XMHK, KS-XM, and KS-HK (Table 2). The UPGMA
tree of these 6 sampled areas could be divided into
3 groups: 1 included the ECS, another comprised
HK, XM, and KS, and the last contained TC and
CD (Fig. 2).
Various groupings of samples were tested
using AMOVA, but only 2 significant groupings
Table 2. FST values among 6 whiskered velvet shrimp populations. Abbreviations for the sampling locations
are defined in table 1
XM
TC
CD
HK
KS
ECS
XM
TC
CD
HK
0.2685**
0.5444**
0.5540**
0.1786**
0.1504**
0.3211**
0.3324**
0.0419ns
-0.0113ns
0.0063ns
0.3018**
0.3638**
0.3189**
0.3772**
-0.0072ns
* p < 0.05; ** p < 0.01, ns; not significant (p > 0.05).
were obtained (Table 3). In the 1st grouping,
the AMOVA for the 6 populations included in a
single group yielded a significant Φ ST value of
0.3531, indicating that at least one of the pair-wise
comparisons showed significant heterogeneity.
None of the values of ΦCT in the 2-group pattern
was significant. One of ΦCT values in the 3-group
pattern was significant (ΦCT = 0.3739, p = 0.0163).
The 1st one included the ECS, the 2nd comprised
TC and CD, and the 3rd contained the XM, HK,
and KS populations.
Among the 187 individuals studied, 101
haplotypes were defined. The median-joining
network of these haplotypes revealed 2 clades (Fig.
3). These 2 clades appeared to have a geographic
structure. Specimens from TC and CD were not
found in clade II, whereas individuals from the ECS
did not appear in clade I. Individuals from HK,
XM, and KS were found in each clade. Haplotype
diversities (h) for clades I and II were 92.8% and
98.2%, respectively. Nucleotide diversities (π)
for clades I and II were 0.746% and 1.362%,
respectively (Table 1).
Tajima’s D and Fu’s Fs statistical tests were
performed to determine departure from neutrality.
Significant Tajima’s D values were obtained for TC
and CD, but not for the other populations. Fu’s
Fs tests were significant for all sampling locations
HK
KS
XM
ECS
TC
CD
(Table 1). The model of population expansion
could not be rejected using Tajima’s D and Fu’s
Fs statistical tests when all populations were
combined. The mismatch distribution including
both clades was bimodal (Fig. 4), with 1 mode
corresponding to the number of differences within
the clades, and the other to differences between
the 2 clades. Separate analyses of clades I and
II in both cases yielded a unimodal distribution,
which did not significantly differ (as measured by
the sum of the squared deviation; p > 0.05) from
that predicted by the growth expansion model (Fig.
4). Both Tajima’s D and Fu’s Fs statistical tests of
the 2 clades were negative and highly significant,
which indicated population demographic expansion
(Table 1).
τ values of clades I and II were 2.346/2u
(95% confidence interval (CI), 1.814-2.930) and
4.338 (95% CI, 3.512-5.115) generations. Because
of the shrimp’s short lifespan, a generation time of
1 yr was used (Tzeng and Yeh 1995). McMillenJackon and Bert (2003) roughly estimated a
Clade I
0.10
0.05
0.00
Fig. 2. UPGMA tree showing relationships among the 6
sampled areas.
Clade II
ECS
CD
TC
KS
XM
0.15
103
1
HK
3
5
9
14
25
Fig. 3. Network of haplotypes for whiskered velvet shrimp from
6 populations. The sizes of the circles are proportional to the
haplotype frequency.
Table 3. Results of the AMOVA. Abbreviations for sampling locations are given in table 1
Grouping
One group for all locations
Group 1 {ECS, XM, CD, TC, HK, KS}
Three groups
Group 1 {ECS}
Group 2 {TC, CD}
Group 3 {XM, HK, KS}
Source of variation
d.f.
Variance component
Φ-statistics
p
Among populations
Within populations
5
181
1.2093
2.2154
ΦST = 0.3531
0.0000
Among groups
Among populations within groups
Within populations
2
3
181
1.1422
0.0289
1.8838
ΦCT = 0.3739
ΦSC = 0.0151
ΦST = 0.3833
0.0163
0.1178
0.0000
104
mutation rate of 19%/106 yr (MY) for the mtDNA
control region of brown shrimp Farfantepenaeus
aztecus and white shrimp Litopenaeus setiferus.
Using this rate, the estimated time of expansion
for clade I was 17,946 (95% CI, 13,877-22,414) yr
ago, and for clade II, was 33,185 (95% CI, 26,86739,129) yr ago.
DISCUSSION
According to the results of FST tests, AMOVA
tests, and the UPGMA tree of these 6 populations,
3 distinct groups appeared to exist in the studied
waters. The 1st group was in the northern ECS,
the 2nd was in the eastern Taiwan Strait (CD and
TC), and the 3rd was in HK, XM (western TS),
and KS. Two environmental factors prevailing
in the studied waters here may have resulted in
genetic differences between the 1st group (ECS)
and the other 2 groups. First, the major source of
nutrients in the northern ECS is the relatively large
freshwater input from the Yangtze River (Tzeng
et al. 2004). Tsang et al. (2008) indicated that the
lack of suitable substratum for settlement restricted
Frequency
2500
2000
1500
1000
500
0
Frequency
2000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Pairwise differences
Clade I
1500
1000
500
0
Frequency
Pooled
600
500
400
300
200
100
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Clade II
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Fig. 4. Observed pair-wise differences (bars) and the expected
mismatch distributions under a sudden expansion model (solid
line) of mitochondrial control-region haplotypes in M. barbata
off China and Taiwan.
the northern distribution limit of Tetraclita japonica
to the mouth of the Yangtze River, and this habitat
discontinuity reduced the effective gene flow,
resulting in genetic differentiation between different
populations. Second, the mean temperature of
the sea in the northern ECS is lower than those in
the TS and South China Sea (SCS) (Tzeng et al.
2004). The whiskered velvet shrimp population
from the northern ECS genetically differed from
that from the eastern TS, and this result was
in agreement with a previous outcome that 2
morphologically distinguishable stocks separately
exist in the TS and ECS (Tzeng et al. 2001).
Similar results were also found for kuruma shrimp
(Penaeus japonicus) (Tzeng et al. 2004) and sword
prawn (Parapenaeopsis hardwickii) populations
(Tzeng 2007, Tzeng et al. 2008).
During summer, the SCS Warm Current (Fig.
1) moves northwards from the SCS to the ECS
(Niino and Emery 1961) and the southwesterly
monsoon drives water masses from Singapore and
Vietnam into the TS (Morton and Blackmore 2001).
The main branch of the warm Kuroshio Current
flows northward along the east coasts of the
Philippines and Taiwan (Chu 1972). In winter, a
small branch can be deflected by the northeasterly
monsoon to flow through the eastern TS (Farris
and Wimbush 1996), but this up-flowing branch is
prevented from flowing further north at the Penghu
Is. in the TS by deeper waters beyond Penghu (Jan
et al. 2002). Multiple oceanic currents meeting
in the eastern TS may thus explain significant
genetic differences between populations from the
eastern TS and the other groups. A similar result
was also found for kuruma shrimp (Pen. japonicus)
populations (Tsoi et al. 2007).
The whiskered velvet shrimp migrates from
inshore to offshore as it grows to a specific size or
life stage, but migratory distances are limited (Dall
et al. 1990). Thus, the dispersal of larvae is the
primary source of gene flow, and ocean currents
play a major role in dispersing this species. The
spawning season of this shrimp lasts from June to
Oct. (Sakaji et al. 1992). Along the east coast of
China, larvae from the ECS may be transported
and pass through the western TS and enter the
SCS with the China Coastal Current (Wu 1982).
This mixing of larvae results in homogeneity
among the ECS and HK/XM populations, but
might not be large enough to eliminate all genetic
differences between them (Table 2). This also
explains why the same haplotypes were found
in HK, XM, and the ECS, why individuals from
HK and XM were included in clade II (Fig. 3),
and why there were larger nucleotide diversities
in XM and HK (1.484% in HK and 1.17% in XM)
than in the eastern TS (about 0.72%) populations.
Moreover, recruitment of larvae from the ECS
into the western TS and SCS might partly explain
genetic differences among the HK, XM, and CD/
TC populations. However, larvae from the ECS
could not be transported into the eastern TS,
and that resulted in individuals from CD and TC
not being included in clade II (Fig. 3), and the
nucleotide diversity in the eastern TS population
being relatively low (Table 1).
During the late spring and summer, warm
water from the SCS dominates the TS and flows
into the ECS (Wang and Chern 1989). Larvae from
the SCS and TS may be transported into adjacent
waters of KS but not into the western ECS. Thus,
specimens in KS were found in 2 different clades
(Fig. 3), and there was relatively larger nucleotide
diversity at KS. This also explains why there
was a larger geographical distance between KS
and HK/XM than between KS and CD/TC, but no
genetic differences between KS and HK/XM were
found (Fig. 1, Table 2). Although the SCS, TS, and
ECS are adjacent waters and high gene flow might
occur among them, the population from the eastern
TS still differed from those from different sampling
locations. We considered that the genetic
variation in the eastern TS population resulted
from adaptation to the severe and complicated
environments as described above. Based on this
continuity of geographic position, gene flow, and
complex oceanic currents meeting in the eastern
TS (TC and CD), the 2nd and 3rd groups should
be considered a single population.
The neutrality of control-region mutations
was rejected on the basis of Tajima’s D and
Fu’s F tests for the total population (Table 1).
These 2 statistics are sensitive to factors such
as bottlenecks and population expansions which
tend to drive Tajima’s D and Fu’s F towards morenegative values (Tajima 1996, Martel et al. 2004).
Indeed, significant negative values of these 2
indices in this study indicated that whiskered velvet
shrimp off China and Taiwan had experienced
a population expansion. All haplotypes were
divided into 2 different clades (clades I and II) in
this study. Values of Tajima’s D and Fu’s F of the
2 distinct clades were also significant (Table 1).
Theoretical studies demonstrated that populations
in long-term stable demographic equilibrium show
a chaotic mismatch distribution, while recent rapid
population expansions or bottlenecks translate
into a unimodal (approximately Poisson) profile,
105
with a steeper wave indicative of a smaller initial
population before the expansion (Rogers and
Harpending 1992, Rogers 1995). The unimodal
mismatch frequency distribution pattern of each
clade accorded well with the predicted distribution
under a model of population expansion (Fig. 4).
An analysis of the demographic history of this
shrimp from the 2 distinct clades (clades I and II)
seems to indicate that clade I displayed a steeper
wave, which is typical of a smaller initial population
prior to the expansion or bottleneck (Fig. 4) (Rogers
and Harpending 1992). This picture also suggests
that clade I could have experienced expansion
in the more-recent past than clade II, the pairwise distribution mode of which was more-clearly
displaced to the right of the distribution pattern (Fig.
4). This was also supported by the 2 estimates of
time of expansion for clades I (13,877-22,414 yr
ago) and II (26,867-39,129 yr ago). Similar results
were also obtained from other marine organisms
in the ECS, TS, and SCS experiencing population
expansions, like Salanx ariakensis (Hua et al.
2009) and Chelon haematocheilus (Liu et al.
2007).
Acknowledgments: We would like to express
gratitude for the funding support by a grant
(NSC97-2313-B-336-001) from the National
Science Council, Taiwan. The authors are also
grateful for the reviewers’ critical comments on the
manuscript.
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Molecular and Morphological Investigations of Shovel-Nosed Lobsters
Thenus spp. (Crustacea: Decapoda: Scyllaridae) in Thailand
Apinan Iamsuwansuk1, Jessada Denduangboripant1,*, and Peter J.F. Davie2
1
2
Department of Biology, Faculty of Science, Chulalongkorn Univ., Phayathai Road, Phatumwan, Bangkok 10330 Thailand
Queensland Museum, PO Box 3300, South Brisbane, Queensland 4101, Australia
Apinan Iamsuwansuk, Jessada Denduangboripant, and Peter J.F. Davie (2012) Molecular and
morphological investigations of shovel-nosed lobsters Thenus spp. (Crustacea: Decapoda: Scyllaridae) in
Thailand. Zoological Studies 51(1): 108-117. Shovel-nosed lobsters (Thenus spp.) (or kang-kradan in Thai)
are the basis of an increasingly important fishery in Thailand and other tropical Indo-West Pacific countries. In
the past, only a single species was recognized, Thenus orientalis. However, a recent taxonomic revision, using
both morphological and DNA sequence analyses, established that at least 3 species occur in Thai waters. The
present work was designed to test the results of that earlier study as applied to the Thai fishery, and extend it
by using a broader sampling regime. Thirty adult Thenus specimens were sampled from 3 provinces: Chonburi
(on the eastern Gulf of Thailand), Phetchaburi (on the western Gulf of Thailand), and Phuket (on the Andaman
Sea). Genomic DNA was extracted from pereiopods, and the mitochondrial cytochrome c oxidase subunit 1 (COI)
gene was amplified and sequenced. A 403-base pair nucleotide data matrix was used to derive phylogenetic
trees using maximum parsimony. The molecular phylogeny clearly separated the Thai specimens into 3 clades:
13 individuals of T. indicus, 7 of T. orientalis, and 10 of T. unimaculatus. The recently proposed morphological
criteria were largely effective in separating Thenus species; however some of the morphometric ratios given in
the previous paper need to be adjusted. New local Thai names are proposed: kang-kradan thammada (common
shovel-nosed lobster) for T. indicus, kang-kradan kha-lai (spotted-leg shovel-nosed lobster) for T. orientalis,
and kang-kradan kha-muang (purple-leg shovel-nosed lobster) for T. unimaculatus. It is evident that there is
some ecological separation of the different species, and we hope this increased knowledge can be used to help
establish sustainable management of their exploitation in Thailand.
Key words: Cytochrome c oxidase subunit I, Molecular phylogenetics, Shovel-nosed lobster, Thailand, Thenus.
T
henus (Leach, 1815) is a genus of marine
scyllarid lobster found in tropical and subtropical
waters in the Indo-West Pacific region, and is
becoming increasing commercially exploited
(Department of Fisheries 1997, FAO 2010).
Common names across the region include shovelnosed lobsters, slipper lobsters, flathead lobsters,
and in Australia, Moreton Bay bugs. The common
names are mostly derived from the peculiar shape
of the broadly flattened cephalothorax. Generic
characteristics were well described and illustrated
in several recent works (Holthuis 1991, Burton and
Davie 2007). The length of the adult body ranges
12-25 cm.
Shovel-nosed lobsters are bottom dwellers
and prefer sand and mud habitats at 10-50 m deep
(Uraiwan 1977, Jones 2007, FAO 2010). They are
widely distributed in Asia and Australia, and Thenus
is the only genus in the family Scyllaridae to be
significantly commercially exploited; and indeed,
it is the main component of many trawl fisheries
(Jones 1993, Burton and Davie 2007). Moreover,
*To whom correspondence and reprint requests should be addressed. Tel: 66-22185378. Fax: 66-2-2185386.
108
Iamsuwansuk et al. – Phylogenetics of Thai Shovel-Nosed Lobsters
they are exported to European countries as frozen
meat (Naiyanetr 1963, Uraiwan 1977, Sungthong
1979). Species of a related, typically deeper-water
genus, Ibacus, also form small fisheries in some
parts of the world (Holthuis 1991).
In Thailand, there is increasing demand for
shovel-nosed lobsters (kang-kradan, in Thai),
because they are considered less common and
very good tasting. In 2003, whole-shelled shovelnosed lobsters were sold for only 140-240 baht
(US$5-8)/kg, but the current price (in 2011) is 400600 baht (US$13-20)/kg, 2.5 times higher than the
past, and the supply cannot keep up with the everincreasing demand. Also, with decreasing stocks
of marine shrimp due to overexploitation, fishermen
are turning to the Thenus fishery to increase their
economic returns. Unfortunately, research into the
mariculture of shovel-nosed lobsters in Thailand
has not yet been successful, and the fishery is
thus still entirely based on wild-caught animals.
Thus, there are significant concerns that natural
populations of Thai shovel-nosed lobsters could
become severely overexploited within a relatively
short period of time.
Most earlier taxonomic studies of Thenus
in Thailand (Naiyanetr 2007) recognized only 1
species: T. orientalis (Lund, 1793). However,
Burton and Davie (2007) recently revised the
genus using a concordance approach involving
3 techniques: morphometrics/morphology, starch
gel isozyme electrophoresis, and mitochondrial
(mt) DNA sequencing of 16S and cytochrome c
oxidase subunit 1 (COI) genes. The COI gene
has successfully been used for barcoding of
several crustaceans such as freshwater shrimp
(Liu et al. 2007) and mud crab (Shih and Suzuki
2008). They concluded that the genus contained
5 species: T. indicus Leach, 1815, T. orientalis
(Lund, 1793), T. australiensis Burton and Davie,
2007, T. unimaculatus Burton and Davie, 2007,
and T. parindicus Burton and Davie, 2007. Of
these, T. indicus, T. orientalis, and T. unimaculatus
were recorded from Thailand. However, they
also stated that, “Despite significant genetic
divergence, several sympatric species are
morphologically similar and identification can be
difficult.” Because of the increasing exploitation
of Thenus in Thailand, we undertook the present
study to try and repeat some aspects of the work
of Burton and Davie (2007) to ensure that we
would be able to identify the local species, and
also to try and better understand the population
ecology and distribution of the species in Thai
waters (since Burton and Davie (2007) examined
109
only a limited number of samples from a few
locations in Thailand). We thus reexamined the
external morphology, morphometric ratios, and
COI sequences of 30 Thenus specimens sampled
from both the Andaman Sea and Gulf of Thailand.
We hope the information gained will be of use
in establishing a sustainable fishery for Thenus
species in Thai waters.
In Apr., May, and Oct. 2010, 30 adult specimens of live or frozen shovel-nosed lobsters were
randomly collected from 3 provinces in Thailand:
10 samples from Sri Racha District, Chonburi
Province (on the eastern Gulf of Thailand), 10
from Cha-am District, Phetchaburi Province (on
the western Gulf of Thailand), and 10 from Muang
District, Phuket Province (on the Andaman Sea)
(Fig. 1). All specimens were photographed to
document the live color patterns, and labeled
as Chon01-10 for Chonburi, Phet01-10 for
Phetchaburi, and Phuk01-10 for Phuket samples.
They were immediately preserved in 95% ethanol
before later examination in the laboratory.
All 30 Thenus samples were identified
THAILAND
Andaman sea
CAMBODIA
Gulf of Thailand
Fig. 1. Sample collecting sites in 3 provinces of Thailand:
Phuket (star), Phetchaburi (circle), and Chonburi (triangle). The
map was taken from http://www.nationsonline.org/oneworld/
map/thailand_map2.htm.
110
following Burton and Davie’s (2007) taxonomic
key to species. Important characters were: 1)
the presence or absence of spots or blotches on
the pereiopods, 2) the presence or absence of
a spine on the merus of the 3rd maxilliped, and
3) the nature of the dentition on the ischium of
the 3rd maxilliped (Fig. 2A). Width and length
measurements of various parts of the body and
legs were made, and morphological ratios were
calculated following Burton and Davie (2007) (Fig.
2B-D, Table 1). These ratios were then also used
as a further aid to discriminate species according
to Burton and Davie’s (2007) criteria (Table 2).
Genomic DNA was extracted from pereiopod
tissue using a QIAamp DNA Mini Kit (QIAgen,
(A)
Hilden, Germany) using the animal tissue protocol
supplied with the extraction kit. An approximately
700-base-pair (bp) fragment of the mitochondrial
COI gene was amplified by a polymerase chain
reaction (PCR) using Folmer’s primers (Folmer et
al. 1994): CO1-1490 (forward primer, 5'-GGTCA
ACAAATCATAAAGATATTGG-3') and CO1-2198
(reverse primer, 5'-TAAACTTCAGGGTGACCAA
AAAATCA-3'). PCR amplification was conducted
on a 50-µl reaction volume containing 1x PCR
optimized buffer, 0.24 mM of mixed dNTP, 2 units
of Dynazyme Taq polymerase (Finnzyme, Vantaa,
Finland), and 0.5 μM of each forward and reverse
primer. The PCR conditions were modified from
Folmer et al. (1994) as follows: 5 min at 95°C for
(B)
CL
(C)
s
m
CW
d
TL
i
TW
PW1
(D)
MW1 (2)
ML3
PL1 (2)
Fig. 2. Morphological characteristics and measurements used in this study. (A) Third maxilliped with spine (s) on the merus (m) and
dentition (d) on the ischium (i); (B) carapace; (C) pereiopods; (D) 6th abdominal segment and telson. For details of other abbreviations
see table 2.
pre-running, then 35 cycles of 60 s at 95°C for
denaturation, 60 s at 49-52°C for annealing, and
90 s at 72°C for extension, followed by 5 min at
72°C for a final extension. Each PCR product was
mixed with a loading dye (0.14% bromophenol
blue) and electrophoresed in a 1.8% agarose gel
stained with ethidium bromide at 80 V for 45 min.
The gel was visualized under ultraviolet light.
The PCR products were purified using a
QIAquick PCR purification kit (QIAgen) before
being sent to an automated cycle-sequencing
service (Macrogen, Seoul, South Korea). All
of these COI sequences were submitted to
GenBank with accession numbers JN16571628 for T. indicus, JN165729-35 for T. orientalis,
and JN165736-45 for T. unimaculatus. The
COI sequences of our Thenus specimens
111
were compared to other Thenus sequences
retrieved from the GenBank nucleotide database
(T. parindicus GenBank no. HM015421, T.
australiensis GenBank accession no. HM015433,
T. orientalis no. HM015440, T. indicus no.
HM015445, and T. unimaculatus no. HM015449)
and aligned using the ClustalX vers. 2.0 program
(Larkin et al. 2007). Ibacus peronii (GenBank
accession no. HM015458) was added as an
outgroup taxon. PAUP* vers. 4.10b (Swofford
2002) was used to reconstruct the phylogenetic
trees (using maximum parsimony (MP), a heuristic
search with random stepwise addition, TBR
branch swapping, and steepest descent options).
Bootstrap support for the clades was determined
using PAUP* with 1000 pseudoreplicates.
Table 1. Abbreviations and their meanings for morphological measurements in this study, following Burton
and Davie (2007)
Abbreviations
Morphological measurements
Carapace (dorsally measured) (Fig. 2B)
CL
CW
Pereiopods 1, 2 and 3 (Fig. 2C)
PL1(2)
PW1
ML3
MW1(2)
Abdomen (dorsally measured) (Fig. 2D)
TL
TW
Length of carapace from base of antennal plate sinus to posterior margin of carapace on
dorsal side
Width of widest section: width of carapace with callipercaliper arms sitting on left and right
postorbital spines
Length of the 1st (and 2nd) propodus: anterior internal protrusion to posterior spine
Width of the 1st propodus: widest posterior dimension at right angles to propodus length
Length of the 3rd merus: anterior spine to posterior spine
Width of the 1st (and 2nd) merus: widest posterior dimension at right angles to merus
length
Length of telson: from mid-anterior margin to posterior margin of the calcified region
Width of telson: from left to right latero-posterior spine
Table 2. Morphological measurement ratios used to distinguish Thenus species following Burton and Davie
(2007)
Morphological ratios
CW/CL
ML3/CL
MW1/CL
MW2/CL
PL1/CL
PL2/CL
PW1/PL1
TL/TW
Suggested species
T. indicus
T. orientalis
T. unimaculatus
> 0.45
< 0.07
-
< 0.079
> 0.31
> 1.29
< 0.23
> 0.39
> 0.35
-
112
RESULTS
The morphological examination suggested
that T. indicus and T. orientalis specimens (Fig. 3A,
D, respectively) possessed numerous dark brown
spots on the carapace, whereas T. unimaculatus
typically had purple spotting (Fig. 3G). The
carapace of T. orientalis also had pink spots and
pink blotches near the orbits. The pereiopods of
T. orientalis were all distinctively spotted (Fig. 3F),
while T. unimaculatus had a purple blotch on the
inner face of the merus of 1 or more pereiopods
(Fig. 3I). Thenus indicus lacked any spots or
blotches on the pereiopods.
The COI sequence data from the 30 Thai
Thenus specimens were combined with 5 existing
Thenus species in GenBank resulting in a 403-bp
alignment. The PAUP* analysis produced 32
most parsimonious trees with a tree length of 210
steps (Fig. 4 shows an example tree). The semistrict consensus phylogenetic tree (Fig. 5) shows
that the present Thai samples were all grouped
into one of 3 major species-clades previously
reported from Thai waters (Burton and Davie
2007). Clade A included all Thenus samples from
Chonburi (Chon01-10) and 3 specimens from
Phetchaburi (Phet01, -07, and -08), and all were
strongly supported (100% bootstrap support) as
T. indicus (reference GenBank sequence). Clade
B strongly supported (98% bootstrap support)
all other specimens from Phetchaburi Province
(Phet02-06, -09, and -10) as being T. orientalis.
Finally, clade C included all Thenus samples from
Phuket (Phuk01-10) as T. unimaculatus (with 97%
bootstrap support).
The morphometric ratios of all 30 Thenus
shovel-nosed lobsters (according to Burton and
Davie 2007) were calculated (Table 2) and used
to identify the Thenus species. The results are
presented in table 3. We found that around
1/2 of all Thenus samples (16 of 30) failed to
be unambiguously identified on the basis of
the calculated ratios, and only 10 samples
(Chon01, -03, -05, -09, -10, Phet01, -03, -07,
-08, and Phuk01) correctly matched the name
given following the morphological and genetic
identifications. Some samples (such as Chon02 T.
indicus) presented with ratios that could place it in
2 separate species according the ratios given by
Burton and Davie (2007), e.g., for Chon02 the ML3
to CL ratio was 0.51 (> 0.45) correctly indicating it
to be T. indicus), but its MW2 to CL ratio was 0.077
(< 0.079) which also placed it as T. orientalis.
This identification problem also occurred with
samples Phet04, -06, -10, and Phuk08, as for
each of these cases, the identification based on
the morphometric ratios differed from that based
on simple morphological characteristics such as
the color pattern. In total, only 33% (10 of 30) of
all Thenus samples were unambiguously identified
using the morphometric ratio technique.
DISCUSSION
We found that morphological identification
of all samples using the key provided by Burton
and Davie (2007) gave the same results as the
COI analysis, thus proving the basic usefulness
of the key for this region (Table 3). From this
finding, morphological examinations combined
with molecular phylogenetic analysis could identify
at least 3 species of Thenus in Thailand, i.e.,
T. orientalis supposedly living only on the west
coast of the Gulf of Thailand, T. indicus on both
eastern and western sides of the Gulf, and T.
unimaculatus distributed only in the Andaman Sea
with the highest endemism among the 3 species.
Our suggestion on the simple recognition of T.
orientalis and T. unimaculatus as 2 additional
Thenus species in Thailand is to look for the
specific appearance of small brown spots on the
pereiopods of T. orientalis and purple-blotched
pereiopods of T. unimaculatus. Furthermore, we
propose a new local Thai name for T. indicus of
kang-kradan thammada (common shovel-nosed
lobster), kang-kradan kha-lai (spotted-leg shovelnosed lobster) for T. orientalis, and kang-kradan
kha-muang (purple-blotched leg shovel-nosed
lobster) for T. unimaculatus.
In Burton and Davie’s study (2007), their morphometric analysis gave strong, unambiguous
results, and showed all species groups to be 100%
discriminated for specimens for which complete
datasets were available. Given a large dataset
for analysis, it is clear that a canonical analysis
can discriminate any given individual, even
though this is not a practical technique for rapid
field or laboratory identification. Unfortunately,
they also stated that there is a large degree of
overlap between species when any specific ratios
were considered on their own. This means that
simple ratios are of limited usefulness for uniquely
identifying a particular species from all others.
When all 5 species were compared, the ratio of the
1st pereiopod propodus width vs. length showed
the best discrimination of means, but also showed
overlapping ratios between some individuals of
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H)
(I)
113
Fig. 3. External morphological characteristics of 3 Thenus species of Thailand. (A-C) Specimen Phet08 as representative of T.
indicus. (A) Dorsal view; (B) ventral view; (C) non-spotted pereiopods. (D-F) Specimen Phet06 as representative of T. orientalis. (D)
Dorsal view; (E) ventral view; (F) brown spots and dots on pereiopods. (G-I) Specimen Phuk02 as representative of T. unimaculatus. (G)
Dorsal view; (H) ventral view; (I) purple blotches on pereiopods.
114
all species. They did however suggest that the
ratios may be of some use for identifying some
individuals if they were above or below certain
threshold values for that species.
Such a suggestion was confirmed in our
present study. We followed Burton and Davie
(2007) in calculating morphometric ratios for all
30 Thai Thenus shovel-nosed lobsters and found
a problem of using these ratios for discriminating
species in the absence of color. However, with
some changes in the values, the success of
identification improved. For example, the ML3/CL
ratio should be adjusted to be > 0.48 (up from
0.45) for T. indicus, and the PW1/PL1 ratio should
be changed to be > 0.33 (from 0.35) to moreaccurately distinguish T. unimaculatus. It is likely
that greater numbers of specimens may be needed
to more-finely calibrate these ratios for maximum
usefulness.
According to the phylogenetic tree in figure 4,
several samples within clade A (T. indicus) show
marked separation from the main group (Chon01,
Phet01, and Chon08), and this perhaps indicates
some populational level genetic divergence that
warrants further investigation. In addition, even
though there was a strong cluster (100% bootstrap
support) for clade B (T. orientalis), samples Phet03
and Phet04 had a 4-6-bp difference from the main
grouping (see branch lengths in Fig. 4).
Biogeographically, it appears from the results
so far that T. orientalis may be more abundant
on the western side of the upper Gulf of Thailand
with all of the present samples being confined to
Phetchaburi Province. However, Burton and Davie
2
16
3
16
60
5 changes
Chon01
Chon02
Chon03
Chon04
Chon05
1
Chon06
30
Chon09
Chon10
Phet08
1
HM015445 T. indicus
1 Phet01
Chon07
Phet07
30
Chon08
37
HM015421 T. parindicus
Phet02
Phet05
2 Phet06
Phet09
Phet10
9
HM015440 T. orientalis
2 Phet04
4
Phet03
2
Phuk01
16
Phuk02
1
Phuk03
Phuk04
Phuk05
7
1 Phuk06
2 Phuk07
Phuk08
1 Phuk09
Phuk10
6
HM015449 T. unimaculatus
10
HM015433 T. australiensis
HM015458
Outgroup
Ibacus peroinii
Fig. 4. One of 32 most-parsimonious trees based on the cytochrome c oxidase subunit 1 (COI) sequence of Thenus spp. (with a tree
length of 210, a consistency index of 0.810, a retention index of 0.952, and a rescaled consistency index of 0.771).
(2007) previously reported 1 sample of this species
from Pattaya, in Chonburi Province (eastern side).
So it is evident that T. orientalis is also present on
the eastern side of the Gulf, as one might expect
given that its broader distribution is as far as
Taiwan.
T. indicus occurs on both the eastern and
western sides of the upper Gulf of Thailand
(Chonburi and Phetchaburi Provinces,
respectively), and the strong clustering of T.
indicus specimens (Chon01-10 with Phet01, -07,
and -08) suggests that there is gene flow between
populations on both sides of the gulf. The oceanic
circulation patterns in the upper Gulf of Thailand
would likely widely distribute the planktonic
phyllosoma larvae throughout the region. Currents
move in a counterclockwise direction under the
influence of the northeasterly monsoon (Nov.
75
< 50
Semi-strict
75
< 50
75
< 50
100
50
100
77
100
100
100
72
100
98
50
< 50
100
95
100
88
100
97
100
80
115
to Jan.) and clockwise and counterclockwise
under the influence of the southwesterly monsoon (May to Aug.) (Buranapratheprat 2008).
Broader population-level studies, perhaps using
microsatellites, are needed to properly investigate
if there are any discrete populations elsewhere in
Thai waters.
Interestingly, T. indicus was also recorded
as far west as Pakistan and India (Burton and
Davie 2007). However, while its distribution
should therefore encompass the Andaman Sea
coast of southern Thailand, no samples have
so far been collected there. All samples from
this area consisted only of T. unimaculatus. T.
unimaculatus, although more-widely distributed
in the Indian Ocean, is still only known from the
Andaman Sea coast (Phuket Province), and has
not been found in the Gulf of Thailand. This may
Chon0
Phet01
Chon02
Chon03
Chon04
Chon05
Chon06
Group A
Chon09
Chon10
Phet08
HM015445 T. indicus
Chon07
Phet07
Chon08
HM015421 T. parindicus
Phet02
Phet05
Phet06
Phet09
Group B
Phet10
HM015440 T. orientalis
Phet04
Phet03
Phuk01
Phuk02
Phuk03
Phuk04
Phuk05
Phuk06
Group C
Phuk07
Phuk08
Phuk09
Phuk10
HM015449 T. unimaculatus
HM015433 T. australiensis
Outgroup
HM015458 Ibacus peronii
Fig. 5. Semi-strict consensus tree of the 32 most-parsimonious trees based on cytochrome oxidase subunit 1 (COI) sequences of
Thenus spp. Numbers above the branches indicate percentages of nodes recovered in the most-parsimonious trees. Numbers below
the branches show bootstrap support based on 1000 pseudoreplicates.
116
be the result of the deep-sea fishing fleet which
trawls in the area dominated by T. unimaculatus,
with T. indicus perhaps occurring in more-inshore
shallower waters and thus not being taken.
Further intensive sampling is needed to resolve
this anomaly.
The regional geographic distribution patterns
of Thenus species are likely to be influenced by
the availability of habitat suitable for each species.
For example, the preferred habitat of T. parindicus
(referred to as the ‘mud bug’ in Australia) is
shallow muddy inshore waters to 20 m deep, while
T. australiensis (the sand bug) inhabits deeper,
rockier areas (Jones 1993 2007). Similarly in
Thailand, T. indicus appears to prefer shallower
water than T. orientalis. Local fish sellers in
Phetchaburi Province we interviewed reported that
T. indicus is normally captured live by small local
fishing boats in areas close to the coast. However,
T. orientalis is usually caught by large commercial
ships working in the open sea, and they must
be frozen before being sent back to port. Moredetailed studies of the ecological preferences of
all 3 Thai species, T. indicus, T. orientalis, and
T. unimaculatus, are urgently needed in order to
enable effective conservation and management
strategies to control their future exploitation.
Table 3. Morphological measurement ratios calculated from 30 live or frozen Thenus samples in this
study. Bold numbers indicate in-range ratios which identify a species (see the criterion in Table 2). The
specific names were suggested by morphological measurement ratios compared to those by morphological
characteristics. Abbreviations of the specific names are: ind for T. indicus, ori for T. orientalis, and uni for T.
unimaculatus
Morphological measurement ratio
Sample name
CW1/CL
ML3/CL
MW1/CL
MW2/CL
PL1/CL
PL2/CL
PW1/PL1
TL/TW
Species identified by
Species identified
measurement ratios
by morphological
characteristics
Chon01 (live)
Chon02 (live)
Chon03 (live)
Chon04 (live)
Chon05 (live)
Chon06 (live)
Chon07 (live)
Chon08 (live)
Chon09 (live)
Chon10 (live)
Phet01 (frozen)
Phet02 (frozen)
Phet03 (frozen)
Phet04 (frozen)
Phet05 (frozen)
Phet06 (frozen)
Phet07 (frozen)
Phet08 (frozen)
Phet09 (frozen)
Phet10 (frozen)
Phuk01 frozen)
Phuk02 frozen)
Phuk03 frozen)
Phuk04 frozen)
Phuk05 frozen)
Phuk06 frozen)
Phuk07 frozen)
Phuk08 frozen)
Phuk09 frozen)
Phuk10 frozen)
1.29
1.29
1.24
1.29
1.23
1.27
1.43
1.20
1.24
1.20
1.25
1.24
1.24
1.22
1.21
1.21
1.28
1.27
1.22
1.24
1.34
1.27
1.27
1.22
1.23
1.21
1.26
1.23
1.20
1.24
0.46
0.51
0.47
0.45
0.48
0.49
0.54
0.47
0.47
0.49
0.56
0.48
0.44
0.46
0.46
0.45
0.53
0.52
0.44
0.46
0.43
0.38
0.40
0.38
0.38
0.36
0.38
0.40
0.35
0.36
-b
0.09
0.10
0.10
0.10
0.09
0.10
0.09
0.10
0.27
0.35
0.10
0.10
0.41
0.09
0.09
0.09
0.10
0.09
0.09
0.13
0.12
0.12
0.11
0.11
0.10
0.11
0.12
0.12
0.11
0.088
0.077
0.083
0.083
0.083
0.078
0.088
0.087
0.084
0.081
0.080
0.093
0.091
0.089
0.084
0.085
0.088
0.080
0.080
0.086
0.110
0.115
0.099
0.100
0.097
0.098
0.097
0.100
0.109
0.097
-b
0.28
0.27
0.27
0.26
0.25
0.28
0.25
0.26
0.27
0.29
0.27
0.24
0.26
0.26
0.26
0.27
0.29
0.25
0.26
0.28
0.24
0.25
0.24
0.23
0.23
0.24
0.27
0.22
0.25
0.27
-b
0.33
0.35
0.34
0.34
0.51
0.36
0.35
-b
0.36
0.33
0.31
0.31
0.35
0.32
0.32
0.38
0.31
0.32
0.35
0.33
0.30
0.31
0.29
0.30
0.30
0.32
0.29
0.31
-b
0.19
0.27
0.30
0.27
0.28
0.28
0.29
0.30
0.27
0.21
0.30
0.34
0.30
0.31
0.33
0.26
0.27
0.30
0.29
0.34
0.38
0.36
0.35
0.42
0.38
0.37
0.33
0.41
0.37
0.27
0.25
0.34
0.25
0.33
0.29
0.27
0.25
0.29
0.30
0.26
0.34
0.33
0.29
0.32
0.29
0.29
0.27
0.28
0.31
0.28
0.34
0.37
0.35
0.36
0.32
0.36
0.34
0.32
0.36
ind
ori, ind
ori, ind
ind
ori, ind
ori, ind
uni, ind
ind
ind
ind
ind
ori, ind
ori
ind
ori, ind
ind
ind
ind
-a
ind
uni
ori, uni
ori, uni
ori, uni
ori, uni
ori, uni
ori, uni
ori
ori, uni
ori, uni
ind
ind
ind
ind
ind
ind
ind
ind
ind
ind
ind
ori
ori
ori
ori
ori
ind
ind
ori
ori
uni
uni
uni
uni
uni
uni
uni
uni
uni
uni
Phet09 specimen was not identifiable to species by the measurement ratio. bSome ratios could not be calculated because of the loss
of the 1st and 2nd pereiopods.
a
Acknowledgments: This project was financially supported by the A1B1-MICO (TRF)
Research Fund and the Research Program
on Conservation and Utilization of Biodiversity
and the Center of Excellence in Biodiversity,
Faculty of Science, Chulalongkorn Univ., the
Plant Genetic Conservation Project under
the Royal Initiative of Her Royal Highness
Princess Maha Chakri Sirindhorn, and the
9 0 t h A n n i v e r s a r y o f C h u l a l o n g k o r n U n i v.
Fund (Ratchadaphiseksomphot Endowment
Fund). Special thanks go to Prof. Emeritus
Phaibul Naiyanetr of Chulalongkorn Univ. for his
suggestions on sample collection and taxonomy.
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(Decapoda, Stomatopoda, Anostraca, Myodopoca and
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chasmagnathus complex (Crustacea: Brachyura:
Varunidae) from East Asia. Zool. Stud. 47: 114-125.
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sand lobsters rearing in aquarium. Bangkok, Thailand:
Department of Fisheries. (in Thai with English summary)
Swofford DL. 2002. PAUP*. Phylogenetic Analysis Using
Parsimony (*and other methods). Vers. 4. Sunderland,
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On Taiwanese species of Baeocera Erichson (Coleoptera: Staphylinidae:
Scaphidiinae)
Ivan Löbl
Muséum d'histoire naturelle, Case postale 6434, 1211 Geneva 6, Switzerland
Ivan Löbl (2012) On Taiwanese species of Baeocera Erichson (Coleoptera: Staphylinidae: Scaphidiinae).
Zoological Studies 51(1): 118-130. The rove beetle subfamily Scaphidiinae of Taiwan is still inadequately
studied. The present paper provides a review of Taiwanese members of Baeocera Erichson, which is one of the
more species-rich genera of this group. While only 5 species were previously reported from Taiwan, 15 species
are recognized at present: B. alesi sp. nov., B. aliena sp. nov., B. alishana sp. nov., B. anchorifera sp. nov., B.
lindae sp. nov., and B. mutata sp. nov. are described as new and illustrated. Baeocera caliginosa, B. cooteri,
B. longicornis, and B. takizawai are reported for the 1st time from Taiwan. A key to the Taiwanese species of
Baeocera is provided. http://zoolstud.sinica.edu.tw/Journals/51.1/118.pdf
Key words: Coleoptera, Staphylinidae, Scaphidiinae, Baeocera, Taiwan.
W
hile many data on Taiwanese phytophagous beetles are available (Lee et al. 2011),
knowledge of the taxa associated with fungi
remains inadequate. The Scaphidiinae is one
of the more species-rich subfamilies of rove
beetles which feed on fungi and myxomycetes.
Miwa and Mitono (1944) gave an overview of
the Japanese and Taiwanese species, but as
far as the Scaphisomatini is concerned, they
seem to have used data previously published by
Achard (1923), and just added a description of
1 new species which they erroneously placed in
Toxidium LeConte, 1860. The latest taxonomic
account of the Taiwanese Scaphisomatini was
given by Löbl (1980). It was based on a study
of the type material of previously described taxa,
on additional collections made by H. Sauter
at the beginning of the 20th century, and on a
few more-recent collections mainly made by
T. Kano, J. and S. Klapperich, T. Nakane, and
Y. Yano. These collections included 5 species
of the genus Baeocera Erichson, 1845. As
members of Baeocera are common in leaf litter
and other vegetation debris in Asian subtropical
and tropical forests, the number of Taiwanese
species of Baeocera so far reported appears low,
particularly compared to the 10 species currently
known from Japan (Löbl 1984) or e.g., to the 10
species reported from Sri Lanka by Löbl (1971; in
fact 11, but one of them, B. mussardi Löbl, 1971,
was subsequently transferred from Baeocera to
Kasibaeocera Leschen and Löbl, 2005).
The genus Baeocera can be readily distinguished from other Scaphisomatini known to
occur in Taiwan by the following characters in
combination: body not compressed laterally;
antennomere III elongate, subcylindrical, and
similar to the following segment; apical segment
of maxillary palpi aciculate; and hind angles of
pronotum prominent apically. A key to the genera
of Scaphisomatini is given in Leschen and Löbl
(2005).
Recently, I examined collections of Taiwanese
Scaphisomatini made by A. Smetana (Ottawa,
*To whom correspondence and reprint requests should be addressed. Tel: 41-22-7843389. E-mail:[email protected]
118
Löbl – The Baeocera of Taiwan
Canada) and S. Vít (Geneva, Switzerland) that
contain a number of Baeocera extracted from
sifted samples of forest litter. This material
consisted of 12 species, 6 of which are recognized
as new, and four of which are reported for the 1st
time from Taiwan.
The examined material is housed in the
Muséum d'histoire naturelle, Geneva, Switzerland
(MHNG) and the Taiwan Agricultural Research
Institute, Taichung, Taiwan (TARI).
The length of specimens was measured from
the anterior pronotal margin to the inner apical
angle of the elytra. The body width was measured
at the widest point of the elytra. The length and
width of the mesepimera and metepisterna refer to
their exposed portion. The number of abdominal
ventrites is that of freely visible ones. The length
of the aedeagus was measured without the
eventually extruded part of the internal sac. The
aedeagi were cleared in isopropanol and mounted
1
2
119
in Canada balsam, on acetate slides fixed on
the same pins as the specimens. The aedeagi
are “lying on the side”, i.e. rotated 90°. Their
respective sides as given refer to the morphological
sides, with the ostium situated dorsally.
RESULTS
Baeocera alesi Löbl sp. nov.
(Figs. 1-3)
Type material: Holotype ♂ : Taiwan: Nantou
Co., Meifeng 2310 m, 3 May 1991, A. Smetana
[T61] (MHNG). Paratypes: 5 ♂♂ , 1 ♀ with same
data as for holotype (MHNG, TARI); 2 ♂♂ , 1 ♀,
with same data but 2130 m [T62] (MHNG); 1 ♀:
Hualien Co., Taroko National Park, Nanhushi Hut
2220 m, 12 May 1990, A. Smetana [T54] (MHNG).
Etymology: The species is named in honor of
my friend and the collector of this and many other
new species, Aleš Smetana, Ottawa, Canada.
Description: Length 1.85-2.0 mm, width 1.301.35 mm. Body very dark, almost black. Apices of
3
Figs. 1-3. Baeocera alesi sp. nov. 1, 2. Aedeagus in dorsal and lateral view, scale bar = 0.2 mm. 3. Apical process of median lobe
with extruded part of the internal sac, in lateral view, scale bar = 0.1 mm.
120
elytra, apex of abdomen, femora, and tibiae rufous,
tarsi and antennae lighter, almost yellowish. Body
strongly convex dorsally. Eyes comparatively
small. Length ratio of antennal segments as III
10: IV 10: V 10: VI 10: VII 12: VIII 8: IX 12: X 11:
XI 14. Segments III-VI even, each about 3-times
as long as wide. Segment VII almost 3-times as
long as wide. Segment VIII slightly narrower than
segment VII, twice as long as wide. Segments IXXI distinctly wider than segment VII, segment IX
about twice as long as wide, following segments
less than twice as long as wide. Pronotum and
elytra with even, very fine, sparse punctation,
distinct pubescence, not microsculptured, or elytra
with barely visible microsculpturing (even at 100x
magnification). Lateral margins of pronotum
strongly convex, lateral keels not visible in dorsal
view. Lateral margins of pronotum and elytra
separately arcuate in dorsal view. Scutellum
completely covered by pronotal lobe. Elytra
almost reaching tip of abdomen. Elytron without
basal stria, with very shallow sutural stria, strongly
shortened, starting posterior to basal 1/4 of sutural
length. Adsutural area flat. Metathoracic wings
completely reduced. Ventral side of body very
finely punctate. Hypomera strongly impressed
on posterior 3/5. Mesepimera conspicuously
large, swollen behind, impressed along anterior
margin, about twice as long as wide, narrowly
separated from mesocoxa. Posterior margin
of mesepisterna strongly arcuate, ridge-like,
and not level with mesepimera. Metaventrite
very finely and sparsely punctate, except for a
few coarse admesal punctures. Submesocoxal
line arcuate, margined by fine puncture row not
extended laterally, nor along section situated
between coxae. Submesocoxal area 0.05 mm
long, shortest interval between its margin and
metacoxa about 0.15 mm. Metepistenum flat,
at widest point 0.07-0.08 mm wide, with distinct,
sinuate, impunctate suture. Abdominal ventrite 1
with fine submetacoxal puncture rows, punctures
not elongate and not separated by wrinkles. Tibiae
straight.
Male: Protarsomeres 1-3 strongly widened,
almost as wide as apex of protibia.
Mesotarsomeres 1 and 2 strongly widened, almost
as wide as apex of mesotibia, mesotarsomere
3 weakly widened. Aedeagus as in figures 1-3,
0.92-0.94 mm long. Basal bulb oval, rather
strongly sclerotized. Apical process of median lobe
strongly sclerotized, slightly asymmetrical in dorsal
view, strongly incurved ventrally and tapering.
Ostium covered by single, asymmetrical, dorsal
plate. Parameres evenly narrow, lacking lobes,
sinuate in lateral view, shorter than basal bulb.
Internal sac very complex, with robust flagellum
and robust flagellar guide-sclerite bifid basally.
Basal part of internal sac strongly widened and
curved. Extruded part of flagellum and flagellar
guide-sclerite complex reaching level of apex of
parameres, consisting of 3 flat rods.
Habitat: Old broadleaf forests, in sifted lush
vegetation, humus, and various debris around
large trees, in debris accumulated at base of an
embankment along old forest road, and in little
mushrooms, moss, and humus around them on a
large fallen tree.
Comments: This and the following 2
species described below are characterized by
the hypomera being strongly impressed near
the basal margin, the large mesepimera, and
the particular type of aedeagus. While the
median lobe is similar to that in species of the B.
curtula group, the internal sac drastically differs.
In members of the B. curtula group, only the
ejaculatory duct is permanently extruded. This
species resembles B. cooteri Löbl, 1999 by the
large size and dark coloration of the body. It can
be easily distinguished by the elytra which almost
completely cover the abdomen, the much-finer
elytral punctation, and the shortened sutural striae.
Baeocera aliena Löbl sp. nov.
(Figs. 4-7)
Type material: Holotype ♂ : Taiwan: Taoyuan
Co., Takuanshan Forest, 17 Apr. 1990, 1650 m,
A. Smetana [T5] (MHNG). Paratypes: 4 ♀♀ with
same data as for holotype (MHNG, TARI).
Etymology: The name is a Latin adjective and
refers to the strange morphological characters of
the species.
Description: Length 1.85-1.95 mm, width
1.25-1.30 mm. Dorsal side of body as in B. alesi
sp. nov., except each elytron possessing complete
basal stria joined to lateral stria. Ventral characters
almost as in B. alesi sp. nov., in particular mesepimera conspicuously large, overlapped by margin
of mesepisterna, but barely swollen, metepisterna
narrower, about 0.05 mm wide, with almost-straight
suture. Shortest interval between submesocoxal
line and metacoxa about 0.12 mm.
Male: Protarsi and mesotarsi similar as in
B. alesi sp. nov. Aedeagus as in figures 4-7,
1.0 mm long. Basal bulb of median lobe weakly
sclerotized, oval. Apical process of median lobe
strongly sclerotized, wide, distinctly asymmetrical
4
6
121
5
7
Figs. 4-7. Baeocera aliena sp. nov. 4, 5. Aedeagus in dorsal and lateral view, scale bar = 0.2 mm. 6. Apical process of median lobe
with extruded part of the internal sac, in dorsal view, scale bar = 0.1 mm. 7. Apical process of median lobe with extruded part of the
internal sac, in lateral view, scale bar = 0.1 mm.
122
Alishan Natl. Scenic Area, env. 2200 m, road no.
18, km 88.5, S. Vít, 7 Jan. 2009 (MHNG).
Etymology : The name is derived from Alishan
where the species was found.
Description: Length 1.85 mm, width 1.30 mm.
With most external diagnostic characters as in B.
alesi sp. nov. and B. aliena sp. nov., but notably
differing by the elytra with complete, joined sutural
and basal striae, punctuation on pronotum, elytra,
and metaventrite less fine, metepisterna 0.07 mm
wide, almost flat, parallel-sided, with weakly curved
to straight suture. Submesocoxal area 0.06 mm
long, shortest interval to metacoxa 0.12 mm long.
Male: Protarsi and mesotarsi similar as in B.
alesi sp. nov. Aedeagus as in figures 8-10, 1.0 mm
long. Median lobe similar to that in B. alesi sp. nov.
but apical process evenly curved in lateral view.
Parameres almost straight and slightly narrowed
apically. Internal sac with 2 robust flagellar guidesclerites thickened basally. Extruded part of
internal sac with serrate rod moderately incurved
and 2 joined rods strongly curved.
Habitat: Mountain forest floor litter.
Comments: The species may be readily
distinguished from its allies by the sutural striae
and structure of the internal sac.
in dorsal view, moderately incurved ventrally and
abruptly narrowed to form a point in lateral view.
Ostium covered by single, asymmetrical, dorsal
plate. Parameres slightly narrowed apically,
lacking lobes, weakly curved in apical 1/3 (lateral
view), about as long as basal bulb. Internal sac
very complex, sclerotized parts forming robust
basal and apical denticles. Extruded part of
internal sac partly formed by long, spine-like
structures with serrate margin and large, weakly
sclerotized hook-like apophysis.
Habitat: Original mixed forest with gigantic
Chamaecyparis trees, in sifted humus and debris
among lush vegetation in a wet area.
Comments: This species may be easily
distinguished from B. alesi sp. nov. and other
congeners by the elytra having complete basal
striae and shortened sutural striae.
Baeocera alishana Löbl sp. nov.
(Figs. 8-10)
Type material: Holotype ♂ : Taiwan: Chiayi
Co., Alishan Natl. Scenic Area, env. 2350 m, road
no. 18, km 102, old Lulin Tree Track, S. Vít, 12
Apr. 2009 # II 2 (MHNG). Paratype ♀: Chiayi Co.,
8
9
10
Figs. 8-10. 8. Baeocera alishana sp. nov. Apical process of median lobe with extruded part of the internal sac, in lateral view, scale
bar = 0.1 mm. 9, 10. aedeagus in dorsal and lateral view, scale bar = 0.3 mm.
Baeocera anchorifera Löbl sp. nov.
(Figs. 11-15)
Ty p e m a t e r i a l : H o l o t y p e ♂ : Ta i w a n :
Kaohsiung Co., road from Tengchih to Chuyunshan
1400 m, 25 Apr. 1990, A. Smetana [T23] (MHNG).
Paratypes: 1 ♂ , 4 ♀♀ with same data as for
holotype (MHNG, TARI); 1 ♀: Kaohsiung Co.,
Tengchih 1610 m, 24 Apr. 1990, A. Smetana [T20]
(MHNG); 1 ♂ : Kaohsiung Co., Tengchih 1565 m,
23 Apr. 1990, A. Smetana [T18] (MHNG).
Etymology : The name is Latin and refers to
the anchor-like shape of the sclerotized base of
the internal sac of the aedeagus.
1.33-1.40 mm. Body black, hypomera, apical
abdominal segments, femora, and tibiae dark
reddish-brown to almost black, antennae and tarsi
lighter, light brown to yellowish. Body strongly
convex dorsally. Eyes large. Length ratio of
antennal segments as III 12: IV 13: V 15: VI 14:
VII 16: VIII 12: IX 15: X 15: XI 19. Segments III-VI
almost evenly narrow, segment V about 5-times as
long as wide. Segments VII and VIII only slightly
wider than segment VI, segment VII almost 5-times
as long as wide. Segment VIII about 3-times as
long as wide. Segments IX-XI distinctly wider than
segment VII, segment IX about 3-times as long
as wide, segment XI about 2.5-times as long as
11
12
123
wide. Pronotum and elytra not microsculptured,
with dense punctation and indistinct pubescence
(even at 100x magnification). Punctures very
shallow and not clearly delimited. Lateral margins
of pronotum and elytra separately arcuate in dorsal
view. Lateral margins of pronotum weakly convex,
lateral keels not or barely visible in dorsal view.
Tip of scutellum exposed. Elytra not covering
abdominal apex. Elytron with deep sutural
stria, curved at base to form basal stria joined to
lateral stria. Adsutural area flat. Metathoracic
wings not reduced. Ventral side of body almost
entirely very finely punctate. Hypomera weakly
impressed posteriorly. Mesepimera not enlarged,
not or barely swollen, about 3-times as long as
wide, twice as long as interval to mesocoxa.
Posterior margin of mesepisterna almost level with
mesepimera, not ridge-like. Submesocoxal line
arcuate, margined by fairly coarse punctures not
extended along section situated between coxae.
Submesocoxal area 0.05 mm long, shortest
interval between its margin and metacoxa about
0.20-0.22 mm. Metepistenum flat, at widest point
0.09-0.10 mm wide, narrowing anteriad, with deep,
almost-straight, impunctate suture. Abdominal
ventrite 1 with fairly coarse submetacoxal puncture
rows, punctures not elongate and not separated by
wrinkles. Tibiae somewhat curved.
Male: Tarsomeres 1 of protarsi and mesotarsi
13
14
15
Figs. 11-15. 11. Baeocera anchorana sp. nov., internal sac, without the extruded apical part of the ejaculatory duct, scale bar = 0.1 mm.
12, 13. Aedeagus in dorsal and lateral view, scale bar = 0.2 mm. 14, 15. Paramere in ventral and lateral view, scale bar = 0.1 mm.
124
strongly widened, narrower than apex of tibiae.
Tarsomeres 2 and 3 of protarsi and mesotarsi
weakly widened. Aedeagus as in figures 11-15,
0.84-0.95 mm long, strongly sclerotized. Median
lobe symmetrical, with slightly asymmetrical and
blunt apex in dorsal view. Apical process in lateral
view oblique and tapering to posterior level of
ostium. Parameres sinuate and with small apical
membranous lamina in dorsal view. Dorsal margin
of parameres widely notched at base, almost
straight posterior to notch, with distinct apical
denticle in lateral view. Flagellum of internal sac
sinuate, enlarged in basal 1/2 and forming a ridge,
at base curved and anchor-like. Flagellar guidesclerite robust, sinuate, widened at apex.
Habitat : Old Taiwania cryptomeroides forest,
in sifted various debris around bases of trees and
along fallen trees; old clearing in an old forest
with lush vegetation, shrubs, and rotting stumps
of trees. In sifted old mushrooms, old bark, and
humus around tree stumps; old broadleaf forest,
in sifted fermenting fruit accumulated on ground
around a large tree.
Comments: The species is a member of
the B. curtula group. Its male genital characters
suggest a close relationship with B. cooteri. This
16
new species can be distinguished from B. cooteri
by the finer elytral punctation, scarce punctation on
the middle part of the metaventrite, not elongate
submetacoxal punctures, distinctive shape of the
parameres of the aedeagus, and the presence of
a tooth-like sclerotized piece at the base of the
internal sac.
Baeocera caliginosa Löbl
(Figs. 16-19)
Baeocera caliginosa Löbl 1980: 186.
Material examined: Taiwan: Taoyuan Co.,
Fusing Township, road no. 118, km 5, 23 Feb.
2010, S. Vít, # 7, 8 ex. (MHNG, TARI).
Habitat: Dead trunks and litter under arborescent ferns.
Distribution: Japan (Okinawa and Kyushu),
Taiwan.
Comments: The description of this species
was based on a single specimen from Yonahedake, Okinawa. Since that time, I have examined
specimens from Mt. Mishime, Okinawa (MHNG).
The species appears to be variable in size. The
length of the Taiwanese specimens ranges 1.27-
17
18
19
Figs. 16-19. 16, 17. Baeocera caliginosa Löbl, aedeagus in dorsal and lateral view, scale bar = 0.1 mm. 18. Internal sac in detail,
dorsal view, scale bar = 0.05 mm. 19. Paramere in lateral view, scale bar = 0.1 mm.
1.50 mm, the width is 0.88-1.02 mm, and the
aedeagi are 0.37-0.41 mm long. The aedeagus
of the holotype was only illustrated in dorsal view.
New illustrations are given (Figs. 16-19) to show
the variability and characters as seen in lateral
view.
Baeocera cooteri Löbl
Baeocera cooteri Löbl 1999: 729.
Material examined : Taiwan: 1 ♀: Nantou
Co., Hwy. 14 below Wushe, 1700 m, 21 Apr. 1990,
A. Smetana [T15] (MHNG); 1 ♂ : Hsinchu Co.,
Jienshih Township, near Hsinkuang Village (Vill.),
km 44 road no. 60, env. 1600 m, 25 Mar. 2008,
S. Vít, Canacea litter, # III 6 (MHNG); 1 ♂ , 1 ♀:
Taitung Co. before Litao, road no. 20, km 180, env.
1000 m, 8 Apr. 2007, S. Vít, vegetative compost,
debris # 4; 1 ♂ , 1 ♀ with same data but “at foot of
rock” (MHNG, TARI); 1 ♂ : Taitung Co. after Litao,
road no. 20, km 174, env. 1300 m, 8 Apr. 2007, S.
Vít, mountain forest litter (MHNG); 1 ♀: Taitung
Co., road no. 20, km 184 before Wulu, env. 600 m,
10 Apr. 2007, S. Vít, mountain forest litter (MHNG);
1 ♀: Chiayi Co., Alishan road no. 18, km 4.5 S.
Lungmei, env. 800 m, 12 Apr. 2009, S. Vít, mixed
forest, litter # ii 6 (MHNG); 1 ♀: Taoyuan Co.,
Fusing Township, road no. 118, km 45, 23 Feb.
2010, S. Vít, dead trunks and arborescent fern, #
7 (MHNG); 1 ♀: Taoyuan Co., Fusing Township, N
Baling Hwy. 7, km 47, 22 Feb. 2010, bush litter, S.
Vít # 1 (MHNG); 1 ♂ : Hualien Co. road no. 23, km
7.5 lateral valley, 10 Apr. 2007, env. 400 m, S. Vít
(MHNG); 1 ♂ : Taipei Co., Beitou (NW Taipei City)
“Yangmingshan cemetery”, 300 m, 22 Oct. 2007, S.
Vít, rotten Pinus trunk, # 2 (MHNG).
Habitat: In rotten Pinus trunks, Canacea litter,
forest floor debris and compost, a plum orchard,
in a sifted accumulated pile of rather-fresh plum
leaves, in forests ranging 300-1600 m in elevation.
Distribution: China (Hong Kong, Zhejiang
Prov.), Taiwan. New to Taiwan.
Baeocera formosana Löbl
Baeocera formosana Löbl 1980: 97.
Distribution: Taiwan: “Pilam” (= Beinan,
Taitung Co.) and Puli (Nantou Co.).
Habitat: Unknown.
Comments: This species was not found within
the recent collections examined. It remains known
only from the 2 localities cited above.
125
Baeocera lindae Löbl sp. nov.
(Figs. 20-22)
Ty p e m a t e r i a l : H o l o t y p e ♂ : Ta i w a n :
Kaohsiung Co., Tengchih 1610 m, 24 Apr. 1990 A.
Smetana [T20] (MHNG). Paratypes: 2 ♂♂ , 1 ♀
and 11 specimens sex not examined: with same
data as for holotype (MHNG, TARI); 3 specimens
sex not examined, with same data but 1565 m, 23
Apr. 1990 [T18] (MHNG); 1 ♂ : Chiayi Co., Alishan
Natl. Scenic Area, env. 2350 m, road no. 18, km
102 old Lulin Tree Track, S. Vít, 11 Apr. 2009, # ii 3
(MHNG).
Etymology: The species is named in honor of
Mrs. Linda Schreyer-Crowe, Sherman Oaks, CA,
USA.
0.85 mm. Body rufous to almost black. Femora
similar to body, antennae, tibiae, and tarsi lighter
to yellowish. Body strongly convex dorsally.
Eyes comparatively large. Length ratio of
antennal segments as III 8: IV 10: V 12: VI 11:
VII 12: VIII 10: IX 13: X 12: XI 14. Segments IIIVI even in width, V and VI each about 5-times
as long as wide. Segments VII and VIII each
about 3-times as long as wide. Segment VIII
slightly narrower than segment VII. Segments
IX-XI distinctly wider than segment VII, segment
XI about twice as long as wide. Pronotum and
elytra with even, very fine, sparse punctation,
barely visible at 100x magnification, indistinct
pubescence, not microsculptured. Lateral
margins of pronotum strongly convex, lateral
keels not visible in dorsal view. Lateral margins
of pronotum and elytra continuously arcuate in
dorsal view. Scutellum completely covered by
pronotal lobe. Elytra in dorsal view completely
or almost completely covering abdomen. Sutural
stria of elytron curved along base to form basal
stria joined to lateral stria. Outer section of basal
stria approximate to basal margin. Adsutural area
flat. Metathoracic wings completely reduced.
Hypomera impunctate, impressed in large middle
part, with carinate anterior margin. Mesepisterna
impunctate. Mesepimera flat, slightly below plan
of mesepisterna, about 3-times as long as wide
and twice as long as interval to mesocoxa. Large
central part of metaventrite flat and smooth,
smooth area delimited by fairly coarse punctures
bearing long setae. Lateral parts of metaventrite
conspicuously coarsely punctate, punctures
well delimited, not or only slightly elongate,
with intervals somewhat smaller than puncture
diameters. Submesocoxal line parallel to coxa,
126
margined by fairly coarse puncture row extending
along lateral part of coxa. Submesocoxal area
about 0.03 mm long, shortest interval between its
margin and metacoxa conspicuously short, about
0.07-0.08 mm long. Metepisterna not clearly
separated, inner suture indicated by impressed
row of particularly coarse punctures. Abdominal
ventrite 1 with coarse submetacoxal puncture
rows, consisting of slightly elongate punctures
not separated by wrinkles. Remaining abdominal
punctation extremely fine. Tibiae straight.
Male: Protarsomeres 1-3 barely widened.
Aedeagus as in figures 20-22, 0.30-0.36 mm long,
moderately sclerotized. Basal bulb comparatively
large, with short, weakly inflexed apical process,
its ventral margin sinuate, tip strongly narrowed
and bent. Parameres barely sinuate and almost
evenly wide in dorsal view, straight in middle, and
distinctly widened at tip in lateral view. Internal sac
with narrow flagellar guide-sclerite, widened and
subtriangular at apex, with concave apical margin.
Habitat : Old Taiwania cryptomeroides forest,
in sifted various debris around bases of trees and
20
along fallen trees; old clearing in an old forest
with lush vegetation, shrubs, and rotting stumps
of trees. In sifted old mushrooms, old bark, and
humus around tree stumps.
Comments: This species is a member of
the B. lenta group. While the structure of the
internal sac of the aedeagus is rather similar to
that in B. caliginosa, the parameres are distinctive,
and their widened apex as seen in lateral view
is diagnostic. The habitus of this new species
reminds one of species of the B. alesi sp. nov.
group because of the long elytra completely or
almost completely covering the abdomen and the
very fine pronotal and elytral punctation. These
species share reduced metathoracic wings and a
short metaventrite.
Additional specimens from the following
localities are possibly conspecific: 1 ♂ , 2 ♀♀:
Hsinchu Co., Jienshih Township, road no. 60 near
Yulao Scenery platform env. 1400 m, S. Vít, 25
Mar. 2008, # iii 7, road side slope litter (MHNG);
2 ♀♀: Hsinchu Co., Wufeng Township km 19 via
Shei-Pai NP (road 122) env. 1200 m, 26 Mar.
21
22
Figs. 20-22. Baeocera lindae sp. nov. 20, 21. Aedeagus in dorsal and lateral view, scale bar = 0.1 mm. 22. Internal sac in detail, in
dorsal view, scale bar = 0.05 mm.
2008, S. Vít # iii 12, mountain forest litter (MHNG);
2 ♂♂ : Pingtung Co., Pietawushan Trail at 2000 m,
23 May 1991, A. Smetana [T91] (MHNG). They
may be distinguished by the elytra with several
coarse punctures situated near the base. Their
aedeagal and other diagnostic characters are as in
specimens from Tengchih and Alishan.
Baeocera longicornis (Löbl)
Eubaeocera longicornis Löbl 1971: 955.
Material examined : Taiwan: Taipei Co., Beitou
(NW Taipei City) “Yangmingshan cemetery” 300 m,
22 May 2007, S. Vít, # 2, 16 ex. (MHNG); Taipei
Co., Beitou Township (Jiantan Metro Station) 2 Jan.
2009, S. Vít, Jiantan Shan Hiking Trail, #3, 1 ex.
(MHNG); Taoyuan Co., Fusing Township, road no.
118, km 5, 23 Feb. 2010, S. Vít # 7, 1 ex. (MHNG);
Taoyuan Co., Upper Baling 1200 m, 18 Apr. 90, A.
Smetana [T6], 2 ex. (MHNG); Kaohsiung Co., road
no. 20, km 117, Yushan NP, env. 1800 m, 13 Apr.
2009, S. Vít, #9, 4 ex. (MHNG); Chiayi Co., Alishan
Natl. Scenic Area, env. 2350 m, road no. 18, km
102 old Lulin Tree Track, S. Vít, 11 Apr. 2009, # ii 3,
1 ex. (MHNG); Chiayi Co., Alishan road no. 18, km
4.5 S. Lungmei, env. 800 m, 12 Apr. 2009, S. Vít
# ii 6, 10 ex. (MHNG, TARI); Hsinchu Co., Jienshih
Township, road no. 60 nr. Yulao Scenery platform,
env. 1400 m, 25 Mar. 2008, S. Vít, # iii 7, roadside
slope litter, 3 ex. (MHNG); Hsinchu Co., Jienshih
Township, near Hsinkuang Vill., km 44 road no.
60, env. 1600 m, 25 Mar. 2008, S. Vít, # III 6, 1 ex.
(MHNG); Hsinchu Co., Hengshan Township, env.
600 m, S. Hengshan road no. 35, 27 Mar. 2008, S.
Vít, 1 ex. (MHNG).
Habitat: In rotten trunks, forest leaf litter, and
under arborescent fern, in forest sites ranging 3002350 m in elevation.
Distribution: From Sri Lanka, India, and Nepal
to Thailand and continental southern China to
Taiwan. New to Taiwan.
Baeocera mutata Löbl sp. nov.
(Figs. 23-26)
Type material: Holotype ♂ : Taiwan: Pingtung
Co., Peitawushan trail at 1500 m, 1 May 1992, A.
Smetana [T110] (MHNG). Paratype ♂ : with same
data as for holotype (MHNG).
Etymology : The name is a Latin adjective,
derived from “mutatio” and refers to the variation in
characters.
Description: Length 1.75 mm, width 1.08 mm.
127
Most of body, femora, and tibiae dark brown,
somewhat reddish. Apices of elytra, apex of
abdomen, tarsi, and antennomeres I-V lighter,
following antennomeres similar to most of body.
Eyes large. Length ratio of antennomeres as
III 11: IV 11: V 12: VI 10: VII 13: VIII 8: IX 14: X
13: XI 21. Segments III and IV similar, slender,
segments V and VI slightly wider. Segments VII
and VIII distinctly wider than segment VI, segment
VII about 3-times as long as wide, segment VIII
almost 3-times as long as wide. Segment XI
3-times as long as wide. Pronotum and elytra
without microsculpturing, pubescence barely
visible at 100x magnification, and lateral contours
continuously arcuate in dorsal view. Pronotum
with evenly rounded lateral margins, lateral keels
not visible in dorsal view, punctation very fine,
punctures shallow, not clearly delimited. Minute tip
of scutellum exposed. Elytra covering almost entire
abdomen, sutural striae deep, entire, curved along
base and forming basal striae joined to lateral
striae; adsutural areas flat. Elytral punctation fine
and fairly dense, most punctures similar to that
on pronotum, slightly larger punctures irregularly
scattered. Hypomera not impressed, almost level
with mesepisterna, impunctate. Mesepisternum
level with mesepimeron. Mesepimeron about
4-times as long as wide and about 3-times as long
as interval to mesocoxa. Middle of metaventrite
convex, very finely punctate. Lateral parts of
metaventrite sparsely and fairly finely punctate,
intervals between punctures much larger than
puncture diameters, punctures not elongate.
Submesocoxal line arcuate, fairly finely punctate,
submesocoxal area 0.05 mm long, about 1/3
of shortest interval to metacoxa. Metepisterna
somewhat convex, 0.04-0.05 mm wide, parallelsided, with straight, deeply impressed, and
punctate suture. First abdominal ventrite with
fine submetacoxal puncture row, lacking basal
wrinkles; remaining abdominal punctation very fine
and sparse. Tibiae straight.
Male: Protarsomeres 1 moderately widened,
2 and 3 weakly widened. Aedeagus as in figures
23-26, 0.57-0.60 mm. Median lobe symmetrical,
moderately sclerotized, with long, tapering distal
process, abruptly bent at apex. Parameres
converging, slightly curved, and except base,
almost evenly wide in dorsal view, slightly arcuate
in lateral view. Internal sac with sinuate flagellum
gradually widened basally, in basal part joined to
short weakly apically sclerotized rod; center with
scale-like membranes.
Habitat: Old broadleaf evergreen forest. In
128
sifted rotting moldy wood and debris under and
around old soft Polyporus-type mushrooms on an
old fallen tree.
Comments: This species is a member of the
B. brevicornis group and similar to B. sordida Löbl,
1980 from Japan. It can easily be distinguished
from B. sordida by the sparse and fine punctation
on the lateral parts of the metaventrite. In addition,
the metepisterna are flat in B. sordida. The
aedeagi of these 2 species are similar, except for
the distinctive shape of the basal portion of the
internal sac. The only Taiwanese member of the
brevicornis group so far known is B. sauteri Löbl.
It can easily be distinguished from both B. sordita
and B. mutata by the light-rufous body, the notably
coarser punctation on the elytra and metaventrite,
the narrow metepisterna, and the parameres of the
aedeagus, which are almost straight in lateral view.
Baeocera myrmidon (Achard)
Scaphosoma myrmidon Achard 1923: 116.
Habitat: Unknown.
Distribution: Japan (Nagasaki, Kiushu), and
Taiwan: “Pilam” (= Beinan, Taitung Co.).
Comments: The description of the species
was based on material from Nagasaki. Löbl
(1966) designated a lectotype, redescribed and
transferred the species to Baeocera, and provided
illustrations of its aedeagus and antenna. Löbl
(1980) reported this species from Taiwan, and
gave a new redescription with a more-detailed
illustration of its aedeagus.
Baeocera nanula Löbl
Baeocera nanula Löbl 1980: 96.
Material examined: Taiwan: Taichung Co.,
Wufeng 100-120 m, 14 Apr. 1990, A. Smetana [T1],
1 ex. (MHNG); Taichung Co., Wufeng 1000 m 16
Apr. 1990, A. Smetana [T2], 1 ex. (MHNG); Taitung
Co., road no. 20, km 202 after Chulai, 300 m, 8
Apr. 2007, S. Vít, 3 ex. (MHNG, TARI).
Habitat: Plain forest floor litter. In sifted fallen
leaves and flower petals under a broadleaf tree
in a predominantly bamboo forest, and in debris
around a rotting Phallus-type mushroom.
Distribution: Taiwan.
Comments: The species was reported from a
single locality, “Akau” (= Pingtung Co.).
25
23
26
24
Figs. 23-26. Baeocera mutata sp. nov. 23, 25. Aedeagus in dorsal and lateral view, scale bar = 0.1 mm. 24, 26. Internal sac in detail,
scale bar = 0.1 mm.
Baeocera semiglobosa (Achard)
Scaphosoma semiglobosa Achard 1921: 87.
Material examined: Taiwan: Taichung Co.,
Wufeng 100-120 m, 14 Apr. 1990, A. Smetana
[T1], 25 ex. (MHNG, TARI); Taichung Co., Wufeng
1000 m 16 Apr. 1990, A. Smetana [T2], 5 ex.
(MHNG); Hsinchu Co., Henshan Township, env.
600 m, S. Hengshan road no. 35, 27 Mar. 2007,
S. Vít, 1 ex. (MHNG); Taitung Co., road no.
20, km 184 before Wului, 600 m, 10 Apr. 2007, S.
Vít, 3 ex. (MHNG); Taitung Co., road no. 11, W.
Tulan, near First Moonlight Inn, env. 200 m, 12
Apr. 2007, S. Vít, 2 ex. (MHNG); Hualien Co.
road no. 23, km 7.5 lateral valley, env. 400 m, 10
Apr. 2007, S. Vít, 2 ex.; Taitung Co. before Litao,
road no. 20, km 180, env. 1000 m, 8 Apr. 2007, S.
Vít, 7 ex. (MHNG); Taitung Co., road no. 20, km
202 after Chulai, 300 m, 8 Apr. 2007, S. Vít, 7 ex.
(MHNG); Chiayi Co., Alishan Natl. Scenic Area,
env. 2200 m, road no. 18, km 84, 7 Jan. 2009, S.
Vít, # 12, 2 ex. (MHNG); Taipei Co., Beitou (NW
Taipei City) “Yangmingshan cemetery”, 300 m, 22
Oct. 2007, S. Vít, # 2, 4 ex. (MHNG); Taoyuan Co.,
Fusing Township, km 45 road no. 118, 23 Feb.
2010, S. Vít, # 7, 4 ex. (MHNG); Taoyuan Co.,
Fusing Township, N. Baling, km 47, road no. 7,
22 Feb. 2010, S. Vít, #1 (MHNG); Kaohsiung Co.,
“Kosempo” (= Chiasien), 1 ex. (TARI).
Habitat: Plains and mountain forest litter, litter
along rocks, under bushes, in rotten trunks and
under arborescent fern, in fallen leaves and flower
petals under a broadleaf tree in a predominantly
bamboo forest, and in sifted debris around a rotting
Phallus-type mushroom.
Distribution: Taiwan, apparently common,
ranging from lowlands to montane forests, found
up to 2200 m in elevation. It was previously
reported in Löbl (1980) from the following localities:
“Koroton” (= Fengyuan, Taitung Co.) (the type
locality), “Pilam” (= Beinan, Taitung Co.), and
“Kosempo” (= Chiasien, Kaohsiung Co.).
Comments: The relationships of the species
is discussed, and its aedeagus illustrated in Löbl
1980.
Baeocera sauteri Löbl
Baeocera sauteri Löbl 1980: 93.
Habitat: Unknown.
Distribution: Taiwan: “Pilam” (= Beinan,
Taitung Co.).
129
Comments: This species was not found in
the recent collections. It is only known by the type
specimens found in Feb. 1908.
Baeocera takizawai Löbl
Baeocera takizawai Löbl 1984: 190.
Material examined: Taiwan: Taitung Co., road
no. 20, km 184 before Wului, 600 m, 10 Apr. 2007,
S. Vít, 1 ♂ (MHNG).
Habitat: In a decaying trunk with termites.
Distribution: Japan (Ryukyus), China (Jiangxi
Prov.); Taiwan. New to Taiwan.
Comments: The Taiwanese specimen is
larger and as in specimens from Jiangxi, darker
than the types from the Ryukyus. The body length
is 2.0 mm, the body width is 1.35 mm, and the
aedeagus is 0.68 mm long. This species is a
member of the B. monstrosa group, and its allies
have a very complex internal sac of the aedeagus.
The latter exhibits minor variations in the position
and shape of the sclerotized pieces that I consider
to be infraspecific variations.
Key to the Taiwanese species of Baeocera
1
2
3
4
5
-
6
7
Elytral punctation even, entirely very fine, similar to pronotal
punctation ......................................................................... 2
Elytral punctation uneven, partly or entirely coarser than
pronotal punctation ........................................................... 5
Elytron with sutural stria shortened, starting well posterior
to pronotal lobe ................................................................. 3
Elytron with sutural stria not shortened, starting at elytral
base .................................................................................. 4
Elytron lacking basal stria .......................... B. alesi sp. nov.
Elytron with basal stria ............................. B. aliena sp. nov.
Larger species, body 1.85 mm long. Lateral parts of
metaventrite impunctate. Metepisternal suture distinct ......
.............................................................. B. alishana sp. nov.
Smaller species, body 1.25-1.30 mm long. Lateral
parts of metaventrite conspicuously coarsely punctate.
Metepisternal suture indistinct ................. B. lindae sp. nov.
Lateral parts of metaventrite and of 1st abdominal ventrite
with even, very fine punctation, submesocoxal and
submetacoxal puncture rows excepted. Metepisterna
large, with distinct suture .................................................. 6
Lateral parts of metaventrite and of 1st abdominal ventrite
with uneven punctation; both or only those of metaventrite
coarsely punctate. Metepisterna usually narrow and with
indistinct suture ............................................................... 10
Basal striae of elytra not joined to lateral striae. Body 1.01.6 mm long ...................................................................... 7
Basal striae of elytra joined to lateral striae. Body 1.752.40 mm long .................................................................... 8
Elytra with coarse punctures covering most of surface,
basal striae reaching outer 1/4 of basal width. Body
length 1.55-1.60 mm. Body light reddish-brown. Exposed
abdominal terga coarsely punctate, diameters of some
130
-
8
9
-
10
11
12
13
14
-
punctures as large as puncture intervals .............................
................................................................ B. formosana Löbl
Elytral with coarse punctures limited to small, well-delimited
lateral area, basal striae reaching about basal mid-width.
Body length 1.0-1.30 mm. Body very dark brown. Exposed
abdominal terga with very fine punctation, puncture
diameters much smaller than puncture intervals .................
.......................................................... B. myrmidon (Achard)
Antennomere XI about 1.7-times as long as antennomere X;
antennomere V < 3-times as long as wide ..........................
.................................................................. B. takizawai Löbl
Antennomere XI about 1.3-times as long as antennomere X;
antennomere V about 5-times as long as wide ................ 9
Submetacoxale punctures not elongate. Metaventrite with
few coarse admesal punctures. Aedeagus with denticulate
apex of parameres in lateral view ........................................
......................................................... B. anchorifera sp. nov.
Submetaxocal punctures elongate. Metaventrite with
numerous coarse admesal punctures. Aedeagus with
lobed apex of parameres in lateral view ..............................
...................................................................... B. cooteri Löbl
Body length not exceeding 1 mm. Elytron with coarse
punctation limited to small, well-delimited anterolateral
area. Metepisternal suture distinct .............. B. nanula Löbl
Body length exceeding 1 mm. Elytron with coarse
punctation extending over inner surface, often over most of
discal surface. Metepisternal suture often indistinct ...... 11
Basal stria of elytron shortened, reaching about to elytral
mid-width. Base of abdominal ventrite 1 wrinkled ......... 12
Basal stria of elytron not shortened, but extending to and
joined with lateral stria. Base of abdominal ventrite 1
usually not wrinkled ........................................................ 13
Body length about 1.10-1.25 mm. Aedeagus with notched
parameres in middle, internal sac with basal tuft of spinelike structures .............................. B. semiglobosa (Achard)
Body length about 1.25-1.45 mm. Aedeagus with narrow
parameres, not notched. Internal sac without tuft of spinelike structures ......................................... B. caliginosa Löbl
Lateral parts of abdominal ventrite 1 with very finely
punctate posterior submetacoxal puncture row, lateral parts
of metaventrite coarsely punctate ................................... 14
Lateral parts of metaventrite and of abdominal ventrite 1
coarsely punctate ........................................ B. sauteri Löbl
Antennomere VIII long and narrow, slightly shorter than
antennomere VII. Lateral contours of pronotum and elytra
separately arcuate. Smaller species, 1.35-1.60 mm long ...
............................................................. B. longicornis (Löbl)
Antennomere VIII short, about as long as 2/3 of
antennomere VII. Lateral contours of pronotum and elytra
continuously arcuate. Larger species, 1.75 mm long .........
................................................................ B. mutata sp. nov.
DISCUSSION
At present, 15 species of Baeocera are
known from Taiwan. This number appears high,
compared to the 22 species reported from China.
The fact that three of the previously described
species, B. formosana Löbl, B. myrmidon (Achard),
and B. sauteri Löbl, were not found in recent
collections, suggests gaps persisting in sampling
and still-higher diversity. A particular feature of
the Taiwanese Baeocera is the presence of an
endemic species group, consisting of B. alesi
sp. nov., B. aliena sp. nov., and B. alishana sp.
nov., characterized by the elytra almost covering
the tip of the abdomen, impressed hypomera,
enlarged mesepimera, and aedeagi with a
complex, permanently extruded apical portion of
the internal sac. The enlarged mesepimera and
the shape of the internal sac are unique within the
genus. Another notable feature of the Taiwanese
Baeocera is the comparatively high number of
species (four of 15 species) that have reduced
metathoracic wings.
Acknowledgments: Cordial thanks are due to
my friend and colleague Ales Smetana, Ottawa,
Canada and to Mr. S. Vít, Geneva, Switzerland for
the material they collected and generously donated
to the Geneva museum. In addition, A. Smetana
commented on a draft of the present paper. C.F.
Lee, Wufeng, Taiwan provided useful information
and material.
REFERENCES
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Achard J. 1923. Revision des Scaphidiidae de la faune
japonaise. Frag. Entomol., pp. 94-120.
Lee CF, HY Chang, CL Wang, WS Chen. 2011. A review
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Chrysomelidae: Galerucinae: Alticini). Zool. Stud. 50:
525-533.
Leschen RAB, I Löbl. 2005. Phylogeny and classification of
Scaphisomatini Staphylinidae: Scaphidiinae with notes
on mycophagy, termitophily, and functional morphology.
Coleopt. Soc. Mon. 3: 1-63.
Löbl I. 1966. Baeocera myrmidon (Achard, 1923) comb.
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Scaphidiiden. Annot. Zool. Bot. 31: 1-3.
Löbl I. 1971. Scaphidiidae von Ceylon (Coleoptera). Rev.
Suisse Zool. 78: 937-1006.
Löbl I. 1980. Beitrag zur Kenntnis der Scaphidiidae
(Coleoptera) Taiwans. Rev. Suisse Zool. 87: 91-123.
Löbl I. 1984. Contribution à la connaissance des Baeocera
du Japon (Coleoptera, Scaphidiidae). Arch. Sci. 37: 181192.
Löbl I. 1999. A review of the Scaphidiinae (Coleoptera:
Staphylinidae) of the People’s Republic of China, I. Rev.
Suisse Zool. 106: 691-744.
Miwa Y, T Mitono. 1943. Scaphidiidae of my country [=
Scaphidiidae of Japan and Formosa]. Trans. Nat. Hist.
Soc. Formosa 33: 512-555. (in Japanese)
131
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Zoological Record
Vol. 51, No. 1
January, 2012
ORIGINAL PAPERS
ANIMAL BEHAVIOR
Prenatal Ethanol Exposure Increases Depressive-Like Behavior and Central
Estrogen Receptor α and Oxytocin Expressions in Adult Female Mandarin
Voles
F.Q. He, J. Zhang, and X. Guo
1
B. Nesa, A.H. Baird, S. Harii, I.
Yakovleva, and M. Hidaka
12
ANIMAL / PLANT INTERACTIONS
Algal Symbionts Increase DNA Damage in Coral Planulae Exposed to
Sunlight
T. Mieczan
18
ECOLOGY
Distributions of Testate Amoebae and Ciliates in Different Types of Peatlands
and Their Contributions to the Nutrient Supply
I. Chan, L.C. Tseng, S. Kâ, C.F.
Chang, and J.S. Hwang
27
An Experimental Study of the Response of the Gorgonian Coral Subergorgia
suberosa to Polluted Seawater from a Former Coastal Mining Site in Taiwan
E. Prato, A. Danieli, M. Maffia, and
F. Biandolino
38
Lipid Contents and Fatty Acid Compositions of Idotea baltica and Sphaeroma
serratum (Crustacea: Isopoda) as Indicators of Food Sources
S. Capello, M. Marchese, and M.L.
de Wysiecki
51
Feeding Habits and Trophic Niche Overlap of Aquatic Orthoptera Associated
with Macrophytes
N.J. Leander, K.N. Shen, R.T.
Chen, and W.N. Tzeng
59
Species Composition and Seasonal Occurrence of Recruiting Glass Eels
(Anguilla spp.) in the Hsiukuluan River, Eastern Taiwan
M.S. Krishna, H.T. Santhosh, and
S.N. Hegde
72
N.V. Wei, H.J. Hsieh, C.F. Dai, C.C.
Wallace, A.H. Baird, and C.A. Chen
85
M. Pichon, Y.Y. Chuang, and C.A.
Chen
93
T.J. Chu, D. Wang, H.L. Huang, F.J.
Lin, and T.D. Tzeng
99
Population Structure and Historical Demography of the Whiskered Velvet
Shrimp (Metapenaeopsis barbata) off China and Taiwan Inferred from the
Mitochondrial Control Region
A. Iamsuwansuk, J. Denduangboripant,
and P.J.F. Davie
108
Molecular and Morphological Investigations of Shovel-Nosed Lobsters
Thenus spp. (Crustacea: Decapoda: Scyllaridae) in Thailand
I. Löbl
118
On Taiwanese species of Baeocera Erichson (Coleoptera: Staphylinidae:
Scaphidiinae)
EVOLUTION
Offspring of Older Males are Superior in Drosophila bipectinata
Reproductive Isolation among Acropora Species (Scleractinia: Acroporidae)
in a Marginal Coral Assemblage
SYSTEMATICS AND BIOGEOGRAPHY
Pseudosiderastrea formosa sp. nov. (Cnidaria: Anthozoa: Scleractinia) a New
Coral Species Endemic to Taiwan

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