Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(4): 3-8, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2439 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Aiumsumang, S., Vidthay- anon, C., Kulabtong, S., Tanomtong, A., & Phimphan, S. (2023). First cytoge- netic study of the Somphong’s rasbora (Trigonostigma somphongsi ) (Perci- formes, Cyprinidae), a critically endan- gered species in Thailand. Caryologia 76(4): 3-8. doi: 10.36253/caryologia-2439 Received: December 30, 2023 Accepted: February 25, 2024 Published: March 14, 2024 Copyright: © 2023 Aiumsumang, S., Vid- thayanon, C., Kulabtong, S., Tanom- tong, A., & Phimphan, S. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) (Perciformes, Cyprinidae), a critically endangered species in Thailand Surachest Aiumsumang1, Chavalit Vidthayanon2, Sitthi Kulabtong3, Alongklod Tanomtong4, Sumalee Phimphan1,* 1 Biology Program, Faculty of Science and Technology, Phetchabun Rajabhat University, Phetchabun 67000, Thailand 2 Scientific Committee. SuebNakasathien Foundation, 140 Tiwanon Road, Bang Kraso Subdistrict, Mueang Nonthaburi District, Nonthaburi Province 11000, Thailand 3 Faculty of Agro-Industrial Technology, Rajamangala University of Technology Tawan-ok Chantaburi Campus, Chantaburi Province 22210, Thailand 4 Program of Biology, Faculty of Science, Khon Kaen University, Muang, Khon Kaen 40002, Thailand *Corresponding author. E-mail: sumalee.phi@pcru.ac.th; joodoof@gmail.com Abstract. The first chromosome study of Trigonostigma somphongsi (Meinken,1958) from Thailand. Specimens were collected from Pak Phli District, Nakhornnayok Prov- ince, central Thailand. The mitotic chromosomes were directly prepared from kidney tissues of two males and two females. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results shown that diploid chromosome number of T. somphongsi was 2n=50, the fundamental numbers (NF) were 92 in both male and female. The types chromosomes consisted of 10 metacen- tric, 20 submetacentric, 12 acrocentric and 8 telocentric chromosomes. The result also exhibited that the interstitial nucleolar organizer regions (NORs) were clearly observed at the short arm of chromosome pair 2. The karyotype formula could be deduced as: 2n (diploid) 50=10m+20sm+12a+8t. Keywords: karyotype, chromosome, Trigonostigma somphongsi. INTRODUCTION The Trigonostigma somphongsi is a critically endangered species in nat- ural habitat of Thailand. It has been discovered in a deep water rice field, floodplain of Bangpakong Basin, Nakhornnayok Province, central Thai- land. (Petsut et al. 2014). The Trigonostigma is a genus of small cyprinid fish found in Southeast Asia. There are currently five recognized species in this genus including T. heteromorpha, T. hengeli, T. somphongsi, T. espei and T. truncate (Tan, 2020). The T. somphongsi was found in the rice field and in a http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2439 https://doi.org/10.36253/caryologia-2439 http://www.fupress.com/caryologia mailto:sumalee.phi@pcru.ac.th mailto:joodoof@gmail.com 4 Surachest Aiumsumang et al. densely vegetated ditch, which was flooded by a nearby river. It differs from the rest of the genus in its dark pat- tern which occurs as a strait horizontal line that extends from the base of the caudal fin and ends just after it passed the posterior part of the dorsal fin, instead of showing the wedge-like marking of other species in the genus (Petsut et al, 2014) (Fig. 1). Although the Som- phong’s rasbora is important for fish biodiversity of Thailand, there were quite scarce data about cytogenetics in these fishes especially banding analysis in fish chro- mosomes. Up to date, information about karyotypes in genus Trigonostigma are rare and usually based on convention- al staining technique. Only T. heteromorpha has been published, and the result showed that the diploid chro- mosome number (2n) was 50 (Khuda 1979). The cytoge- netic studies using conventional staining technique pro- vided valuable information on the great karyotype diver- sity shown by these animals. Analyses of cytogenetic markers, included the number and karyotype formula, number and location of nucleolar organizer regions (NORs). The present study is the first report on the chro- mosomal characteristics of T. somphongsi determined using conventional staining and Ag-NOR banding tech- niques. T. somphongsi is a critically endangered species in natural habitats of Thailand. The results enhance the level of cytogenetic information available and enable future comprehensive studies to be conducted on taxon- omy and evolutionary relationships. Moreover, the data provide useful basic information for conservation and on breeding practices as well as an analysis of the chro- mosomal evolution of this species of Trigonostigma. MATERIALS AND METHODS Sample collection Two males and two female of T. somphongsi were collected from agricultural land, Pak Phli District, Nak- hornnayok Province, central Thailand, and were grown starting from May 2023 (permission from an ethi- cal committee ID U1-04498-2559). All specimens were maintained in aerated, flowing seawater acquaria until the analysis. Chromosome preparation Chromosomes preparations were obtained from kidney by cell suspension technique. Briefly, the kidney was cut into small pieces and then mixed with 0.075 M potassium chloride (KCl). After discarding all large piece tissues, cell sediments were transferred to a centrifuge tube and incubated for 30 minutes, then centrifuged at 1,500 rpm for 5 minutes. The KCl was discarded from the supernatant after centrifugation at 1,500 rpm for 5 minutes. Cells were fixed in fresh cool fixative (3 metha- nol:1 glacial acetic acid) and gradually made up to 8 ml before centrifuging again at 1,500 rpm for 5 minutes, whereupon the supernatant was discarded. Fixation was repeated until the supernatant was clear. The mixture was dropped onto a clean and cold slide by micropipette followed by air-drying technique. Chromosome staining Conventional staining of the chromosomes in the air-dried slides was done using 10% Giemsa solution for 10 minute (Rooney 2001). Ag-NOR banding was car- ried out following method of Howell and Black (1980) by adding 4 drops of 50% silver nitrate and 2% gelatin on slides. The slides were then sealed with cover glasses and incubated at 60°C for 5 minutes. After that the slides were soaked in distilled water until the cover glasses were separated. Chromosome checking Twenty clearly observable cells with well spread chromosomes of each male and female were selected and photographed under Olympus Bx63 microscope. Meta- phase figures were analyzed according to the chromo- some classification of Turpin and Lejeune (1965). The length of the short arm chromosome (Ls) and the long arm chromosome (Ll) were measured and the length of the total arm chromosome (LT, LT = Ls + Ll) was cal- culated. The relative length (RL), the centromeric index (CI), and standard deviation (SD) of RL and CI were estimated. The CI (q/p + q) between 0.50-0.59, 0.60-0.69, 0.70-0.89, and 0.90-0.99 were described as metacentric, submetacentric, acrocentric and telocentric chromo- Figure 1. The characteristic of T. somphongsi. Scale bar indicate 0.2 cm. 5First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) somes, respectively. The fundamental number (NF, num- ber of chromosome arms) was obtained by assigning a value of 2 to metacentric, submetacentric and acrocen- tric chromosomes and 1 to telocentric chromosome. All parameters were used in karyotyping and idiogram. RESULTS AND DISCUSSION This is the first report on cytogenetic characteriza- tion using conventional staining and Ag-NOR band- ing techniques for T. somphongsi. The results indicated diploid chromosome number (2n) were found 50 in all studies samples as show in Fig. 2. This result is coinci- dent with T. heteromorpha reports by Khuda 1979, and similar to another species in Rasbora (Post 1965; Man- na and Khuda-Bukhsh 1977; Khuda-Bukhsh et al. 1979; Donsakul and Magtoon 1995; Donsakul and Magtoon 2002; Seetapan and Moeikum 2004; Donsakul et al. 2005; Donsakul et al. 2009; Yeesaem et al. 2019; Aium- sumang et al. 2021; 2022). From the previous report, most of cyprinid species have 2n=50, chromosome con- sisting of both mono- and bi-arm chromosomes. There is no observation of strange size chromosomes related to sex, which is in accordance to the author of this genus (Khuda 1979). The types chromosomes of T. somphongsi were 10 metacentric, 20 submetacentric, 12 acrocentric and 8 telocentric chromosomes. The mean values calcu- lated from twenty mitotic metaphases showed the cen- tromeric index of chromosome complements ranging from 0.569±0.001 to 1.000±0.000 (Table 1). To our results, NORs could be observed in one pair of chromosomes in both male and female of T. som- phongsi. The result demonstrated that the chromosome marker shows in the chromosome pair 2, which is meta- centric chromosome (Fig. 3). An important character- istic of Nucleolar Organizer Regions (NORs) in fish is related to that it has inter- and intra-species polymor- phism. NORs characters can be a cytogenetic marker for cytotaxonomic studies and also have been used for studying of phylogenetic relationships among the Cypri- nid fishes (Amemyia and Gold 1988; Galetti Jr 1998; Almeida-Toledo et al. 2000). The important karyotype feature of T. somphongsi is the symmetrical karyotype, which were found in four types of chromosomes (meta- centric, submetacentric, acrocentric, and telocentric chromosomes). Figure 4 show the idiograms from con- ventional staining and Ag-NOR banding techniques. The karyotype formula could be deduced as: 2n (dip- loid) 50=10m+20sm+12a+8t. The study on fish chromo- somes is the basic knowledge which can be applied for the several fields such as classification, evolution, hered- ity, systematic (Gold et al. 1990; Ueda et al. 2001; Barat et al. 2002; Barat and Sahoo 2007), breeding, rapid pro- duction of inbred lines and cytotaxonomy (Kirpichnikov 1981). Furthermore, cytogenetic studies on fish have also been used as biological indicator to determine the eco- logical toxicology (Klinkhardt, 1993) and cytogenetic techniques have been widely applied to improve farmed stocks in many aquaculture species in the World (Beard- more et al. 2001; Desprez et al. 2003). Here, we have that the karyotype of T. somphongsi is 2n=50 and might represent a derived character, probably also shared by all members of the Trigonostigma clade. Besides, our study is the first cytogenetic karyotype data to describe in detail the karyotypic features of the Som- Figure 2. Metaphase chromosome plates and karyotypes of the T. somphongsi by conventional staining. Species share the karyotype com- posed of 50 chromosomes. Scale bar indicate 5 µm. 6 Surachest Aiumsumang et al. Table 1. Karyomorphological details of T. somphongsi from 20 metaphases chromosome, 2n (diploid)=50. Chro. pair Ls Ll LT RL ± SD CI ± SD Chro. size Chro. type 1 2.651 2.965 5.616 0.047±0.002 0.569±0.001 Large metacentric 2 2.547 2.882 5.429 0.045±0.002 0.574±0.001 Large metacentric 3 2.433 2.716 5.149 0.043±0.003 0.571±0.002 Large metacentric 4 2.337 2.709 5.046 0.040±0.001 0.583±0.002 Medium metacentric 5 2.927 2.609 5.536 0.039±0.001 0.583±0.002 Medium metacentric 6 2.800 3.982 6.782 0.045±0.002 0.688±0.004 Large submetacentric 7 2.692 3.237 5.929 0.044±0.001 0.680±0.001 Large submetacentric 8 2.764 3.753 6.517 0.042±0.004 0.682±0.002 Large submetacentric 9 2.985 3.706 6.691 0.040±0.004 0.674±0.002 Large submetacentric 10 2.597 3.646 6.243 0.037±0.005 0.694±0.003 Medium submetacentric 11 2.564 3.480 6.044 0.035±0.003 0.692±0.005 Medium submetacentric 12 2.488 2.842 5.330 0.030±0.003 0.653±0.001 Medium submetacentric 13 2.488 2.842 5.330 0.030±0.003 0.653±0.001 Medium submetacentric 14 2.293 4.470 6.763 0.029±0.008 0.669±0.002 Medium submetacentric 15 2.121 3.942 6.063 0.025±0.003 0.664±0.003 Medium submetacentric 16 0.549 3.998 4.547 0.041±0.001 0.898±0.004 Large acrocentric 17 0.404 3.565 3.969 0.038±0.001 0.894±0.004 Large acrocentric 18 0.354 3.445 3.799 0.038±0.001 0.871±0.004 Large acrocentric 19 0.344 3.014 3.358 0.037±0.001 0.874±0.004 Medium acrocentric 20 0.175 2.985 3.160 0.035±0.001 0.872±0.004 Medium acrocentric 21 0.168 2.014 2.182 0.031±0.001 0.890±0.004 Medium acrocentric 22 0.000 4.624 4.624 0.029±0.001 1.000±0.000 Large telocentric 23 0.000 3.615 3.615 0.027±0.001 1.000±0.000 Large telocentric 24 0.000 3.550 3.550 0.026±0.001 1.000±0.000 Medium telocentric 25 0.000 2.989 2.989 0.026±0.001 1.000±0.000 Medium telocentric Remarks: Ls=short arm chromosome, Ll=length of long arm chromosome, LT=length of total chromosomes, RL=relative length, CI=centromeric index, SD=standard deviation. Figure 3. Metaphase chromosome plates and karyotypes of the T. somphongsi. The arrows indicate NOR banding by Ag-NOR staining tech- nique. Scale bar indicate 5 µm. 7First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) phong’s rasbora species. The data can be a support for the investigation of chromosomal cytotaxonomy evo- lutionary history of relationships, conservation and on breeding practices within Trigonostigma. ACKNOWLEDGEMENTS This work was financially supported by Critical Ecosystem Partnership Fund (CEPF). We would like to thank Phetchabun Rajabhat University for available help. REFERENCES Aiumsumang S, Phimphan S, Suwannapoom C, Chai- yasan P, Supiwong W, Tanomtong A. 2021. A com- parative chromosome study on five Minnow fishes (Cyprinidae, Cypriniformes) in Thailand. Caryolo- gia 74(1): 89 –196. https://doi.org/10.36253/caryolo- gia-1017. Aiumsumang S, Phimphan S, Tanomtong A, Supiwong W. 2022. Chromosome study of Rasbora triline- ata and Rasbora borapetensis (Cyprinidae, Cyprini- formes): Reveal by conventional staining technique. Science Technology and Engineering Journal 8(2): 29–39. Almeida-Toledo, LF, Foresti F. Toledo-Filho SA. 1984. Complex sex chromosome system in Eigenmannia sp. (Pisces, Gymnotiformes). Genetica 64: 165–169. https://doi.org/10.1007/BF00115340. Amemiya CT, Gold JR. 1988. Chromosomal NORs as taxonomic and systematic characters in North Amer- ican cyprinid fishes. Genetica 76: 81–90. https://doi. org/10.1007/BF00058806. Barat A, Sahoo PK. 2007. Karyotype analysis of Channa punctatus (Pisces) using restriction endonucleases. Cytologia 72: 471–473. https://doi.org/10.1508/cyto- logia.72.471. Barat A, Sahoo PK, Ponniah AG. 2002. Karyotype and Nucleolar Organizer Regions (NORs) in a few hill stream fishes. In: Ayyappan, S., Jena, J. K. and Joseph, M. M. (Eds.), The Fifth Indian Fisheries Forum Pro- ceedings, AFSIB, Mangalore and AoA, Bhubaneswar, pp.111–114. Beardmore JA, Mair GC, Lewis RI. 2001. Monosex male production in finfish as exemplified by tilapia: appli- cations, problems, and prospects. Aquaculture 197: 283–301. doi:10.1016/S0044-8486(01)00590-7. Desprez D, Mélard C, Hoareau MC, Belleme`ne Y, Bosc P, Baroiller JF. 2003. Inheritance of sex in two ZZ pseudofemale lines of tilapia, Oreochromis aureus. Aquaculture 218: 131–140. https://doi.org/10.1016/ S0044-8486(02)00572-0. Figure 4. Idiogram showing lengths and shape of chromosomes of the T. somphongsi, n (haploid)=50, by conventional staining (A) and Ag- NOR staining technique (B). The arrows indicate NOR banding. https://doi.org/10.36253/caryologia-1017 https://doi.org/10.36253/caryologia-1017 https://doi.org/10.1007/BF00115340 https://doi.org/10.1007/BF00058806 https://doi.org/10.1007/BF00058806 https://doi.org/10.1508/cytologia.72.471 https://doi.org/10.1508/cytologia.72.471 https://doi.org/10.1016/S0044-8486(02)00572-0 https://doi.org/10.1016/S0044-8486(02)00572-0 8 Surachest Aiumsumang et al. Donsakul T, Magtoon W. 1995. Karyological studies of Puntius brevis (Bleeker, 1850) and Cyclocheilichthys enoplos in Thailand. Proceeding of 21st Congress on Science and Technology of Thailand, pp. 292–293. Donsakul T, Magtoon W. 2002. Karyotype of Rasbora Caudimaculata, R. myersi, R. retrodorsalis, R. paviei from Thailand. Academic conference seminar for research papers of Srinakharinwirot University (pp. 1–7). Bangkok. Donsakul T, Magtoon W, Rangsiruji W. 2005. Karyotype of five species of fish (Pla siew) in subfamily Rasbo- ranae. 31st Congress on Science and Technology of Thailand. Suranaree University of Technology. Nak- hon Ratchasima Province Thailand. Donsakul T, Rangsiruji A, Magtoon W. 2009. Karyotypes of five cyprinid fishes (Cyprinidae, DanioninaeDan- ionini): Rasbora agilis, R. dorsiocellata, R. rubrodor- salis, Boraras maculate and B. urophthalmoides from Thailand. In Proceedings of the 47th Kasetsart Uni- versity Annual Conference (pp. 320–327). Kasetsart University, Thailand Galetti Jr PM. 1998. Chromosome diversity in neotropi- cal fish NOR studies. Italian Journal of Zoology. 65: 53–56. https://doi.org/10.1080/11250009809386795. Gold JR, Li YC, Shipley NS, Powers PK. 1990. Improved methods for working with fish chromosomes with a review of metaphase chromosome banding. Journal of Fish Biology 37: 563–575. https://doi. org/10.1111/j.1095-8649.1990.tb05889.x. Howell WM, Black DA. 1980. Controlled silver-staining of nucleolus organizer regions with a protective col- loidal developer: A 1-step method. Experientia 36: 1014–1015. https://doi.org/10.1007/BF01953855. Khuda-Bukhsh AR. 1979. Karyology of two species of hillstream fishes, Barilius bendelisis and Rasbora daniconius (Fam: Cyprinidae). Current Sci. 48(17): 793–794. Manna GK, Khuda-Bukhsh, A.R. 1977. Karyomorpholo- gy of cyprinid fishes and cytological evaluation of the family. The Nucleus, 20: 119–127 Petsut N, Panitvong N, Kulabtong S, Petsut J, Nonpay- om C. 2014.  The first record of  Trigonostigma som- phongsi  (Meinken,1958), a critically endangered spe- cies, in its natural habitat of Thailand (Cypriniformes Cyprinidae). Biodiversity Journal 5(4): 471–474. Post A. 1965. Vergleichede Untersuchungen der chromo- somenzahlen bei Susswasser-Teleosteern. In: Gylden- holm, A.O. and Scheel, J.J. Chromosome number of fishes I. Journal Fish Biology, 3: 47–93. Rooney DE. 2001. Human Cytogenetics: Constutition- al analysis: A Practical approach. 3rd ed. London: Oxford University Press. Seetapan K, Moeikum T. 2004. Karyotypes of ten Cypri- nid fishes (Family Cyprinidae). Journal Agricultural Extension, 22: 92–101 Tan HH. 2020.  Trigonostigma truncata, a new species of harlequin rasbora from Malay Peninsula (Teleostei: Danionidae). Raff. Bull. Zool. 68: 421–433. https:// doi.org/10.26107/RBZ-2020-0058 Turpin R, Lejeune J. 1965. Les Chromosomes Humains. Gauthier Pillars. Ueda T, Naoi H, Arai R. 2001. Flexibility on the karyotype evolution in bitterlings (Pisces, Cyprinidae). Genetica 111: 423–432. https://doi.org/10.1023/a:1013703717626 Yeesaem N, Jantarat S, Yeesin P. 2019. Cytogenetic Char- acterigation of Rasbora einthovenii in Sirindhorn Peat Swamp Forest, Narathiwat Province. Journal of Fish- eries Technology Research, 13: 58–68. https://doi.org/10.1080/11250009809386795 https://doi.org/10.1111/j.1095-8649.1990.tb05889.x https://doi.org/10.1111/j.1095-8649.1990.tb05889.x https://doi.org/10.1007/BF01953855 https://doi.org/10.26107/RBZ-2020-0058 https://doi.org/10.26107/RBZ-2020-0058 https://doi.org/10.1023/a:1013703717626 First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) (Perciformes, Cyprinidae), a critically endangered species in Thailand Surachest Aiumsumang1, Chavalit Vidthayanon2, Sitthi Kulabtong3, Alongklod Tanomtong4, Sumalee Phimphan1,* Evaluation of the evolutionary process within Populus caspica species from Hyrcanian forests by karyotype analysis Fereshteh Asadi-Corom1,*, Farhad Asadi2, Hossein Mirzaie-Nodoushan1 Chromosomal and genome size variations in Opium poppy (Papaver somniferum L.) from Afghanistan Sayed Zia Rasekh, Ghasem Karimzadeh* Karyological analyses in several Algerian populations of six species of the genus Vicia L. 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