Dascyllus Trimaculatus (Perciformes, Pomacentridae) in Thailand

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 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: at t 2n () 48 =L2 +L 36 +M 10

Key words Dascyllus trimaculatus, Damselﬁsh, NOR, Chromosome, Cytogenetics.

The family Pomacentridae (Perciformes) contains land (Fig. 1). 28 genera and approximately 320 species known as Karyological studies of fish can contribute signifi- damselfishes. This family is one of the most diverse cantly to a better understanding of many problems in among marine teleosts, which are widely distributed areas of research ranging from taxonomy, systematic or in tropical seas of the world (Nelson 2006). Species of genetics to phylogenetics, or environmental toxicology the family Pomacentridae are found in coastal waters (Al-Sabti 1985). However, the small size and large num- associated with rocky substrates, usually occurring at ber of chromosomes in fish and the lack of a standard low depths and often assembling in large fish schools. technique for fish chromosome preparation make their The taxonomy of damselfishes is complicated by the evaluations difficult (Denton 1973, Thorgaard and Dis- large number of complex species and the color patterns ney 1990). Chromosomal analysis is of interest in fish that vary among individuals and populations of the breeding from the viewpoint of genetic control, the rapid same species. Several species are of growing economic production of inbreed lines, taxonomy and evolutionary interest because of their diverse color patterns, and this studies (Al-Sabti 1987). has led to their exploitation (Molina and Galetti 2004). Of about 13000 marine fish species that have been There are only two genera in the Chrominae subfamily, recorded, fewer than 5% have been studied cytoge- Chromis and Dascyllus. The genus Dascyllus comprises netically (Arai 2011). Most marine fish studied have nine species (Nelson 2006). The three-spot damselfish, a diploid complement of 48 acrocentric chromosomes D. trimaculatus, is popular as an aquarium fish in Thai- (Brum 1996). For the family Pomacentridae, there are not only color pattern variations among individuals, but * Corresponding author, e-mail: [email protected] also inter-individual variations of chromosome number DOI: 10.1508/cytologia.82.51 and karyotype complements. In this family, 48 species 52 N. Getlekha et al. Cytologia 82(1) Special Issue

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 tissues, 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 Fig. 1. General characteristic of the three-spot damselfish, being centrifuged again at 1200 rpm for 8 min, at which Dascyllus trimaculatus. time the supernatant was discarded. The fixation was repeated until the supernatant was clear and the pellet have been cytogenetically studied (Hinegardner and was mixed with 1 mL fixative. The mixture was dropped Rosen 1972, Rishi 1973, Arai and Inoue 1976, Arai onto a clean and cold slide by a plastic pipette followed et al. 1976, Alvarez et al. 1980, Ojima and Kashiwagi by air-dry technique (Kasiroek et al. 2017). 1981, Ojima 1983, Takai and Ojima 1986, 1987, 1991a, b, 1995, 1999, Aguilar et al. 1998, Brum et al. 2001, Chromosome staining Molina and Galetti 2002, 2004, Hardie and Hebert 2004, The slide was conventionally stained with 20% stock Kashiwagi et al. 2005, Galetti et al. 2006, Nagpure Giemsa’s solution for 30 min. Ag-NOR banding was et al. 2006, Takai and Kosuga 2007, Tanomtong et al. conducted by adding four drops of 50% silver nitrate 2012, Kasiroek et al. 2014). In these results, the diploid and 2% gelatin on slides, in order. The slides were then numbers ranged from 27 to 50 chromosomes while most sealed with cover glasses and incubated at 60°C for of them (43 species) had 2n=48 chromosomes, and the 5 min. After that, the slides were soaked in distilled wa- fundamental numbers ranged from 48 to 96. Karyotypes ter until the cover glasses were separated (Howell and involved in Robertsonian rearrangements with diploid Black 1980, Sangpakdee et al. 2017). chromosomes less than 2n=48 and large bi-armed chromosomes were found in three Dascyllus (Ojima Chromosome checking, karyotyping and idiograming and Kashiwagi 1981, Kashiwagi et al. 2005). Dascyllus Standardized karyotypes and idiograms of this fish species showed inter- and intraspecific Robertsonian were constructed. Chromosome checking was per- polymorphism; the diploid chromosome numbers were formed on mitotic metaphase cells under a light micro- 2n=47 and 48 in D. trimaculatus, 2n=34, 35, 36, and 37 scope. The frequencies of chromosome number per cell in D. reticulatus, 2n=27, 28, 29, 30, 31, 32 and 33 in D. were counted. The maximum frequency of chromosome aruanus, and 2n=48 in D. melanurus, these fundamen- number per cell is the diploid chromosome number of tal numbers presenting 48 in all specimens. In another this fish. Ten cells of each male and female with clearly genus, Chromis, only one species showed intra-specific observable and well-spread chromosome were selected Robertsonian polymorphism; the diploid chromosome for karyotyping. numbers were 2n=46 and 47 in C. insolata, both the The length of short arm chromosome (Ls) and long fundamental numbers being 56 (Molina and Galetti arm chromosome (Ll) were measured and calculated to 2002). the length of total arm chromosome (LT, LT=Ls+Ll). In the present research, we report the standardized The relative length (RL), the centromeric index (CI) karyotype and idiogram of D. trimaculatus from Thai and standard deviations (S.D.) of RL and CI were cal- population by conventional and Ag-NOR staining tech- culated. The CI (q/p+q) between 0.50–0.59, 0.60–0.69, niques. In the future, basic knowledge on cytogenetics of 0.70–0.89 and 0.90–0.99 were representing the metacen- D. trimaculatus would be useful for the studies of breed- tric, submetacentric, acrocentric and telocentric chromo- ing, conservation and chromosome evolution in this fish. somes, respectively (Chaiyasut 1989). The fundamental number (number of chromosome arm, NF) was obtained Materials and methods by assigning a value of two to metacentric, submetacentric and acrocentric chromosomes and one to telocentric Chromosome preparation chromosome. All parameters were used in karyotyping Four males and four females of D. trimaculatus were (Chooseangjaew et al. 2017). The idiogram was con- obtained from the Institute of Marine Science, Burapha structed using a model drawing of karyotype and ac- University, Chonburi Province, Thailand. A solution complished by a computer program. 2017 Chromosomal Characteristics of the Three-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacentridae) in Thailand 53

Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) of three-spot damselﬁsh (Dascyllus trimaculatus, 2n=48) by conventional staining technique (scale bars indicate 10 µm).

Results and discussion Promacentrids indicates a high degree of chromosomal conservation in which a large number of species show Chromosome number, fundamental number and karyo- only minor deviations in the chromosomal organiza- type of D. trimaculatus tion and NF (Molina and Galetti 2004). A karyotype The diploid chromosome number (2n) of D. tri- with 2n=48 is considered the ancestral condition for maculatus was 48 chromosomes in all specimens. It the Teleosts and occurs in 43 of the 48 Promacentrid is in agreement with the previous reports from Japan species analyzed so far. Nevertheless, half of all of the (Ojima and Kashiwagi 1981, Hardie and Hebert 2004) subfamily Chrominae that have been analyzed cytoge- but it differs from reports of Arai and Inoue (1976) netically have 2n=48 with NF=48 (Molina and Galetti and Kashiwagi et al. (2005), which reported diploid 2004, Kashiwagi et al. 2005, Arai 2011). A karyotype chromosome numbers of 47 and 48 in D. trimaculatus. with 2n=48 (NF=48), considered ancestor in the group, The obtained result is the same as most species in the has been observed in Chromis chromis (Alvarez et al. family Pomacentridae and most marine fishes. The 1980), C. multilineata (Molina and Galetti 2002), C. ter- fundamental number (NF) of D. trimaculatus was 50 natensis (Takai and Ojima 1999), D. melanurus (Ojima in both sexes and the karyotype comprises 2 large acro- and Kashiwagi 1981, Kashiwagi et al. 2005), and D. centric, 36 large terocentric and 10 medium telocentric trimaculatus (Ojima and Kashiwagi 1981, Hardie and chromosomes, or two bi-armed and 46 mono-armed Hebert 2004, Kashiwagi et al. 2005). The other species chromosomes (Fig. 2). These results differ from the in the genus Chromis have NF more than 48 while those studies of Ojima and Kashiwagi (1981) and Hardie and in the genus Dascyllus have NF=48 with the 2n varia- Hebert (2004) that reported the fundamental number tion below the basal number (Table 1). The fundamental of D. trimaculatus as 48 with 48 mono-armed chromo- number has considerable variation in the 2n values in the somes. Moreover, Arai and Inoue (1976) and Kashiwagi Dascyllus species and variation in the NF values in the et al. (2005) exhibited the NF and karyotype of D. tri- Chromis species. These findings provide support that maculatus as 48 with one metacentric and 47 acrocentric pericentric inversions and Robertsonian translocation chromosomes (mono-armed chromosome). The causes or centric fusion play an important role in karyotypic of these differences are related the intraspecific Robert- diversification of Chromis and Dascyllus, respectively. sonian polymorphism which can be found in D. aruanus and D. reticulates (Ojima and Kashiwagi 1981, Kashi- Chromosome markers of D. trimaculatus wagi et al. 2005). This is similar to the other species of The present study was accomplished by using the the family Pomacentridae which have no cytologically Ag-NOR staining technique. The objective of this tech- distinguishable sex chromosome for D. trimaculatus. nique is to determine NORs, which represent the loca- The cytogenetic data currently available for marine tion of genes (loci) that function in ribosome synthesis 54 N. Getlekha et al. Cytologia 82(1) Special Issue

Table 1. Cytogenetic reviews of the subfamily Chominae (Pomacentridae).

Species 2n NF 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) 31 48 ̶ 17 m/sm+14a Japan Kashiwagi et al. (2005) 30 48 ̶ 18 m/sm+12a Japan Ojima and Kashiwagi (1981) 30 48 ̶ 18 m/sm+12a Japan Kashiwagi et al. (2005) 29 48 ̶ 19 m/sm+10a Japan Ojima and Kashiwagi (1981) 29 48 ̶ 19 m/sm+10a Japan Kashiwagi et al. (2005) 28 48 ̶ 20 m/sm+8a Japan Ojima and Kashiwagi (1981) 28 48 2 20 m/sm+8a Japan Kashiwagi et al. (2005) 27 48 ̶ 21 m/sm+6a Japan Ojima and Kashiwagi (1981) D. melanurus 48 48 ̶ 48a Japan Ojima and Kashiwagi (1981) 48 48 2 48a Japan Kashiwagi et al. (2005) D. reticulatus 36 48 2 12 m/sm+24a Japan Hardie and Hebert (2004) 36 48 2 12 m/sm+24a Japan Kashiwagi et al. (2005) 35 48 ̶ 13 m/sm+22a Japan Ojima and Kashiwagi (1981) 35 48 ̶ 13 m/sm+22a Japan Kashiwagi et al. (2005) 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 Hebert (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: 2 n=diploid chromosome number, NF=fundamental number (number of chromosome arm), m=metacentric, sm=submetacentric, st=subtelocentric, a=acrocentric, t=telocentric chromosome, NORs=nucleolar organizer regions and ̶=not available.

Fig. 3. Metaphase chromosome plates of male (A) and female (B) of three-spot damselﬁsh (Dascyllus trimaculatus, 2n=48) by Ag-NOR banding technique; scale bars indicate 10 µm. The short arm of acrocentric chromosome pair 1 showed clearly observable nucleolar organizer regions (NORs) (arrows). 2017 Chromosomal Characteristics of the Three-Spot Damselﬁsh, Dascyllus trimaculatus (Perciformes, Pomacentridae) in Thailand 55

Table 2. Mean length of short arm chromosome (Ls), length long arm chromosome (Ll), length total arm chromosome (LT), relative length (RL), centromeric index (CI) and standard deviation (SD) of RL, CI from 20 metaphase cells of the male and 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 chromosome.

(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 intraspecific 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 rollandi (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 Fig. 4. Standardized idiogram showing lengths and shapes of NORs, this does not appear to be the main evolutionary chromosomes of the three-spot damselfish (Dascyllus tendency in the karyotype of this subfamily, but may be trimaculatus, 2n=48) by conventional staining technique. characteristic of species groups (Kasiroek et al. 2014). The arrow indicates nucleolar organizer regions. The chromosome of mitotic metaphase cells and the karyotypes of D. trimaculatus by conventional staining of long arm chromosome (Ll), total length of arm chro- and Ag-NOR staining techniques are shown in Figs. 2 mosome (LT), relative length (RL), centromeric index and 3. The length of chromosomes of 20 cells (males (CI), standard deviation of RL, CI, size and type of and females) in the mitotic metaphase was measured. chromosomes are shown in Table 2. The idiogram of The mean length of short arm chromosome (Ls), length D. trimaculatus shows a gradually decreasing length of 56 N. Getlekha et al. Cytologia 82(1) Special Issue

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 studies 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 conventional 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- Fig. 5. Standardized idiogram of chromosomes of the three-spot lus species provides remarkable karyotype features for damselfish (Dascyllus trimaculatus, 2n=48) by Ag-NOR chromosome evolutionary discussion. Further studies banding technique. The arrow indicates nucleolar organizer of other species as well as additional information and regions on the short arm of acrocentric chromosome pair 1. molecular techniques for chromosome analyses are ex- pected to clarify and explain the reasons to support the the chromosomes (Figs. 4 and 5). For D. trimaculatus, karyotype polymorphism and chromosome evolution in the chromosome markers are chromosome pairs 1 and these fishes. 24, which are the large acrocentric chromosome and the smallest telocentric chromosome, respectively. The im- Acknowledgements portant karyotype feature is the symmetrical karyotype, which was found in almost telocentric chromosomes. This work was supported by the Development and The karyotype formula of D. trimaculatus could be de- Promotion of Science and Technology talents project duced as: (DPST) and the Toxic Substances in Livestock and at t Aquatic Animals Research Group, Khon Kaen Univer- 2n () 48 =L2 +L 36 +M 10 sity. We would like to thank the cytogenetic research group for the accuracy check of the report and valuable Chromosome evolution of the Chrominae subfamily help. Karyotypic studies suggest that the karyotype with 48 acrocentric chromosomes (48 mono-armed) was the References ancestral karyotype in the Pomacentridae, and other karyotypes have been differentiated mainly by the pres- Aguilar, C. T., Oliveira-Corrêa, M. M. and Galetti, P. M. Jr. 1998. ence of pericentric inversions and/or Robertsonian re- Chromosome associations by centromeric heterochromatin in marine fishes. Chromosome Sci. 2: 73–76. arrangements (Takai and Ojima 1987, 1991a, b, 1995, Alvarez, M. C., Cano, J. and Thode, G. 1980. DNA content and chro- Kashiwagi et al. 2005). Previous studies on cytogenet- mosome complement of Chromis chromis (Pomacentridae, Perci- ics of the Chrominae subfamily and the present study formes). Caryologia 33: 267–274. demonstrated that there are four patterns of its chromo- Arai, R. 2011. Fish Karyotypes: A Check List. Springer Japan, Tokyo. some evolution. Firstly, the conserved 2n and karyotype Arai, R. and Inoue, M. 1976. Chromosomes of seven species of Poma- centridae and two species of Acanthuridae from Japan. Bull. were shown as ancestor. This was evident in studies of Natl. Mus. Nat. Sci. Ser. A Zool. 2: 73–78. C. chromis (2n=48, NF=48), C. multilineata (2n=48, Arai, R., Inoue, M. and Ida, H. 1976. Chromosome of four species of NF=48), C. ternatensis (2n=48, NF=48) and D. mel- coral fishes from Japan. Bull. Natl. Mus. Nat. Sci. Ser. A Zool. 2: anurus (2n=48, NF=48) (Alvarez et al. 1980, Ojima 137–141. and Kashiwagi 1981, Takai and Ojima 1999, Molina and Al-Sabti, K. 1985. Chromosomal studies by blood leukocyte culture Galetti 2002, Kashiwagi et al. 2005). Secondly, the oc- technique on three Salmonids from Yugoslavian water. J. Fish Biol. 26: 5–12. currence of pericentric inversion at telocentric chromo- Al-Sabti, K. 1987. Cytogenetic studies on five species of Pisces from somes provides the bi-arms chromosomes (metacentric, Yugoslavia. Cytobios 49: 175–188. submetacentric and acrocentric chromosome), which Brum, M. J. I. 1996. Cytogenetic studies of Brazilian marine fish. 2017 Chromosomal Characteristics of the Three-Spot Damselfish, Dascyllus trimaculatus (Perciformes, Pomacentridae) in Thailand 57

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