Chromosomal Characteristics of the Three-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacentridae) in Thailand

Abstract

The present study aims to analyze concerned karyotyping and idiograming of the three-spot damselfish (Dascyllus trimaculatus) in Thailand. Chromosomes were prepared from kidney tissues of fish reared at Institute of Marine Science, Burapha University, Chonburi Province. The mitotic chromosomes were harvested by the colchicine-hypotonic-fixation-air drying method. Conventional and Ag-NOR staining techniques were applied to stain the chromosomes. The results showed that D. trimaculatus had 2n=48, and the fundamental number (NF) was 50 without heteromorphic sex chromosomes. The types of chromosomes are 2 large acrocentric, 36 large terocentric and 10 medium telocentric chromosomes. The Ag-NOR banding exhibited that a single pair of NORs was presented on the short arm region of the large acrocentric chromosome. Basic knowledge on cytogenetics of D. trimaculatus would be applied for support of further studies on breeding, conservation and chromosome evolution in this fish. The karyotype formula of D. trimaculatus is as follows:

Full text

© 2017 The Japa n Mendel Societ y Cytologia 82(1) Special Issue: 51–57
Chromosomal Characteristics of the Three-Spot
Damselfish, Dascyllus trimaculatus
(Perciformes, Pomacentridae) in Thailand
Nuntaporn Getlekha1, Weerayuth Supiwong2*, Pun Yeesin3, Puan Pengseng4,
Wannapa Kasiroek5 and Alongklod Tanomtong1
1 Toxic Substances in Livestock and Aquatic Animals Research Group, Department of Biology, Faculty
of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand
2 Faculty of Applied Science and Engineering, Khon Kaen University, Nong Khai Campus,
Muang, Nong Khai 43000, Thailand
3 Department of Technology and Industries, Faculty of Science and Technology, Prince of Songkla
University (Pattani), Muang, Pattani 94000, Thailand
4 School of Agricultural of Technology, Walailak University, Thasala, Nakhon Si Thammarat 80160,
Thailand
5 Institute of Marine Science, Burapha University, Muang, Chonburi 20131, Thailand
Received February 11, 2015; accepted January 5, 2016
Summary The present study aims to analyze concerned karyotyping and idiograming of the three-spot dam-
selfish (Dascyllus trimaculatus) in Thailand. Chromosomes were prepared from kidney tissues of fish reared at
Institute of Marine Science, Burapha University, Chonburi Province. The mitotic chromosomes were harvested
by the colchicine-hypotonic-fixation-air drying method. Conventional and Ag-NOR staining techniques were ap-
plied to stain the chromosomes. The results showed that D. trimaculatus had 2n=48, and the fundamental num-
ber (NF) was 50 without heteromorphic sex chromosomes. The types of chromosomes are 2 large acrocentric, 36
large terocentric and 10 medium telocentric chromosomes. The Ag-NOR banding exhibited that a single pair of
NORs was presented on the short arm region of the large acrocentric chromosome. Basic knowledge on cytoge-
netics of D. trimaculatus would be applied for support of further studies on breeding, conservation and chromo-
some evolution in this fish. The karyotype formula of D. trimaculatus is as follows:
at t
2 36 10
2 48 =L +L +M()n
Key words Dascyllus trimaculatus, Damselfish, NOR, Chromosome, Cytogenetics.
The family Pomacentridae (Perciformes) contains
28 genera and approximately 320 species known as
damselfishes. This family is one of the most diverse
among marine teleosts, which are widely distributed
in tropical seas of the world (Nelson 2006). Species of
the family Pomacentridae are found in coastal waters
associated with rocky substrates, usually occurring at
low depths and often assembling in large fish schools.
The taxonomy of damselfishes is complicated by the
large number of complex species and the color patterns
that vary among individuals and populations of the
same species. Several species are of growing economic
interest because of their diverse color patterns, and this
has led to their exploitation (Molina and Galetti 2004).
There are only two genera in the Chrominae subfamily,
Chromis and Dascyllus. The genus Dascyllus comprises
nine species (Nelson 2006). The three-spot damselfish,
D. trimaculatus, is popular as an aquarium fish in Thai-
land (Fig. 1).
Karyological studies of fish can contribute signifi-
cantly to a better understanding of many problems in
areas of research ranging from taxonomy, systematic or
genetics to phylogenetics, or environmental toxicology
(Al-Sabti 1985). However, the small size and large num-
ber of chromosomes in fish and the lack of a standard
technique for fish chromosome preparation make their
evaluations difficult (Denton 1973, Thorgaard and Dis-
ney 1990). Chromosomal analysis is of interest in fish
breeding from the viewpoint of genetic control, the rapid
production of inbreed lines, taxonomy and evolutionary
studies (Al-Sabti 1987).
Of about 13000 marine fish species that have been
recorded, fewer than 5% have been studied cytoge-
netically (Arai 2011). Most marine fish studied have
a diploid complement of 48 acrocentric chromosomes
(Brum 1996). For the family Pomacentridae, there are
not only color pattern variations among individuals, but
also inter-individual variations of chromosome number
and karyotype complements. In this family, 48 species
* Corresponding author, e-mail: supiwong@hotmail.com
DOI: 10.1508/cytologia.82.51

52 N. Getlekha et al. Cytologia 82(1) Special Issue
have been cytogenetically studied (Hinegardner and
Rosen 1972, Rishi 1973, Arai and Inoue 1976, Arai
et al. 1976, Alvarez et al. 1980, Ojima and Kashiwagi
1981, Ojima 1983, Takai and Ojima 1986, 1987, 1991a,
b, 1995, 1999, Aguilar et al. 1998, Brum et al. 2001,
Molina and Galetti 2002, 2004, Hardie and Hebert 2004,
Kashiwagi et al. 2005, Galetti et al. 2006, Nagpure
et al. 2006, Takai and Kosuga 2007, Tanomtong et al.
2012, Kasiroek et al. 2014). In these results, the diploid
numbers ranged from 27 to 50 chromosomes while most
of them (43 species) had 2n= 48 chromosomes, and the
fundamental numbers ranged from 48 to 96. Karyotypes
involved in Robertsonian rearrangements with diploid
chromosomes less than 2n= 48 and large bi-armed
chromosomes were found in three Dascyllus (Ojima
and Kashiwagi 1981, Kashiwagi et al. 2005). Dascyllus
species showed inter- and intraspecific Robertsonian
polymorphism; the diploid chromosome numbers were
2n=47 and 48 in D. trimaculatus, 2n=34, 35, 36, and 37
in D. reticulatus, 2n=27, 28, 29, 30, 31, 32 and 33 in D.
aruanus, and 2n= 48 in D. melanurus, these fundamen-
tal numbers presenting 48 in all specimens. In another
genus, Chromis, only one species showed intra-specific
Robertsonian polymorphism; the diploid chromosome
numbers were 2n= 46 and 47 in C. insolata, both the
fundamental numbers being 56 (Molina and Galetti
2002).
In the present research, we report the standardized
karyotype and idiogram of D. trimaculatus from Thai
population by conventional and Ag-NOR staining tech-
niques. In the future, basic knowledge on cytogenetics of
D. trimaculatus would be useful for the studies of breed-
ing, conservation and chromosome evolution in this fish.
Materials and methods
Chromosome preparation
Four males and four females of D. trimaculatus were
obtained from the Institute of Marine Science, Burapha
University, Chonburi Province, Thailand. A solution
of 0.01% colchicine (1 mL per 100 g body weight) was
injected into the abdominal cavity and left for one hour.
Chromosomes were prepared from the kidney cells
of the fish by the squash technique (Chen and Ebel-
ing 1968, Nanda et al. 1995). Kidney tissues were cut
into small pieces then mixed with hypotonic solution
(0.075 M KCl). After discarding all large pieces of tis-
sues, 7 mL of cell sediments were transferred to a 15-mL
centrifuge tube and incubated for 45 min. Hypotonic
solution was discarded from the supernatant after cen-
trifugation at 1200 rpm for 8 min. Cells were fixed in a
fresh cool fixative (3 absolute methanol : 1 glacial acetic
acid) to which up to 7 mL were gradually added before
being centrifuged again at 1200 rpm for 8 min, at which
time the supernatant was discarded. The fixation was
repeated until the super natant was clear and the pellet
was mixed with 1 mL fixative. The mixture was dropped
onto a clean and cold slide by a plastic pipette followed
by air-dry technique (Kasiroek et al. 2017).
Chromosome staining
The slide was conventionally stained with 20% stock
Giemsaʼs solution for 30 min. Ag-NOR banding was
conducted by adding four drops of 50% silver nitrate
and 2% gelatin on slides, in order. The slides were then
sealed with cover glasses and incubated at 60C for
5 min. After that, the slides were soaked in distilled wa-
ter until the cover glasses were separated (Howell and
Black 1980, Sangpakdee et al. 2017).
Chromosome checking, karyotyping and idiograming
Standardized karyotypes and idiograms of this fish
were constructed. Chromosome checking was per-
formed on mitotic metaphase cells under a light micro-
scope. The frequencies of chromosome number per cell
were counted. The maximum frequency of chromosome
number per cell is the diploid chromosome number of
this fish. Ten cells of each male and female with clearly
observable and well-spread chromosome were selected
for karyotyping.
The length of short arm chromosome (Ls) and long
arm chromosome (Ll) were measured and calculated to
the length of total arm chromosome (LT, LT=Ls+ Ll).
The relative length (RL), the centromeric index (CI)
and standard deviations (S.D.) of RL and CI were cal-
culated. The CI (q/p+q) between 0.50–0.59, 0.60–0.69,
0.70–0.89 and 0.90–0.99 were representing the metacen-
tric, submetacentric, acrocentric and telocentric chromo-
somes, respectively (Chaiyasut 1989). The fundamental
number (number of chromosome arm, NF) was obtained
by assigning a value of two to metacentric, submetacen-
tric and acrocentric chromosomes and one to telocentric
chromosome. All parameters were used in karyotyping
(Chooseangjaew et al. 2017). The idiogram was con-
structed using a model drawing of karyotype and ac-
complished by a computer program.
Fig. 1. General characteristic of the three-spot damselfish,
Dascyllus trimaculatus.

2017 Chromosomal Characteristics of the Th ree-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacent ridae) in Thailand 53
Results and discussion
Chromosome number, fundamental number and karyo-
type of D. trimaculatus
The diploid chromosome number (2n) of D. tri-
maculatus was 48 chromosomes in all specimens. It
is in agreement with the previous reports from Japan
(Ojima and Kashiwagi 1981, Hardie and Hebert 2004)
but it differs from reports of Arai and Inoue (1976)
and Kashiwagi et al. (2005), which reported diploid
chromosome numbers of 47 and 48 in D. trimaculatus.
The obtained result is the same as most species in the
family Pomacentridae and most marine fishes. The
fundamental number (NF) of D. trimaculatus was 50
in both sexes and the karyotype comprises 2 large acro-
centric, 36 large terocentric and 10 medium telocentric
chromosomes, or two bi-armed and 46 mono-armed
chromosomes (Fig. 2). These results differ from the
studies of Ojima and Kashiwagi (1981) and Hardie and
Hebert (2004) that reported the fundamental number
of D. trimaculatus as 48 with 48 mono-armed chromo-
somes. Moreover, Arai and Inoue (1976) and Kashiwagi
et al. (2005) exhibited the NF and karyotype of D. tri-
maculatus as 48 with one metacentric and 47 acrocentric
chromosomes (mono-armed chromosome). The causes
of these differences are related the intraspecific Robert-
sonian polymorphism which can be found in D. aruanus
and D. reticulates (Ojima and Kashiwagi 1981, Kashi-
wagi et al. 2005). This is similar to the other species of
the family Pomacentridae which have no cytologically
distinguishable sex chromosome for D. trimaculatus.
The cytogenetic data currently available for marine
Promacentrids indicates a high degree of chromosomal
conservation in which a large number of species show
only minor deviations in the chromosomal organiza-
tion and NF (Molina and Galetti 2004). A karyotype
with 2n= 48 is considered the ancestral condition for
the Teleosts and occurs in 43 of the 48 Promacentrid
species analyzed so far. Nevertheless, half of all of the
subfamily Chrominae that have been analyzed cytoge-
netically have 2n= 48 with NF= 48 (Molina and Galetti
2004, Kashiwagi et al. 2005, Arai 2011). A karyotype
with 2n= 48 (NF = 48), considered ancestor in the group,
has been observed in Chromis chromis (Alvarez et al.
1980), C. multilineata (Molina and Galetti 2002), C. ter-
natensis (Takai and Ojima 1999), D. melanurus (Ojima
and Kashiwagi 1981, Kashiwagi et al. 2005), and D.
trimaculatus (Ojima and Kashiwagi 1981, Hardie and
Hebert 2004, Kashiwagi et al. 2005). The other species
in the genus Chromis have NF more than 48 while those
in the genus Dascyllus have NF=48 with the 2n varia-
tion below the basal number (Table 1). The fundamental
number has considerable variation in the 2n values in the
Dascyllus species and variation in the NF values in the
Chromis species. These findings provide support that
pericentric inversions and Robertsonian translocation
or centric fusion play an important role in karyotypic
diversification of Chromis and Dascyllus, respectively.
Chromosome markers of D. trimaculatus
The present study was accomplished by using the
Ag-NOR staining technique. The objective of this tech-
nique is to determine NORs, which represent the loca-
tion of genes (loci) that function in ribosome synthesis
Fig. 2. Metaphase chromosome plates and ka ryoty pes of male (A) and female (B) of three-spot damselfish (Dascyllus
trimaculatus, 2n=48) by conventional staining technique (scale bars indicate 10 µm).

54 N. Getlekha et al. Cytologia 82(1) Special Issue
Fig. 3. Metaphase chromosome plates of male (A) and female ( B) of three-spot d amselfish (Dascyllus trimaculatus, 2n=48) by
Ag-NOR banding technique; scale bars indicate 10 µm. The short ar m of acrocentric chromosome pai r 1 showed clearly
observable nucleolar organizer regions (NORs) (arrows).
Table 1. Cytogenet ic reviews of the subfamily Chominae (Pomacentridae).
Species 2nNF NORs Formula Locality Reference
Chromis chromis 48 48 48a Spain Alvarez et al. (1980)
C. chrysura 48 50 2m+ 46st/a Japan Ojima (1983)
C. flavicauda 39 54 2 9m+ 6sm +24a Brazil Molina and Galetti (2002)
C. insolata 47 56 2 3m+ 6sm +38a Brazil Molina and Galetti (2002)
46 56 2 4m+ 6sm +36a Brazil Molina and Galetti (2002)
C. multilineata 48 48 2 48a Brazil Molina and Galetti (2002)
C. ternatensis 48 48 48a Pacific Takai and Ojima (1999)
Dascyllus aruanus 33 48 15 m/sm+18a Japan Ojima and Kashiwagi (1981)
32 48 16 m/sm+16a Japan Ojima and Kashiwagi (1981)
32 48 2 16 m/sm+16a Japan Kashiwagi et al. (2005)
31 48 17 m/sm+14a Japan Ojima and Kashiwagi (1981)
Kashiwagi et al. (2005)
31 48 17 m/sm+14a Japan
30 48 18 m/sm+12a Japan Ojima and Kashiwagi (1981)
Kashiwagi et al. (2005)
30 48 18 m/sm+12a Japan
29 48 19 m/sm+10a Japan Ojima and Kashiwagi (1981)
Kashiwagi et al. (2005)
29 48 19 m/sm+10a Japan
28 48 20 m/sm+8a Japan Ojima and Kashiwagi (1981)
Kashiwagi et al. (2005)
28 48 2 20 m/sm+8a Japan
27 48 21 m/sm+6a Japan Ojima and Kashiwagi (1981)
D. melanurus 48 48 48a Japan Ojima and Kashiwagi (1981)
Kashiwagi et al. (2005)
48 48 2 48a Japan
D. reticulatus 36 48 2 12 m /sm+ 24a Japan Hardie and Hebert (2004)
Kashiwagi et al. (2005)
36 48 2 12 m/sm+24a Japan
35 48 13 m/sm+22a Japan Ojima and Kashiwagi (1981)
Kashiwagi et al. (2005)
35 48 13 m/sm+22a Japan
34 48 14 m/sm+20a Japan Ojima and Kashiwagi (1981)
D. trimaculatus 48 48 48a Japan Ojima and Kashiwagi (1981)
48 48 48a Japan Hardie and Heber t (2004)
48 48 2 48a Japan Kashiwagi et al. (2005)
47 48 1m+ 46a Japan Arai and Inoue (1976)
47 48 1m+ 46a Japan Kashiwagi et al. (2005)
48 50 2 2a+ 46t Gulf of Thailand Present study
Remarks: 2n= diploid chromosome number, NF= fundamental number (number of chromosome ar m), m= metacentric, sm=submetacentric,
st =subtelocent ric, a= acrocentric, t=telocentric chromosome, NORs= nucleolar organizer regions and =not available.

2017 Chromosomal Characteristics of the Th ree-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacent ridae) in Thailand 55
(18S and 28S ribosomal RNA), and a positive NOR is
detected when these genes are functioning (Sharma
et al. 2002). From the result, the short arm of acrocentric
chromosome pair 1 showed clearly observable NORs
in both sexes without size heteromorphism (Fig. 3). It
is the same as previous studies in the subfamily Chro-
minae with two NOR bearing chromosomes (Molina
and Galetti 2002, Kashiwagi et al. 2005). Kashiwagi
et al. (2005) suggested that NOR-bearing chromosomes
showed size variation among the species, but no intra-
specific variation, and these chromosomes have been
differentiated involving heterochromatin amplifications
or translocation of NORs in the genus Dascyllus. How-
ever, some representatives of the subfamilies Poma-
centrinae and Chrominae show marked heteromorphic
NORs (two NORs from non-homologous chromosomes),
such as Microspathodon chrysurus, Chrysiptera rol-
landi (Kasiroek et al. 2014), Chromis insolata and C.
flavicauda (Molina and Galetti 2002). This pattern sug-
gests simple translocation in the karyotypic evolution
of this group. Despite the importance of heteromorphic
NORs, this does not appear to be the main evolutionary
tendency in the karyotype of this subfamily, but may be
characteristic of species groups (Kasiroek et al. 2014).
The chromosome of mitotic metaphase cells and the
karyotypes of D. trimaculatus by conventional staining
and Ag-NOR staining techniques are shown in Figs. 2
and 3. The length of chromosomes of 20 cells (males
and females) in the mitotic metaphase was measured.
The mean length of short arm chromosome (Ls), length
of long arm chromosome (Ll), total length of arm chro-
mosome (LT), relative length (RL), centromeric index
(CI), standard deviation of RL, CI, size and type of
chromosomes are shown in Table 2. The idiogram of
D. trimaculatus shows a gradually decreasing length of
Table 2. Mean length of short arm chromosome (Ls), length long arm chromosome (Ll), length total ar m chromosome (LT), relative length (RL),
centromeric index (CI) and stand ard deviation (SD) of RL, CI from 20 metaphase cells of the male a nd female three -spot damselfish
(Dascyllus trimaculatus), 2n= 48.
Chromosome pair Ls Ll LT RLSD CI SD Chromosome size Chromosome type
1* 1.61 4.03 5.63 0.0470.004 0.7130.060 Large Acrocentric
2 0.00 5.92 5.92 0.0480.002 1.000.000 Large Telocentric
3 0.00 5.89 5.89 0.048 0.003 1.000.000 Large Telocentric
4 0.00 5.77 5.77 0.0470.002 1.00 0.000 Large Telocentric
5 0.00 5.64 5.64 0.0460.003 1.000.000 Large Telocentric
6 0.00 5.57 5.57 0.046 0.002 1.000.000 Large Telocentric
7 0.00 5.51 5.51 0.045 0.002 1.000.000 Large Telocentric
8 0.00 5.46 5.46 0.045 0.002 1.000.000 Large Telocentric
9 0.00 5.34 5.34 0.044 0.003 1.000.000 Large Telocentric
10 0.00 5.31 5.31 0.043 0.002 1.000.000 Large Telocentric
11 0.00 5.25 5.25 0.043 0.003 1.000.000 Large Telocentric
12 0.00 5.21 5.21 0.043 0.002 1.000.000 Large Telocentric
13 0.00 5.19 5.19 0.0430.002 1.00 0.000 Large Telocentric
14 0.00 5.11 5.11 0.0430.002 1.00 0.000 Large Telocentric
15 0.00 5.04 5.04 0.0410.002 1.000.000 Large Telocentric
16 0.00 5.03 5.03 0.0410.002 1.000.000 Large Telocentric
17 0.00 4.98 4.98 0.0410.002 1.000.000 Large Telocentric
18 0.00 4.87 4.87 0.040 0.001 1.000.000 Large Telocentric
19 0.00 4.71 4.71 0.0390.002 1.00 0.000 Large Telocentric
20 0.00 4.60 4.60 0.0380.002 1.000.000 Medium Telocentric
21 0.00 4.43 4.43 0.036 0.001 1.000.000 Medium Telocentric
22 0.00 4.30 4.30 0.035 0.002 1.000.000 Medium Telocentric
23 0.00 3.97 3.97 0.0330.002 1.000.000 Medium Telocentric
24 0.00 3.40 3.40 0.028 0.002 1.000.000 Medium Telocentric
Remark: * NOR-bearing ch romosome.
Fig. 4. Standardized id iogram showing lengt hs and shapes of
chromosomes of the th ree-spot damselfish (Dascyllus
trimaculatus, 2n=48) by conventional staining technique.
The arrow indicates nucleolar organizer regions.

56 N. Getlekha et al. Cytologia 82(1) Special Issue
the chromosomes (Figs. 4 and 5). For D. trimaculatus,
the chromosome markers are chromosome pairs 1 and
24, which are the large acrocentric chromosome and the
smallest telocentric chromosome, respectively. The im-
portant karyotype feature is the symmetrical karyotype,
which was found in almost telocentric chromosomes.
The karyotype formula of D. trimaculatus could be de-
duced as:
at t
2 36 10
2 48 =L +L +M()n
Chromosome evolution of the Chrominae subfamily
Karyotypic studies suggest that the karyotype with
48 acrocentric chromosomes (48 mono-armed) was the
ancestral karyotype in the Pomacentridae, and other
karyotypes have been differentiated mainly by the pres-
ence of pericentric inversions and/or Robertsonian re-
arrangements (Takai and Ojima 1987, 1991a, b, 1995,
Kashiwagi et al. 2005). Previous studies on cytogenet-
ics of the Chrominae subfamily and the present study
demonstrated that there are four patterns of its chromo-
some evolution. Firstly, the conserved 2n and kar yotype
were shown as ancestor. This was evident in studies of
C. chromis (2n= 48, NF = 48), C. multilineata (2n=48,
NF=48), C. ternatensis (2n=48, NF= 48) and D. mel-
anurus (2n= 48, NF= 48) (Alvarez et al. 1980, Ojima
and Kashiwagi 1981, Takai and Ojima 1999, Molina and
Galetti 2002, Kashiwagi et al. 2005). Secondly, the oc-
currence of pericentric inversion at telocentric chromo-
somes provides the bi-arms chromosomes (metacentric,
submetacentric and acrocentric chromosome), which
does not affect the chromosome number but provides an
increment of fundamental numbers. This was evident in
studies of C. chrysura (2n=48, NF=50) (Ojima 1983).
Thirdly, the occurrence of the combination of pericentric
inversions and centric fusions results in decrement of 2n
and increment of NF values. This was evident in stud-
ies of C. flavicauda (2n=39, NF =54) and C. insolata
(2n= 46–47, NF=56) (Molina and Galetti 2002). Fourth-
ly, it is apparent from the chromosomal features that the
karyotypes of Dascyllus species have been differentiated
from the ancestral 48A karyotype involving only Rob-
ertsnian rearrangements. From the data of the distribu-
tional patterns of centromeric C-bands and NOR-bearing
chromosomes, Kashiwagi et al. (2005) suggested that
among the four Dascyllus species, the 48A karyotype
of D. melanurus looked like the ancestral form in con-
ventional staining, but it has been most differentiated in
constitutive heterochromatin distribution.
Up to the present, there are four of nine species of
the genus Dascyllus cytogenetically analyzed. Dascyl-
lus species provides remarkable karyotype features for
chromosome evolutionary discussion. Further studies
of other species as well as additional information and
molecular techniques for chromosome analyses are ex-
pected to clarify and explain the reasons to support the
karyotype polymorphism and chromosome evolution in
these fishes.
Acknowledgements
This work was suppor ted by the Development and
Promotion of Science and Technology talents project
(DPST) and the Toxic Substances in Livestock and
Aquatic Animals Research Group, Khon Kaen Univer-
sity. We would like to thank the cytogenetic research
group for the accuracy check of the report and valuable
help.
References
Aguilar, C. T., Oliveira-Corrêa, M. M. and Galetti, P. M. Jr. 1998.
Chromosome associations by cent romeric heterochromatin i n
marine fishes. Ch romosome Sci. 2: 73–76.
Alvarez, M. C., Cano, J. and Thode, G. 1980. DNA content and chro-
mosome complement of Chromis chromis (Pomacentridae, Perci-
formes). Caryologia 33: 267–274.
Arai, R. 2011. Fish Karyotypes: A Check List. Springer Japan, Tokyo.
Arai, R. and Inoue, M. 1976. Chromosomes of seven species of Poma-
centridae and t wo species of Acanthuridae from Japan. Bull.
Natl. Mus. Nat. Sci. Ser. A Zool. 2: 73–78.
Arai, R., Inoue, M. and Id a, H. 1976. Chromosome of four species of
coral fishes from Japan. Bull. Natl. Mus. Nat. Sci. Ser. A Zool. 2:
137–141.
Al-Sabti, K. 1985. Chromosomal studies by blood leu kocyte culture
technique on three Salmonids from Yugoslavia n water. J. Fish
Biol. 26: 5–12.
Al-Sabti, K. 1987. Cytogenetic st udies on five species of Pisces from
Yugoslavia. Cytobios 49: 175–188.
Brum, M. J. I. 1996. Cytogenetic studies of Brazilian marine fish.
Fig. 5. Standardized id iogram of chromosomes of the three -spot
damselfish (Dascyllus trimaculatus, 2n=48) by Ag-NOR
banding technique. The ar row indicates nucleolar organizer
regions on the short arm of acrocentric ch romosome pair 1.

2017 Chromosomal Characteristics of the Th ree-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacent ridae) in Thailand 57
Rev. Bras. Genet. 19: 421– 427.
Brum, M. J. I., Corrêa, M. M. O. and Aguilar, C. T. 2001. Kar yotype
analysis in South Atlantic percifor ms Micropogonias furnieri
and Abudefduf saxatilis. Chromosome. Sci. 5: 83–89.
Chaiyasut, K. 1989. Cytogenetics and Cytotaxonomy of the Family
Zephyranthes. Depart ment of Botany, Faculty of Science, Chul-
alongkorn University, Bangkok.
Chen, T. R. and Ebeling, A. W. 1968. Karyological evidence of female
heterogamety in the mosquito fish, Gambusia affinis. Copeia 1:
70–75.
Chooseangjaew, S., Tanyaros, S., Maneechot, N., Buasriyot, P., Getle-
kha, N. and Tanomtong, A. 2017. Chromosomal cha racteristics
of the tropical oyster, Crassostrea belcheri Sowerby, 1871 (Os-
treoida, Ostreidae) by conventional and Ag-NOR banding tech-
niques. Cytologia 82: 3–8.
Denton, E. T. 1973. Fish Chromosome Methodology. Charles Thomas
Publisher, Springfield. p. 166.
Galetti, P. M. Jr., Molina, W. F., Affonso, P. R. A. M. and Aguilar, C.
T. 2006. Assessing genetic diversit y of Brazilian reef fishes by
chromosomal and DNA markers. Genet ica 126: 161–177.
Hardie, D. C. and Hebert, P. D. N. 2004. Genome-size evolution in
fishes. Can. J. Fish. Aquat. Sci. 61: 1636–1646.
Hinegardner, R. and Rosen, D. E. 1972. Cellular DNA content and the
evolution of teleostean fishes. A m. Nat. 106: 621–644.
Howell, W. M. and Black, D. A. 1980. Controlled silver-stai ning of
nucleolus organizer regions with a protective colloidal developer:
A 1-step met hod. Experientia 36: 1014–1015.
Kashiwagi, E., Takai, A. and Ojima , Y. 2005. Chromosomal distribu-
tion of constitutive heterochromatin and nucleolus organ izer
regions in four Dascyllus fishes (Pomacentridae, Perciformes).
Cytologia 70: 345–349.
Kasiroek, W., Luangoon, N., Getlek ha, N., Saowakoon, S., Phin-
rub, W. and Tanomtong, A. 2014. First report on heteromor-
phic NORs and chromosome analysis of Rolland ʼs demoiselle,
Chrysiptera rollandi (Perciformes, Pomacentrinae) by conven-
tional and Ag-NOR staining techniques. Cy tologia 79: 1–10.
Kasiroek, W., Indanand a, C., Luangoon, N., Pinthong, K., Supiwong,
W. and Tanomtong, A. 2017. First chromosome analysis of the
humpback cardinalfish, Fibramia lateralis (Perciformes, Apogo-
nidae). Cytologia 82: 9–15.
Molina, W. F. and Galetti, P. M. Jr. 2002. Robertsonian rearrange-
ments i n the reef fish Chromis (Perciformes, Pomacent ridae)
involving chromosomes bearing 5S rRNA genes. Genet. Mol.
Biol. 25: 373–377.
Molina, W. F. and Galetti, P. M. Jr. 2004. Karyotypic changes associ-
ated to the dispersive potential on Pomacentridae (Pisces, Perci-
formes). J. Exp. Mar. Biol. Ecol. 309: 109–119.
Nagpure, N. S., Kumar, R., Srivastava, S. K., Kushwaha, B., Gopal-
akrishnan, A. and Basheer, V. S. 2006. Cytogenetic character-
ization of two marine ornamental fishes, Haetodon collare and
Stegastes insularis. J. Mar. Biol. Assoc. India 48: 267–269.
Nanda, I., Schsrtl, M., Fiechtinger, W., Schlupp, I., Parzefall, J. and
Schmid, M. 1995. Chromosomal evidence for laboratory syn-
thesis of tr iploid hybrid between the g ynogenetic teleost Poecilia
Formosa and its host species. J. Fish Biol. 47: 619–623.
Nelson, J. S. 2006. Fishes of the World. 4th edition. John Wiley and
Sons, Inc., New York.
Ojima, Y. 1983. Fish cy togenetics. In: Shar ma, A. K. and Sharma , A.
(eds.). Chromosomes in Evolution of Eukar yotic Groups. Vol. I.
CRC Press, Boca Raton. pp. 111–145.
Ojima, Y. and Kashiwagi, E. 1981. Chromosomal evolution associated
with Rober tsonian fusion i n the genus Dascyllus (Chrom in ae ,
Pisces). Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 57: 368–370.
Rishi, K. K. 1973. A preliminar y repor t on the karyoty pes of eighteen
marine fishes. Res. Bu ll. Panjab Univ., (N.S.) 24: 161–162.
Sangpa kdee, W., Phimphan, S., Tengjaroenkul, B., Pinthong, K.,
Neeratanaphan, L. and Tanomtong, A. 2017. Cytogenetic study
of tree microhylid species (Anura, Microhylidae) from Thailand.
Cytologia 82: 67–74.
Sharma, O. P., Tripathi, N. K. and Shar ma, K. K. 2002. A review of
chromosome banding in fishes. In: Sobti, R. C., Obe, G. and
Athwal, R. S. (eds.). Some Aspects of Chromosome Str ucture
and Funct ions. New Narosa Publishing House, New Del hi. pp.
109–122.
Takai, A. and Ojima, Y. 1986. Some features on nucleolus organizer
regions in fish chromosomes. In: Uyeno, T., Arai, R., Taniuchi, T.
and Matsuura, K. (eds.). Indo -Pacific Fish Biology. Ichthyologi-
cal Society of Japan, Tokyo. pp. 899–909.
Takai, A. and Ojima, Y. 1987. Comparative stud ies of karyotypes
and dist ributions of nucleolus organizer reg ions in pomacentr id
fishes. I. Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. 63: 17–20.
Takai, A. and Ojima, Y. 1991a. Comparative studies of karyotypes
and dist ribution of nucleolus organizer regions in pomacentrid
fish. 2. Cytobios 65: 199–205.
Takai, A. and Ojima, Y. 1991b. Karyoty pes and nucleolar organizer
regions in two pomacentrid fishes. Chromosome Inform. Serv.
50: 28–30.
Takai, A. a nd Ojima , Y. 1995. Chromosome evolution associated with
Robertsonian rearrangements in pomacent rid fish (Percifor mes).
Cytobios 84: 103–110.
Takai, A. and Ojima , Y. 1999. Constit utive heteroch romatin distribu-
tion in the chromosomes of pomacentrid fishes (Perciformes).
Cytologia 64: 87–91.
Takai, A. and Kosuga, S. 2007. Kar yotypes and banded chromosomal
features in t wo anemonefishes (Pomacentridae, Perciformes).
Chromosome Sci. 10: 71–74.
Tanomtong, A., Supiwong, W., Chaveerach, A., Khak hong, S., Ta-
nee, T. and Sanoamu ang, L. 2012. First report of chromosome
analysis of saddleback anemonefish, Amphiprion polymnus (Pe r-
ciformes, Amphiprioninae), in Thailand. Cytologia 77: 441–446.
Thorgaard, G. H. and Disney, J. E. 1990. Methods for Fish Biology.
American Fisheries Society, Bethesda. pp. 171–187.